extfieldsofstudy: Biology Medicine

text:
Identification and Gene Expression Analysis of a Large Family of Transmembrane Kinases Related to the Gal/GalNAc Lectin in Entamoeba histolytica

ABSTRACT We identified in the Entamoeba histolytica genome a family of over 80 putative transmembrane kinases (TMKs). The TMK extracellular domains had significant similarity to the intermediate subunit (Igl) of the parasite Gal/GalNAc lectin. The closest homolog to the E. histolytica TMK kinase domain was a cytoplasmic dual-specificity kinase, SplA, from Dictyostelium discoideum. Sequence analysis of the TMK family demonstrated similarities to both serine/threonine and tyrosine kinases. TMK genes from each of six phylogenetic groups were expressed as mRNA in trophozoites, as assessed by spotted oligoarray and real-time PCR assays, suggesting nonredundant functions of the TMK groups for sensing and responding to extracellular stimuli. Additionally, we observed changes in the expression profile of the TMKs in continuous culture. Antisera produced against the conserved kinase domain identified proteins of the expected molecular masses of the expressed TMKs. Confocal microscopy with anti-TMK kinase antibodies revealed a focal distribution of the TMKs on the cytoplasmic face of the trophozoite plasma membrane. We conclude that E. histolytica expresses members of each subgroup of TMKs. The presence of multiple receptor kinases in the plasma membrane offers for the first time a potential explanation of the ability of the parasite to respond to the changing environment of the host.

The Gal/GalNAc lectin of Entamoeba histolytica mediates parasite adherence to the host and signals the initiation of cytolysis (41,44,45,49). It is a heterotrimer consisting of covalently linked heavy (Hgl) and light (Lgl) subunits with a noncovalently linked intermediate (Igl) subunit (9,36,37,43,46). The Igl subunit of the Gal/GalNAc lectin has two known family members, Igl1 and Igl2. The Igl subunit has sequence similarity to the variant surface protein (VSP) of Giardia. We have previously identified a large number of proteins in the genome of E. histolytica containing CXXC motifs similar to those of Igl (8). Here we show that these CXXC-rich proteins form a large family of E. histolytica transmembrane kinases (TMKs) with highly variable extracellular domains homologous to Igl and VSPs of Giardia and with cytoplasmic kinase domains.
Amebic trophozoites have been demonstrated to persist in humans for longer than 6 months (21,22). This prolonged period of infection suggests that the amebae evade the immune system. Other protozoan parasites, such as Plasmodium, Giar-dia, and Trypanosoma brucei, are also able to infect the host for long periods in spite of inducing robust immune responses. The mechanism(s) of persistence of these organisms is thought in part to be due to the variation of surface proteins. Plasmodium falciparum has three families of var genes that are independently expressed (29). The highest variation rate of these families is 2% per generation (52). Giardia encodes a family of 100 to 150 VSPs whose surface expression changes at a rate of one variation every 5 to 13 generations (38). T. brucei has a family of over 1,000 variant surface glycoproteins that change at a rate of 10 Ϫ2 to 10 Ϫ7 variations per generation (13,51).
The discovery of the large family of CXXC-containing TMKs is of interest not only for their potential role in antigenic variation but also for their role in cell signaling. E. histolytica must respond to a wide variety of environmental stimuli as it excysts into a trophozoite in the intestinal lumen and enters the host by invasion of the intestinal mucosal epithelium. Invasion involves attaching to the epithelium and responding to that attachment event through signaling events via the E. histolytica Gal/GalNAc adherence lectin that lead to host cell killing. The changing host environment should necessitate having a variety of ways of sensing and responding to the host.
Here we report sequence and expression analysis of the TMKs in laboratory-cultured trophozoites. An oligoarray and real-time PCR were used to measure the expression in cultured trophozoites of the TMK genes. We demonstrate that there are six families of TMKs, with each having one or more family members expressed. In addition, anti-TMK antibodies were used to localize the TMKs to the plasma membrane of trophozoites, consistent with their proposed function in sensing the environment. microtiter plates. The panel of probes, including control (housekeeping) oligonucleotides, was printed with two spot replicates on Corning UltraGAPS coated slides using an Affymetrix 417 arrayer (Affymetrix, Santa Clara, CA). Slide quality control was analyzed by hybridizing two randomly selected slides per batch (up to 40 slides/batch) with Cy3-labeled universal oligonucleotide probes. The hybridized slides were then washed and scanned with a ScanArray 4000 scanner (PerkinElmer Life Sciences Inc., Boston, MA). If the spotting quality standards were met, the batch was deemed satisfactory for analysis.
Erythrophagocytosis. Human erythrocytes were isolated using Mono-Poly resolving medium (ICN Biomedicals, Aurora, OH) according to the manufacturer's directions. Erythrocytes were washed twice in 10 mM HEPES (pH 7.0), 140 mM sodium chloride, and 0.1% bovine serum albumin and then resuspended in the same buffer until use. One million log-phase trophozoites were grown in 50 ml of TYI-S-33 medium for 24 h in the presence or absence of 24 million erythrocytes per ml of medium.
Isolation of RNA. Amebae were lysed with 2 ml of buffer RLT containing ␤-mercaptoethanol (the first component of the RNeasy kit from Qiagen, Valencia, CA). Samples were processed immediately or flash-frozen in liquid nitrogen and stored at Ϫ80°C until processing using QIAshredders, followed by the RNeasy mini kit, including all optional steps and a 5-min incubation with buffer RWI. Samples were treated on the columns with RNase-free DNase from Qiagen according to the manufacturer's directions (Qiagen). Samples were analyzed for residual DNA contamination by PCR using primers for Jacob (conditions are described below). Samples that contained residual DNA were retreated with DNase I (Roche, Indianapolis, IN) for 1 h at 37°C in a 100-l total volume with 10 l of 10ϫ DNase I buffer (100 M Tris [pH 7.5], 25 mM MgCl 2 and 5 mM CaCl 2 ) and 3 l DNase I, repurified on RNeasy columns and rescreened for residual DNA contamination. The Agilent BioAnalyzer (Agilent Technologies, Palo Alto, CA) was used to assess RNA quality. The results were inspected to ensure that both ribosomal peaks were intact and that no degradation had occurred. Acceptable 260/280 ratios ranged from 1.8 to 2.1.
Sample labeling, hybridization, and scanning. DNA oligoarray assay of gene expression used cohybridization of two fluorescently labeled cDNA targets, prepared from different samples. For routine oligoarray expression analysis, a previously described (24) indirect labeling procedure was used. Approximately 10 g of RNA per sample and random hexamers were used for synthesis of cDNA containing amino-allyl-labeled nucleotides. The newly synthesized cDNA was then labeled by a covalent coupling of an appropriate cyanine fluor (CyDye postlabeling reactive dye pack; Amersham Biosciences Corp., Piscataway, NJ). In a typical oligoarray assay, the cDNA of one preparation (control) was labeled with Cy5, while the second cDNA (experiment) was labeled with Cy3. Both reactions were purified with a QIAquick PCR purification kit (Qiagen) for removal of the uncoupled dye. The labeling efficiencies of the purified target preparations were examined by spectrophotometry, as well as by calculations of the mass of cDNA and Cy5 or Cy3 dye incorporation. The nucleotide-to-dye molecular ratios were considered suitable for oligoarray experiments with a ratio of less then 50 nucleotides/dye molecule. Both targets were equalized based on the total amount of dyes incorporated before hybridization (24). The samples were mixed, dried by speed vac, and dissolved in hybridization buffer solution (50% formamide, 5x SSC [1ϫ SSC is 0.15 M NaCl plus 0.015 M sodium citrate] and 0.1% sodium dodecyl sulfate [SDS]). The cDNA-containing hybridization solution was then denatured, applied to the oligoarray (prehybridized in 5x SSC, 0.1% SDS and 1% bovine serum albumin), and hybridized at 42°C for 18 h. Following 5-min washes in 2ϫ SSC-0.1% SDS and in 0.1ϫ SSC, the slide was scanned using a ScanArray 4000 scanner (PerkinElmer, Wellesley, MA). Both Cy5 and Cy3 images of one experiment were analyzed with QuantArray 3.0 microarray analysis software. Normalization to median between both channels was used.
RT-PCR primer design. Real-time PCR (RT-PCR) primers (Table 1) were designed using Beacon Designer 2.0 (Premier Biosoft International, Palo Alto, CA). RT-PCR primers and oligoarray probes were designed independently; thus, the PCR fragment and the oligoarray probe represented different regions of the same gene. Each primer was analyzed against the TIGR E. histolytica database, and any primer that had significant sequence similarity to multiple genes was rejected. Thus, both the forward and reverse primers were specific for one gene, except actin, Jacob, and Hgl, which detected all family members and/or alleles in the genome. Optimal annealing conditions (determined by gradient PCR) were VOL. 4,2005 E. HISTOLYTICA TRANSMEMBRANE KINASE GENE FAMILY used to ensure specificity, and any PCR primer pair that produced more than one melt peak was discarded. PCR products that produced single melt peaks were analyzed by gel electrophoresis in 1.5% agarose-Tris-borate-EDTA, and if multiple bands were observed, the primer pair was discarded. Finally, all PCR products were sequenced using the forward amplification primer to verify specificity.
Real-time PCR validation of oligoarray results. RNA was reverse transcribed using iQsuperscript (Bio-Rad, Hercules, CA) according to the manufacturer's directions. cDNA levels were measured using RiboGreen (Molecular Probes, Eugene, OR) with DNA of known quantity as a standard in a SPECTRAmax Gemini EM fluorescent plate reader, according to the manufacturer's directions. Before proceeding to analysis of cDNA samples, a no-reverse-transcriptase control PCR was done using primers to the cyst-specific gene Jacob to verify that there was no residual DNA contamination of the samples. All samples were analyzed in duplicate, and all time points were analyzed in triplicate. Each time point was thus represented in six wells during the real-time PCR assays, and the six wells were averaged after normalization to the RNA polymerase II gene's average (47). Two sequentially performed growth curves were analyzed in the real-time PCR assays to ensure reproducibility. All real-time PCR assays were quantitative to allow direct comparison of gene expression levels.
A PCR master mix consisted of 1,100 l of iQSYBRGreen super mix (Bio-Rad, Hercules, CA), 1,100 l of distilled H 2 O, 88 l of forward primer (50 pmol/l), and 88 l of reverse primer (50 pmol/l). To each well containing 2 l of cDNA was added 25 l of master mix. Duplicate assays were performed on each sample. Each assay included standards, no-DNA-control wells, and no-RTcontrol wells. The cycling conditions were 95°C for 5 min; 30 cycles of 95°C for 30 s, annealing for 30 s (see Table 1 for annealing temperatures), and 72°C for 30 s; and 1 cycle of 72°C for 2 min 30 s followed by a 90-step melt curve increasing 0.2°C with a 5-s hold.  EUKARYOT. CELL Production of anti-⌬TMK96 rabbit serum production. The kinase region of Tmk96 (⌬TMK96) was PCR amplified with the primers 5Ј-CAATTTAGAGAA GGAATTCCT-3Ј (5Ј primer) and 5Ј-TCACATTAATTGAAGATGTTTTAAA ACAACA-3Ј (3Ј primer). This 1,000-bp fragment was cloned into TOPO NT/T7 (Invitrogen), in frame with an amino-terminal six-His tag via TA cloning. Bacteria were grown at 37°C to an optical density at 600 nm of 0.5 and induced with isopropyl-␤-D-thiogalactopyranoside for 4 h, and the recombinant protein was purified with nickel agarose beads (Qiagen). Antibodies were raised to this purified recombinant protein by a 90-day protocol including three inoculations of New Zealand White rabbits with recombinant ⌬TMK96 (Covance, Princeton, NJ), and the antibodies were purified from serum with a protein A column.
Western blots using ⌬TMK96. Soluble proteins were extracted from amebae by harvesting 5 ϫ 10 7 trophozoites by incubation on ice for 10 min, followed by centrifugation (200 ϫ g at 4°C for 5 min). The amebae were lysed in 10 mM sodium phosphate buffer with protease inhibitor cocktail I (Sigma, St. Louis, MO) per the manufacturer's directions. Membranes were then cleared by centrifugation (100,000 ϫ g at 4°C for 1 h). Whole-cell lysates were prepared by sonication of 10 6 amebae in three 5-min pulses on ice. Large intact particles were eliminated by centrifugation (20,000 ϫ g at 4°C for 30 min). All samples were then separated on 10% polyacrylamide gels and then electrotransferred to polyvinylidene difluoride membranes (Millipore, Bedford, MA). Nonspecific binding was blocked by preincubation with Tris-buffered saline with 5% bovine serum albumin and 0.05% Tween 20 (TBST). In order to detect proteins on the blot, either anti-⌬TMK96 rabbit serum (a dilution of 1:5,000) or preimmune serum (1:2,500) was added in TBST for 1 h at room temperature. Interactions were detected by the addition of peroxidase-conjugated goat anti-rabbit IgG (Sigma) and development with ECL (Amersham) per the manufacturer's directions.
Confocal microscopy. E. histolytica trophozoites (10 6 ) were bound to glass coverslips in a 24-well plate for 30 min at 37°C in TYI-S-33 medium. Adherent amebae were washed twice in phosphate-buffered saline (PBS) and fixed in 3% paraformaldehyde for 30 min at room temperature. To make the plasma membrane permeable, amebae were treated with 0.2% Triton X-100 in PBS for 1 min. Nonspecific binding was blocked by incubation with 20% goat serum and 5% bovine serum albumin (Sigma) in PBS for 1 h at 37°C. After incubation with either the anti-⌬TMK96 rabbit polyclonal antibody (200 g/ml) or anti-Gal/ GalNAc lectin antibody (6 g/ml) for 1 h at 37°C, the coverslips were washed twice before Cy3-conjugated goat anti-rabbit secondary antibodies (Jackson Laboratories, Bar Harbor, ME) were added at a 1:160 dilution for 1 h at 37°C. The coverslips were washed twice and mounted on slides with Gel/Mount (Biomeda, Foster City, CA). Confocal images were visualized using a Zeiss LSM 510 laser scanning microscope (Carl Zeiss, Inc., Thornwood, NY).

RESULTS
Phylogenetic analysis of the E. histolytica TMK family. Previously we reported a family of CXXC-containing proteins with sequence similarity to Igl1 and Igl2 (8). With the completion of the genome project, it became apparent that the majority of these CXXC-containing proteins were members of an approximately 80-gene family with intracellular kinase domains. The extracellular domains shared sequence similarity to Igl and to VSPs of Giardia. The kinase domain of these proteins in most cases contained all five conserved kinase motifs: the key conserved glycine-rich motif, the K residue, and the HRDL, DFG, and APE motifs (Table 2). However, a few lacked the glycinerich motif (Tmk01, Tmk24, Tmk45, and Tmk73), which helps coordinate the second phosphate of ATP (14). The glycine, K, and DFG motifs are implicated in the binding and orientation of ATP. The HRDL motif is involved in catalysis. The APE motif is responsible for anchoring the substrate and thereby influencing the specificity of interaction with the protein that is being phosphorylated (14,20,60). Despite the conservation of the essential kinase motifs, overall the kinase domains were , which are the TGF␤ receptor and activin family of serine/ threonine kinases, OPK VIII, which are a Raf family of serine/ threonine kinases, and OPK XII, which are the casein kinase I family of kinases (Fig. 1). It was not possible to determine by sequence analysis alone if the TMKs were serine/threonine or tyrosine kinases. Sp1A from Dictyostelium discoideum was the most closely related kinase identified by sequence similarity in the NCBI database that has been functionally characterized. The Sp1A kinase has been shown to be a dual-specificity kinase that phosphorylates both tyrosine and serine/threonine residues (40). The Sp1A kinase was found to be phylogenetically within the TMK family (Fig. 1). Unlike the TMK family, SplA is a cytoplasmic kinase rather than a transmembrane kinase.
Two TMKs (Tmk58 and Tmk89) clearly did not group with the TMK family but grouped closely with other protein kinase groups VI and VII (data not shown), suggesting that these may represent serine/threonine transmembrane kinases that have evolved separately from the TMK family. The TMK family members were grouped based on sequence and phylogenetic analysis of their cytoplasmic kinase domains. A specific signature motif between the conserved HRDL and APE motifs was identified for each family ( Table 2) I  II  III  IV  V   Tyrosine LGxGxFGxVxxG VA(I/V)K ExxxM(R/K) GxCooo GxL(F/Y)lx

Ser/Thr
LGxGxFGxVxxx VAIK Gsgxdx gxossoL a Subdomain is according to Hanks's alignment of kinases (20). Underlined in tyrosine and serine/threonine kinases indicate highly conserved residues. Letters in uppercase are conserved. Letters in lowercase indicate moderate conservation. x, any residue; o, hydrophobic residue; -, sequence of varying length.

726
BECK ET AL. EUKARYOT. CELL dues N-terminal to the APE motif (group F) ( Table 2 and Fig.  2). Tmk58 and Tmk89, which did not group with the rest of the TMKs, contained an ITDFGLAKK motif. Group G TMKs lacked a conserved motif in the kinase domain and also lacked one or more of the conserved kinase domain motifs. Sequence similarity between family members was not limited to the kinase domain. Additional motifs were found in some families that can be used to identify group members between the cytoplasmic and kinase domain ( Table 2 footnotes). Additionally, group D TMKs had a serine-rich region N-terminal to their kinase domain and a serine/threonine region C-terminal to their kinase domain. Serine/threonine rich regions are often found in kinases and typically regulate kinase activity (30). The kinase domains of many of the E. histolytica TMKs were a mix of serine/threonine and tyrosine kinase signature motifs, further complicating efforts to predict their kinase activity (Table  3). However, the sequence similarity to SplA of D. discoideum suggests that they likewise may be dual-specificity kinases. An overview of the sequence motifs of the different subfamilies of kinases is shown in Table 2 (all domains except kinase domains), Table 3 (kinase domains), and Fig. 2. The TMKs had extracellular domains of 36 to 2,117 amino acids (with 11 to 98% similarity between family members), a transmembrane domain, and a kinase domain. Groups B, C, and D shared significant sequence similarity in their extracellular domain to Igl1 (Fig. 2). Most extracellular domains were rich in CXC/ CXXC/CXXXC motifs. Almost all of the TMKs had at least one CXXCXXGYY motif beginning approximately 25 amino acids N-terminal to the transmembrane domain. Like Igl1 most of the TMKs had many additional CXXCXXGYY motifs ( Table 2 and Fig. 2). A very similar C(D/E)XCXXG(Y/F) (Y)(G) motif was found in Igl, VSP of Giardia (1,38), and laminin LE domains (27,57,63). In VSP of Giardia the CXXC motifs have not been crystallized but have been shown to bind zinc (38), to have their N terminus at the host parasite inter-face (38), and have nonreactive cysteines (42). In laminin the LE domains form a rod-like structure of mini-globular folds (57,63). This is consistent with the CXXCXXGYY motifs forming a linear array of mini-globular folds. By analogy to the laminin LE domains, the TMKs may have a rod-like structure.
Gene expression analysis of E. histolytica during growth. To characterize the expression profile of these genes, we constructed an oligoarray of the TMK genes, Gal/GalNAc lectin genes, and other putative surface virulence genes. Few changes in gene expression were seen when early phase (12 h) was compared with mid-log phase (48 h) and late log phase (96 h) was compared with nonadherent phase (144 h) ( Table 4). When mid-log-phase (48 h) and late-log-phase (96 h) amebic cultures were compared, very little change was seen (data not shown). actin was clearly growth regulated (Table 4), as were the Gal/GalNAc lectin hgl genes, many but not all of which decreased significantly during late log phase (Table 4 and data not shown). This decrease is consistent with previous observations of hgl1, hgl2, and hgl3 gene expression (48). Expression of known genes was, in general, consistent with RT-PCR, Western, and/or Northern analysis (data not shown). We concluded that E. histolytica did not appear to growth-phase regulate expression of most putative virulence genes when grown under lab culture conditions. Expression analysis of the TMKs. The expression profile of 67 of the TMK genes during mid-log phase (72 h) is shown in Fig. 3. At this time point, 19 genes (group A, tmk61, tmk65, and tmk72; group B2, tmk02, tmk08, and tmk74; group B3, tmk21 and tmk28; group C, tmk39 and tmk63; group D1, tmk40 and tmk56; group D2, tmk19, tmk44 and tmk46; group E, tmk22 and tmk54; group F, tmk59; group G, tmk06) (Fig. 3) showed hybridization values in trophozoites significantly greater than that for the cyst-specific transcript jacob (P Ͻ 0.05).
Analysis of TMK expression by RT-PCR. Real-time PCR was conducted on jacob, tmk31, tmk80, tmk96, and tmk98, all of  which were predicted by oligoarray not to be expressed, and tmk19, tmk21, tmk63, tmk65, tmk71, tmk75, sa, hgl, and actin, all of which were predicted to be expressed. Gene expression was monitored sequentially in trophozoites in laboratory culture over a 12-day period. To allow comparison between time points, results were normalized to the average of three RNA polymerase II genes (Fig. 4). All real-time PCR results of the TMK genes were consistent with the oligoarray results. Significant variations in expression during laboratory culture were observed for tmk19, tmk63, and tmk79 (P Ͻ 0.01) (Fig. 4D, 4F, and 4I). Expression analysis of the TMKs during erythrophagocytosis. One million trophozoites were grown in 50 ml of medium with or without a vast excess of erythrocytes (24 million/ml of medium) for 24 h. We did not observe significant changes in TMK gene expression during erythrophagocytosis (data not shown).

728
BECK ET AL. EUKARYOT. CELL TMKs that do not have an extracellular domain or whose extracellular domain has been cleaved off. Localization of TMK kinase domains to plasma membrane microdomains. Permeabilized amebae showed a focal plasma membrane staining pattern with anti-TMK kinase domain antibodies (Fig. 5B). No staining was seen with nonpermeabilized cells or with permeabilized cells stained with preimmune sera (data not shown). We concluded that the kinase domain was on the cytoplasmic side of the plasma membrane. The TMKs therefore appeared to be typical type I transmembrane proteins with an amino-terminal signal sequence and a predicted transmembrane domain preceding the kinase domain. The focal staining pattern contrasted with the uniform plasma membrane staining pattern seen with anti-Gal/GalNAc lectin antisera (Fig. 5C).

DISCUSSION
The most important finding of this work is the identification of a large family of over 80 transmembrane kinases in E. histolytica. Although eukaryotic-type transmembrane kinases are found in organisms from bacteria to humans (4,64,68), large families of TMKs have been previously described only in multicellular organisms (20). For example, we were not able to find any TMKs in the partially completed genome of the ameba D. discoideum (17), and only a few TMKs were found in the apicomplexan P. falciparum (4). The TMKs were also found in Entamoeba invadens, Entamoeba dispar, and Entamoeba moshkovskii, indicating that this family is conserved within Entamoeba (data not shown) (66). Multiple TMKs were observed to be expressed at the mRNA and protein levels, The existence of multiple different extracellular domains of the TMKs suggests that each interacts with the host environment and signals into the parasite in distinct ways. The extracellular domains of the group B, C, and D TMKs had sequence similarity to Igl of E. histolytica (8), laminin LE domains (27,57,63), and VSPs of Giardia lamblia (1,38). The sequence similarity is largely limited to a repeated CXXCXXGYY motif. In laminin the LE domains function as mini-globular folds arranged in tandem to form a rod-like structure. If the CXX CXXGYY motifs in the TMKs take on a similar conformation, then these motifs may function to help the extracellular domain of the TMKs project off the surface of the cell in a pilus-like manner. This may make them available for interaction with host cell factors. In each TMK subfamily there was one or more members expressed; however, most family members appeared not to be expressed under the conditions of laboratory culture. By RT-PCR we did observe that the expression of some TMKs varied between growth curves. This indicates that the expression of these genes may be dynamic. Whether the TMKs share with the Giardia VSPs the process of antigenic variation under different biological conditions remains to be determined.
The most significant feature of the TMKs is the kinase domain that, with the exception of two TMKs, is distinct from other known kinases. It is not possible, based on sequence analysis, to predict activity, as most have similarity to both the serine/threonine and tyrosine kinases. Interestingly, a closely related kinase, SplA from D. discoideum, is a dual-specificity kinase with both tyrosine and serine/threonine kinase activity (40). All of the essential kinase motifs were conserved, suggesting that these are functional kinases. We were not able to demonstrate kinase activity when the kinase domain was expressed in E. coli. Since all of the functional residues were conserved, the most likely explanation for this is that the kinase is not functional in E. coli or that the kinase domain is not able to phosphorylate the substrates we have used. It has been previously shown that some kinases were not functional when expressed in E. coli (18). Additionally, even if the kinase is functional, identification of a substrate is often the rate-limiting step in characterizing a kinase (5,23). Further experimentation will be necessary to identify the substrate or interacting partners of the TMKs and determine if the TMKs are serine/ threonine and/or tyrosine kinases. Phylogenetic and sequence analysis shows that there are six subfamilies of kinases with distinct motifs within the kinase domains. Some families had additional conserved motifs outside of the kinase domain. This would imply that the subfamilies may represent functionally different families of kinases in sensing (differences in extracellular domains) and signaling (differences in kinase domains). The focal staining pattern of the TMKs distinctly contrasts with the uniform plasma membrane staining pattern seen with the Gal/GalNAc lectin of E. histolytica or VSPs in G. lamblia, both of which lack cytoplasmic kinase domains (38,42,45). This localization suggests that the TMKs form a focal multimolecular signaling complex in the plasma membrane (31,33).
In conclusion, the work presented here may begin to explain how E. histolytica is able to persist in the host for long periods of time despite immune surveillance, as well as sensing and responding to host stimuli. The large families of TMKs described here could serve in both biological sensing and antigenic variation. The distinct extracellular and kinase domains of the TMKs suggest that each TMK may sense or interact with different host factors and cause a distinct signaling event in response to that environmental cue.


extfieldsofstudy: Medicine Biology

s2fieldsofstudy: Biology Chemistry

text:
A Structural Basis for 14-3-3σ Functional Specificity*♦

The 14-3-3 family of proteins includes seven isotypes in mammalian cells that play numerous diverse roles in intracellular signaling. Most 14-3-3 proteins form homodimers and mixed heterodimers between different isotypes, with overlapping roles in ligand binding. In contrast, one mammalian isoform, 14-3-3σ, expressed primarily in epithelial cells, appears to play a unique role in the cellular response to DNA damage and in human oncogenesis. The biological and structural basis for these 14-3-3σ-specific functions is unknown. We demonstrate that endogenous 14-3-3σ preferentially forms homodimers in cells. We have solved the x-ray crystal structure of 14-3-3σ bound to an optimal phosphopeptide ligand at 2.4 Å resolution. The structure reveals the presence of stabilizing ring-ring and salt bridge interactions unique to the 14-3-3σ homodimer structure and potentially destabilizing electrostatic interactions between subunits in 14-3-3σ-containing heterodimers, rationalizing preferential homodimerization of 14-3-3σ in vivo. The interaction of the phosphopeptide with 14-3-3 reveals a conserved mechanism for phospho-dependent ligand binding, implying that the phosphopeptide binding cleft is not the critical determinant of the unique biological properties of 14-3-3σ. Instead, the structure suggests a second ligand binding site involved in 14-3-3σ-specific ligand discrimination. We have confirmed this by site-directed mutagenesis of three σ-specific residues that uniquely define this site. Mutation of these residues to the alternative sequence that is absolutely conserved in all other 14-3-3 isotypes confers upon 14-3-3σ the ability to bind to Cdc25C, a ligand that is known to bind to other 14-3-3 proteins but not to σ.

14-3-3 proteins participate in a wide variety of signal transduction processes including Ras-Raf-mediated activation of the mitogen-activated protein kinase pathway, regulation of apoptosis, adhesion-dependent integrin signaling, and cell cycle control in response to genotoxic stress (1)(2)(3)(4)(5). All of these processes appear to involve multiple 14-3-3 isotypes. Although some isotype-specific differences in signaling clearly exist, the molecular basis for these differences remain obscure and difficult to rationalize given the strong sequence conservation and broad heterodimerization observed among the individual 14-3-3 proteins.
We have been particularly interested in understanding the roles of different 14-3-3 proteins in cell proliferation, cell cycle control, and human tumorigenesis (5,13,14). In epithelial cells, one particular 14-3-3 isoform, 14-3-3, appears to play a particularly important role in this regard. Hermeking et al. (15) demonstrated that 14-3-3 was a major p53 response gene in HCT116 colon carcinoma cells following exposure to DNA damaging agents and suggested a role for 14-3-3 in G 2 /M checkpoint function. Chan et al. (16) found that 14-3-3Ϫ/Ϫ HCT116 cells were unable to maintain the G 2 /M checkpoint arrest following exposure to adriamycin and subsequently died by mitotic catastrophe. One well established function of 14-3-3 in negatively regulating M-phase entry involves functional sequestration of the mitotic promoting phosphatase Cdc25C (17)(18)(19)(20). Intriguingly, however, 14-3-3 does not bind to Cdc25C (16,21), suggesting that alternative -specific ligands, whose identities remain unknown, must also play important roles in controlling progression from G 2 into M by some Cdc25C-independent mechanism.
Given the importance of 14-3-3 in mitotic regulation, it is paradoxical that many different types of epithelial cancers have recently been found to down-regulate 14-3-3 expression at the mRNA and protein level, either through promoter methylation and gene silencing (22)(23)(24)(25)(26) or through up-regulation of a specific E3 ubiquitin ligase that targets 14-3-3 for destruction (27). This may be related to an alternative function of 14-3-3 in promoting cell senescence, although to date this has only been demonstrated in human keratinocytes (28).
Why is 14-3-3 apparently unique among the mammalian isoforms? What is structural basis for the distinct function of and its ligand discrimination? Why are 14-3-3 effects not diluted out through heterodimerization with other 14-3-3 isotypes? This study was designed to investigate structural differences between 14-3-3 and other 14-3-3 family members in an attempt to provide a molecular rationalization for some of these aspects of -specific function.
Bacterial expression of recombinant proteins was performed by transforming BL21(DE3) E. coli with the appropriate plasmids, followed by growth in LB medium containing ampicillin. Cultures were induced at an A 600 of 0.6 -0.8 by addition of isopropyl ␤-D-thiogalactopyranoside to a final concentration of 0.4 mM followed by growth for 4 h at 37°C prior to cell harvesting by centrifugation.
Immunoprecipitations were performed using 1 mg of whole cell lysates and 4 g of the appropriate antibodies overnight at 4°C. Following binding to protein-A-Sepharose beads for 1 h at 4°C, the immunoprecipitates were washed three times with modified radioimmune precipitation assay buffer, suspended in Laemmli sample buffer, resolved on SDS-PAGE gels, and transferred to polyvinylidene difluoride membranes. The membranes were blocked for 1 h at 4°C using 5% nonfat dry milk (Bio-Rad) in phosphate-buffered saline containing 0.05% Tween 20, followed by incubation for 1-2 h at room temperature or overnight at 4°C with mouse monoclonal antibodies specific for the HA epitope (clone 12CA5; Roche Applied Science) or 14-3-3 (clone CS-112 (29)) or with rabbit or goat polyclonal antibodies against 14-3-3␤ (C-20), c-Raf-1 (C-12), and Cdc25C (C- 19) or an antibody that recognizes all 14-3-3 isotypes (K-19), all from Santa Cruz Biotechnology (Santa Cruz, CA). Primary antibodies were used at dilutions between 1:1000 and 1:2500. Blots were washed three times for 15 min each with phosphate-buffered saline containing 0.05% Tween 20, followed by incubation with the appropriate horseradish peroxidase-conjugated secondary antibodies at 1:2500 -1:5000 dilution for 60 min at room temperature. Following washing, immunoreactive bands were visualized by enhanced chemiluminescence (PerkinElmer Life Sciences) and the blots exposed to x-ray film (Eastman Kodak Co.).

FIG. 2. 14-3-3 primarily forms homodimers in cells.
A, homodimerization revealed by two-dimensional gel Western blot analysis. U2OS whole cell lysates were immunoprecipitated with antibodies against 14-3-3␤ (panels a-c) or 14-3-3 (panels d-f) and analyzed for co-associated 14-3-3 isotypes by two-dimensional gel electrophoresis and Western blotting. The two spots observed for 14-3-3␤ and -indicate post-translational modifications. A fraction of 14-3-3␤ is known to be phosphorylated on Ser-185 (6); the post-translational modification of 14-3-3 is not known. B, antibody controls. Bacterial lysates containing recombinant glutathione S-transferase-14-3-3 fusion proteins were separated by SDS-PAGE and blotted with the antibodies used in A. A duplicate gel was stained with Coomassie Blue (lower panel). C, homodimerization confirmed by epitope tagging. U2OS cells were transfected with 2ϫFLAG-tagged 14-3-3. Total cell lysates (TCL) were prepared 24 h later, incubated with bead-immobilized anti-FLAG antibodies, and the immunoprecipitated proteins (IP) released under nonreducing conditions to minimize interfering signals from the antibody light chain. Under these conditions, the 2ϫFLAG-tagged 14-3-3 remained bound to the antibody-immobilized beads. Total cell lysate and immunoprecipitated protein samples, along with unbound material (Flow-thru) in the anti-FLAG-depleted lysates, were analyzed by blotting with the indicated antibodies.
system device (Amersham Biosciences). Strips were washed in re-equilibration buffer (50 mM Tris-HCl, pH 8.8, 6 M urea, 30% glycerol, 2% SDS) in the presence of 20 mg/ml dithiothreitol for 10 min and then in the presence of 25 mg/ml iodoacetamide for 10 min to irreversibly modify all cysteine residues. The strips were then loaded onto 12% SDS-PAGE gels and electrophoresed at 120 V for 1 h. Gels were transferred onto polyvinylidene difluoride membranes and used for Western blot analyses using the appropriate antibodies in conjunction with enhanced chemiluminescence detection.
14-3-3 Purification, Crystallization, and Structure Solution-Pellets of bacteria expressing untagged 14-3-3 corresponding to 3 liters of culture were suspended in 200 ml of lysis buffer (50 mM Tris-HCl pH 8.0, 300 mM NaCl, 2 mM DTT, 1 mM AEBSF) and lysed by sonication. The lysate was clarified by centrifugation, diluted to 100 mM NaCl with dilution buffer (50 mM Tris-HCl, pH 8.0, 2 mM DTT), and loaded onto a Q-Sepharose column equilibrated with buffer QA (100 mM Tris-HCl, pH 8.0, 50 mM NaCl, 2 mM DTT). The column was washed extensively in the same buffer and the protein then eluted with a 100 -500 mM NaCl gradient. Fractions containing 14-3-3, determined by SDS-PAGE analysis, were pooled and dialyzed against gel filtration buffer (50 mM Tris-HCl, pH 8.0, 200 mM NaCl, 2 mM DTT) and concentrated by ultracentrifugation to ϳ2 ml for loading onto a Superdex-75 gel filtration column. Appropriate fractions from the gel filtration column were dialyzed into buffer HA (10 mM Tris-HCl, pH 8.0, 10 mM NaCl, 2 mM DTT) and loaded onto a hydroxyapatite column equilibrated with buffer HA. Protein was eluted with a linear gradient from buffer HA to buffer HB (250 mM sodium phosphate, pH 7.0, 50 mM NaCl, 2 mM DTT). Purified 14-3-3 (ϳ8 mg) was dialyzed into 10 mM Tris-HCl, pH 8.0, 10 mM NaCl, concentrated to ϳ10 mg/ml by ultrafiltration, divided into 250-g aliquots, and flash-frozen in liquid nitrogen.
An optimal mode-1 phosphopeptide was synthesized using N-␣-Fmoc (N-(9-fluorenyl)methoxycarbonyl)-protected amino acids with standard BOP/HOBt coupling chemistry and purified by reverse phase high performance liquid chromatography. Protein-peptide complexes were prepared at 5 mg/ml protein by combining freshly thawed protein stock with a 2-fold molar excess of phosphopeptide and dilution with water. Crystals were grown at 18°C by vapor diffusion in hanging drops over 1-2 weeks. 1-2 l of complex was mixed with an equal volume of well solutions containing 50 mM HEPES, pH 7.0, 6 -10% polyethylene glycol 3350, 100 -150 mM NaF, and 40 mM CaCl 2 . Crystals were frozen in liquid nitrogen after a brief soak in synthetic mother liquor containing 15% glycerol, which was made by adding a 50% solution of glycerol in well solution directly to the hanging drop.
Crystals were screened on a Rigaku rotating anode source with Yale mirror optics and a R-AXIS IV detector. A 2.4 Å resolution data set was collected at 100 K using the NE-CAT 8BM beamline at the Advanced Photon Source, Argonne National Laboratory (Chicago, IL). The space group of the crystals was C222 l , with a single 14-3-3 dimer and its two bound peptides in the asymmetric unit. The data were indexed, integrated, and scaled using HKL2000 (30), and the structure was solved by molecular replacement using AmoRe (31) with the 14-3-3 dimer (with the peptide removed) from the 14-3-3-mode-2 peptide complex struc-ture (Protein Data Bank code 1QJA) as the search model. Phase refinement was performed using CNS (32). The structure was built using the O program (33), and figures were prepared using PyMOL (www. pymol.org). Coordinates have been deposited in the Protein Data Bank as 1YWT.

RESULTS AND DISCUSSION
A comparison of the sequence of 14-3-3 with that of other human 14-3-3 isotypes, as well as with those in the evolutionarily distant budding and fission yeast (Fig. 1A), reveals many regions of considerable sequence conservation. Despite this strong global sequence preservation, there are a number of short regions in 14-3-3 that differ in general from those in other 14-3-3 proteins. A particularly striking example is a unique 3 amino acid substitution in the ␣H-␣I linker. Phylogenetic analysis (Fig. 1B) reveals that is the most divergent of all 14-3-3 proteins, consistent with its apparently unique function in vivo. All other 14-3-3 proteins can be clustered into three major clades, one containing 14-3-3⑀ together with the yeast isoforms, a second clade containing the and ␥ isoforms, and a third clade containing the , ␤, and isoforms. The structure of 14-3-3 gene on chromosome 1, together with its complete lack of introns (a feature unique to ), support a hypothesis that 14-3-3 may represent a functional retrotransposed gene.
These data suggest that endogenous 14-3-3 and ␤ do not form mixed heterodimers. When the anti-␤ immunoprecipitates were blotted with an antibody that recognizes all of the 14-3-3 isotypes, at least 10 distinct spots were observed (panel c), consistent with extensive heterodimerization between ␤ and other 14-3-3 isotypes. In contrast, when the anti-immunoprecipitates were probed with this same antibody (panel f), there were very few additional spots seen. These data indicate that 14-3-3 preferentially forms homodimers within cells. The specificity of the antibodies used in Fig. 2A is shown in Fig. 2B. To verify that the lack of 14-3-3 heterodimerization we observed was not an artifact of dimer disruption by the anti-antibody, we transfected cells with a 2ϫFLAG-tagged 14-3-3 construct and looked at its co-association with endogenous and ␤ in anti-FLAG immunoprecipitates. As shown in Fig. 2C, endogenous 14-3-3 dimerized with the 2ϫFLAGtagged , while endogenous 14-3-3␤ did not. These findings confirm the conclusion, drawn from our two-dimensional gel results, that 14-3-3 preferentially forms homodimers.
To understand the basis of preferential homodimerizaton, and to shed light on mechanisms of functional specificity for 14-3-3, we solved the x-ray crystal structure of 14-3-3 bound to the mode-1 phosphopeptide MARSHpSYPAKKK at 2.4 Å resolution (Table I). As shown in Fig. 3A, the general shape of 14-3-3 is essentially identical to that of 14-3-3 and - (34,35), consisting of nine antiparallel ␣-helices forming a horseshoeshaped dimer. The basis for dimer formation in 14-3-3 is essentially the same as that of these other isotypes and involves a series of distributed interactions from residues in helices ␣A and ␣B in one monomer with residues in helices ␣CЈ and ␣DЈ in the other (Figs. 1A and 3B). For simplicity, we focus further comparisons of the 14-3-3 structure to that of 14-3-3 bound to the same mode-1 phosphopeptide (12), although the conclusions apply equally to those obtained by comparing 14-3-3 with the unliganded FIG. 3. The structure of 14-3-3 and the basis for homodimerization. A, overview of the 14-3-3-mode-1 phosphopeptide complex. Two orthogonal views are shown with the protein in ribbons representation with one monomer colored orange and the other colored pink. The dimer 2-fold axis is indicated in each view. Side chains on either side of the interface that differ between the and isoforms are shown as yellow and green ball-and-stick representations. The phosphopeptide, shown in stick representation, is colored blue. B-E, differences between the dimer interfaces of 14-3-3 and -facilitate homodimerization of the isoform. B, a close-up bottom view of the dimer rotated 180 o from the view in the right panel of A, highlighting sequence differences at the interface. C, alignment of the structures of the and isoforms reveals subtle differences in helix packing and register at the interface. The 14-3-3 ribbon is colored as described for A-C, and the two monomers of the isoform are colored cyan and blue, respectively. Sequence differences in the loop linking helices ␣B and ␣C (orange/blue) may be responsible for the different orientations of helix B. On the opposite side of the interface, 14-3-3 is disordered in the loop connecting helices ␣CЈ and ␣DЈ (pink/cyan) where there is a two-amino acid insertion relative to the sequence, and the ␣DЈ helix has one less turn at its NH 2 -terminal end. D, at the 14-3-3 dimer interface a substitution of Phe () for Cys () at residue 25 produces a ring-ring interaction between Phe-25 (␣B) and Tyr-84 (␣DЈ). The aromatic ring of Phe-25 prevents the alkyl chain of Lys-9 (␣A) from participating in van der Waals interactions with Tyr-84 as seen at the interface. Instead, Lys-9 makes a salt bridge across the interface with the side chain of Glu-83 at the end of the ordered part of helix ␣DЈ. E, in a modeled structure of the 14-3-3/ heterodimer interface, Glu-80 in 14-3-3 ␣DЈ (pink) would be juxtaposed against Glu-5 in ␣A of 14-3-3 (blue). This destabilizing interaction would similarly disrupt heterodimerization of with the ␤, ⑀, and isoforms as well. structure of 14-3-3 (34). In 14-3-3 the angle between ␣A and ␣B is different from that in the dimer. This difference may be related to the unique sequence of the BC loop in 14-3-3 compared with that in other human isoforms. Across the dimer interface in the opposing monomer, the loop connecting helices ␣CЈ and ␣DЈ, which contains two extra amino acids in 14-3-3, is disordered and helix ␣DЈ is one turn shorter at its aminoterminal end than in the 14-3-3 structure. Helices ␣A and ␣DЈ in the 14-3-3 structure are observed to pack slightly closer together across the interface than in (Fig. 3C), perhaps as a consequence of these changes.
There are seven amino acid differences between 14-3-3 and 14-3-3 at the dimer interface (six between and ). Five of these amino acid substitutions are -specific and must provide a structural basis for homodimerization through stabilizing homodimeric interactions and destabilizing heterodimeric in -FIG. 4. Ligand recognition by 14-3-3. A, the phosphopeptide-binding pocket. Stereo view of the mode-1 phosphopeptide bound to one monomeric subunit of the 14-3-3 dimer. The phosphopeptide is shown in stick representation with carbon atoms colored yellow, nitrogens blue, oxygens red, and phosphorus purple. 14-3-3 is shown in surface representation shaded by similar atom-type color coding except with carbon colored green. B, molecular basis for phosphospecificity. The phosphate group of the phosphoserine is coordinated by four conserved 14-3-3 side chains. The protein backbone is represented by green ribbons; the phosphopeptide and the side chains that interact with the phosphate group are shown in stick representation. Key interactions observed between the phosphopeptide and 14-3-3 are the same as those in the crystal structure of the 14-3-3-phosphopeptide complex (12). C, a novel binding surface unique to 14-3-3. Surface representations of the 14-3-3 dimer are shown along with a ball-and-stick representation of the phosphopeptide (green) in the same orientations as in Fig. 3A. The surfaces are color coded by sequence conservation with highly conserved residues shaded magenta and non-conserved residues yellow. Three exposed residues of 14-3-3 (Met-202, Asp-204, and His-206), which differ from the absolutely conserved sequence in all other known isoforms, are shaded blue. These highly exposed residues protrude from the top of a prominent ridge above the concave face that contains the phosphopeptide-binding grooves. D, mutation of the novel binding surface of 14-3-3 allows CDC25C ligand binding. U2OS cells were transfected with HA-14-3-3 wild-type (wt) or a 14-3- teractions. In the 14-3-3 structure, Phe-25, which is unique to , makes a ring-ring interaction across the interface with Tyr-84 on ␣DЈ (Fig. 3D). The aromatic ring of the Phe-25 side chain prevents Lys-9 (on ␣A) from adopting the conformation it has in the 14-3-3 structure. Instead, in the 14-3-3 structure Lys-9 participates in a salt bridge with Glu-83 located at the NH 2 -terminal end of ␣DЈ. Because of 2-fold symmetry, these -specific ring-ring and salt-bridge interactions occur twice in the dimer. Thus, the 14-3-3 homodimer has four stabilizing interactions that cannot occur in heterodimers that include 14-3-3.
Furthermore, Ser-5 and Glu-80, both unique to 14-3-3 (Fig.  1A), lie opposite each other at the dimer interface (Fig. 3B), near the disordered NH 2 terminus of ␣D of 14-3-3. In 14-3-3-containing heterodimers with the , , ␤, or ⑀ isoforms, Glu-80 in would be in close proximity to the Asp or Glu side chain that replaces Ser-5 of (Fig. 3E), resulting in a highly unfavorable interaction between these two opposing negative charges. Thus, in addition to the loss of the two stabilizing homodimeric interactions mentioned above, heterodimers of 14-3-3 with these isoforms would be required to tolerate an additional highly destabilizing interaction across the dimer interface. Furthermore, favorable packing of the bulky hydrophobic Met or Leu sidechains corresponding to Glu-80 in the ⑀, ␤, , , and ␥ isoforms may be incompatible with the subtle shifts in the positions of ␣A and ␣B in 14-3-3. Taken together, these observations suggest a structural basis for our results showing that the isoform preferentially forms homodimers.
Each of the C2 symmetry related monomers of the 14-3-3 dimer contains a phosphopeptide binding pocket running in opposite directions, in which residues 5-10 of the peptide (Ser-His-Ser(P)-Tyr-Pro-Ala) are clearly visible. On one side, poor but interpretable density is present for residues 2-4 (Gly-Ala-Arg). The conformation of the bound phosphopeptide is essentially the same as that in the corresponding complex with 14-3-3. For each monomer, the peptide binds in a narrow cleft formed primarily by the surface-exposed residues from ␣A,␣B, ␣C, and ␣D on the concave surface of the molecule (Fig. 4A). The topography of the cleft requires that the peptide have a distinct kink that allows it to exit the channel after which it becomes disordered in the structure. This kink is produced by a cis-Pro residue in the ϩ2 position. The details of the interaction between the protein and the phosphopeptide are the same as in the 14-3-3-mode1 peptide complex. All of the key interactions, including those involved in coordinating the phosphate group of the phosphoserine (Fig. 4B), are essentially identical. This is not surprising, as the sequence identity of the residues that comprise the phosphopeptide-binding site are strictly conserved (Fig. 4C). Consequently, the selectivity of 14-3-3 selectivity for specific ligands must involve other sites besides the major phosphopeptide-binding groove.
Most of the non-conserved residues are exposed on the opposite face of the dimer from the concave depression that contains the phosphopeptide binding site. However, a patch of three residues in the ␣H-␣I linker (Met-202, Asp-204, and His-206), strictly conserved in every sequence in Fig. 1A except 14-3-3, is exposed at the extreme ends of the ridges at the tops of either side of the U-shaped dimer. These three residues form a small island of exposed non-conserved sequence surrounded mostly by conserved sequence (Fig. 4C). This observation suggests that this site might constitute a 14-3-3-specific binding site for other protein ligands. Intriguingly, the patch formed by these three residues lies adjacent to the invariant Phe-198 (position 204 in the alignment in Fig. 1A), whose mutation in the Drosophila epsilon isoform appears to impair Raf/14-3-3 interactions required for Ras-mediated signaling (36). In addi-tion, the ␣H-␣I linker in 14-3-3 makes direct contact with part of serotonin N-acetyltransferase in the 14-3-3-AANAT co-crystal structure (37). These findings further suggest that this region of 14-3-3 may indeed be part of a second ligand binding surface.
To investigate this, and determine whether this site was involved in -specific ligand discrimination, we mutated Met-202, Asp-204, and His-206 in to the Ile, Glu, and Asp residues that are present in all other non-isotypes and transfected the resulting HA-tagged construct into U2OS cells (Fig. 4D). Both wild-type and the triple mutant bound to c-Raf-1 in cells. In contrast, wild-type did not bind significantly to Cdc25C, whereas the triple mutant form of showed robust binding, as well as a slight increase in Cdc25C binding after irradiationinduced DNA damage, particularly when normalized to the amount of HA-14-3-3 in the IPs. These findings strongly suggest that this region in 14-3-3 normally constitutes a second phospho-independent ligand-binding site that is likely to be involved in mediating selection for and against specific ligands. Similar phospho-independent ligand-binding surfaces that are spatially separate from the phosphopeptide-binding pocket have also been observed in other phosphoserine/threoninebinding domains (38).
In conclusion, we have shown that endogenous 14-3-3 preferentially forms homodimers in cells, a finding that can be rationalized at the structural level by sequence differences and selective interactions at the dimer interface. The structural basis of 14-3-3 phosphopeptide-binding is essentially identical to that seen in 14-3-3 phosphopeptide and protein complexes (12,37,39), suggesting that 14-3-3-specific substrate discrimination involves alternative secondary surfaces. We have identified one of these surfaces in the ␣H-␣I linker involving residues Met-202, Asp-204, and His-206. Mutation of this -specific surface patch to the corresponding sequence in other 14-3-3 proteins causes 14-3-3 to bind to Cdc25C, a molecule that normally binds to other 14-3-3 isoforms but not to . These findings may assist in understanding the molecular basis for 14-3-3-specific function in cell cycle control and cancer.


text:
Interaction between Constitutively Expressed Heat Shock Protein, Hsc 70, and Cysteine String Protein Is Important for Cortical Granule Exocytosis in Xenopus Oocytes*

In many species, binding of sperm to the egg initiates cortical granule exocytosis, an event that contributes to a sustained block of polyspermy. Interestingly, cortical granule exocytosis can be elicited in immature Xenopus oocytes by the protein kinase C activator, phorbol-12-myristate-13-acetate. In this study, we investigated the role of cysteine string protein (csp) in phorbol-12-myristate-13-acetate-evoked cortical granule exocytosis. Prior work indicated that csp is associated with cortical granules of Xenopus oocytes. In oocytes exhibiting >20-fold overexpression of full-length Xenopus csp, cortical granule exocytosis was reduced by ∼80%. However, csp overexpression did not affect constitutive exocytosis. Subcellular fractionation and confocal fluorescence microscopy revealed that little or none of the overexpressed csp was associated with cortical granules. This accumulation of csp at sites other than cortical granules suggested that mislocalized csp might sequester a protein that is important for regulated exocytosis. Because the NH2-terminal region of csp includes a J-domain, which interacts with constitutively expressed 70-kDa heat shock proteins (Hsc 70), we evaluated the effect of overexpressing the J-domain of csp. Although the native J-domain of csp inhibited cortical granule exocytosis, point mutations that interfere with J-domain binding to Hsc 70 eliminated this inhibition. These data indicate that csp interaction with Hsc 70 molecular chaperones is vital for regulated secretion in Xenopus oocytes.

Eggs of many species exhibit cortical granule exocytosis, an early postfertilization event that leads to a sustained block of polyspermy (1). Although oocytes of Xenopus laevis must undergo maturation before sperm can elicit cortical granule exocytosis, immature oocytes (and eggs) of Xenopus secrete in response to activators of protein kinase C (2). Somewhat unexpectedly (given that the majority of regulated secretory events are initiated by an increase of cytosolic Ca 2ϩ , Refs. 3 and 4), cortical granule exocytosis in Xenopus oocytes is insensitive to changes of cytosolic Ca 2ϩ . For instance, this secretory event is not triggered by Ca 2ϩ ionophores (using physiological concentrations of Ca 2ϩ ); it cannot be evoked by direct injection of Ca 2ϩ into the cytoplasm, and it is unaffected by removal of extracellular Ca 2ϩ or by buffers that clamp cytosolic Ca 2ϩ below the resting level (5)(6)(7). Thus, oocytes offer an empirical avenue for assessing the function of proteins in a secretory pathway where calcium ions play no direct role. This issue is of consid-erable importance with respect to cysteine string proteins (csp 2 (s)), a class of proteins found associated with a broad range of regulated secretory organelles (8 -10).
Csp was originally identified as a novel synaptic antigen in Drosophila (11), and several subsequent investigations indicated that csp might be important as a modulator of presynaptic Ca 2ϩ channels (12)(13)(14)(15)(16)(17). However, it also became evident that regulation of presynaptic Ca 2ϩ channels was not the sole function of csp (8 -10). For instance, with the recognition that the majority of the cysteine residues of csp were fatty acylated (18), it was proposed that this unusual hydrophobic domain of csp might participate directly in membrane fusion (19). Empirical evidence for a role of csp in membrane fusion emerged from amperometric recordings of chromaffin cells overexpressing csp. This study revealed a change in the rise time of amperometric spikes, thereby implicating csp in fusion pore expansion (20). Complementing this work, genetic experiments indicated that the cysteine string of csp could not be truncated or functionally substituted by serine residues (21). Concurrently, strategies to perturb csp function in pancreatic and adrenal cells suggested that csp was involved at an undisclosed step of secretion downstream of Ca 2ϩ entry into the cell (22)(23)(24). Studies of a csp null mutant Drosophila yielded a specific hypothesis for this downstream role of csp. Based on an apparent change of Ca 2ϩ homeostasis and the level of Ca 2ϩ needed to trigger quantal secretion, csp was ascribed a role in regulating the Ca 2ϩ sensitivity of the exocytotic machinery (25). In view of these disparate and occasionally conflicting ideas regarding the molecular role(s) of csp, we initiated studies of csp function using Xenopus oocytes.
Xenopus oocytes offer several advantages for studying regulated secretion. As noted, cortical granule exocytosis is a one-time secretory event that can be triggered in a Ca 2ϩ -independent manner by the protein kinase C activator, PMA (phorbol-12-myristate-13-acetate). Secretion can be assayed using single oocytes (7,26). Moreover, oocytes readily express protein from injected mRNA (27)(28)(29)(30), and protein expression and secretion can be correlated for the same cell (7,26). Finally, oocytes possess efficient mechanisms for the delivery of proteins to the plasma membrane (and the extracellular compartment, Refs. 28 -30), which enables one to compare the machinery underlying the regulated and constitutive secretory pathways. Thus, we evaluated the impact on cortical granule exocytosis of overexpressing full-length Xenopus csp. Although we surmised that an elevated cellular content of csp might enhance the kinetics of secretion, instead, we observed a profound reduction of cortical granule exocytosis. Further investigation revealed that little or none of the overexpressed csp was localized to cortical granules. This result raised the possibility that the inhibition of cortical granule exocytosis was because of a non-productive association * This work was supported by Grant NS31934 from the National Institutes of Health (to between the overexpressed csp and one or more csp-interacting proteins. We present evidence that the interaction of overexpressed csp with Hsc 70 (constitutively expressed heat shock protein of 70 kDa) figures prominently in the inhibition of cortical granule exocytosis in these cells.

MATERIALS AND METHODS
Isolation and Culturing of Oocytes, RNA Preparation, and Injection-Stage V-VI X. laevis oocytes were obtained by collagenase treatment of fragments of ovary as described (31). RNA transcripts were prepared from linearized constructs subcloned into the plasmid pCS2 ϩ using the SP6 mMessage Machine from Ambion. Oocytes were injected with 30 -60 ng of RNA and cultured for 2-4 days at 18°C prior to the analysis of secretory events.
Preparation of csp Constructs-Linearized (NotI) full-length Xenopus csp cDNA (32) was either used directly to prepare RNA (Xcsp RNA), or it served as a template for the generation by PCR of the J-domain construct (amino acid residues 1-70). Site-directed mutagenesis was used to generate the J-domain mutations H43Q and D45A. The green fluorescent protein (GFP)-csp construct was kindly supplied by H. E. Yu and W. M. Bement (University of Wisconsin, Madison). All constructs were verified by nucleotide sequence analysis.
Regulated Secretion Assay-Cortical granule exocytosis was evoked by exposing single oocytes to PMA (50 -100 nM in Barths solution) for 20 -30 min followed by Coomassie staining and densitometric analysis of released protein after resolution on SDS-polyacrylamide gels (7,31).
Independently, we developed a procedure to trigger cortical granule exocytosis that appears to involve a protein-independent effect of Ca 2ϩ at the plasma membrane of the oocyte. This was achieved by exposing immature, stage V-VI oocytes to the calcium ionophore, ionomycin (10 M), in Barths solution with added calcium chloride (20 mM). Secretion triggered in this manner is unaffected by overexpression of a dominantnegative fragment of Xenopus myosin IC, or by pretreatment of oocytes with N-ethylmaleimide (10 mM) or phenylarsine oxide (50 M), all of which completely block exocytosis triggered by PMA in immature oocytes (data not shown). We used this high Ca 2ϩ -ionomycin method of evoking cortical granule exocytosis to assess whether intact cortical granules were still present in oocytes expressing recombinant csp.
Immunoblot Analysis of Oocytes-To detect the expression of csp, csp constructs or Hsc 70 (using Hsp 70 monoclonal antibody, clone BRM-22 from Sigma) in oocytes, individual cells were extracted in 20 -40 l of 1% Triton X-100 in 0.1 M NaCl, 0.05 M Tris-HCl (pH 7.4), 1 mM EDTA, and the extract was centrifuged at 15,000 ϫ g for 1 min. The supernatant was mixed with 5ϫ concentrated Laemmli sample buffer and resolved on a SDS-polyacrylamide gel for immunoblot analysis as before (7,26,31). An antibody specific for the amino terminus of csp (N-end antibody) was obtained by covalently conjugating recombinant Xenopus csp 1-112 to Pierce's aminolink resin (following the manufacturer's instructions) and adsorbing csp antiserum (16) to this matrix, washing with phosphate-buffered saline, and eluting the bound antibody using 0.1 M glycine-HCl (pH 3.0). After adjusting the pH to 7.5 with 1 M Tris base, the antibody capable of binding the carboxyl terminus of csp was removed by passing the N-end antibody through an aminolink resin to which the recombinant carboxyl end of Xenopus csp (residues 137-197) was coupled. This protocol yields an antibody that selectively identifies the amino terminus of csp but not the carboxyl terminus of csp. Quantification (by densitometry) of immunoblot signals used one-dimensional image analysis software from Kodak. Results are expressed as the mean Ϯ S.D., and significant results were assessed by Student's t test. In many experiments, overexpression of csp constructs was sufficiently robust that the signal for csp in control oocytes was only detected when the signal for the overexpressed csp constructs exhibited saturation. Although our routine quantification of the extent of overexpression of csp constructs is likely to be underestimated, independent analyses of diluted extracts from csp overexpressing oocytes set this error at Ͻ30%. Subcellular Fractionation of Oocytes-As described (7), oocytes were gently disrupted and centrifuged (3 min at 720 ϫ g) to yield a pellet enriched in cortical granules, plasma membrane, yolk, and pigment granules, whereas most other subcellular constituents (including ␤-tubulin, which was detected by immunoblot using monoclonal antibody E7 from the Developmental Studies Hybridoma Bank) remain in the low speed supernatant (7,26,31). Surface labeling of oocytes prior to this fractionation protocol used water-soluble, polyethylene oxide-maleimide biotin (0.1 mM in Barths solution; Pierce) for 15 min to alkylate thiol residues that had been reduced by dithiothreitol (2 mM). Detection used streptavidin-peroxidase (Sigma) and enhanced chemiluminescence.
Confocal Fluorescence Microscopy-Isolated cortical regions of oocytes were prepared (as in Ref. 26) from control oocytes or oocytes expressing either GFP-csp or GFP-syntaxin 1a (from rat; kindly provided by Dr. T. Coppola, University of Nice, France) and fixed for 10 min in 3% paraformaldehyde in Barths solution prior to mounting (using Fluoromount) on glass slides. Specimens were examined using a Zeiss 510 Meta laser scanning confocal microscope equipped with an argon laser and a 40ϫ 1.3 na Plan-Neofluar objective (Carl Zeiss Microimaging, Inc., Thornwood, N.Y.) with 488 nm excitation and a band pass filter setting of 505-530 nm. Image stacks (5 m) were collected in 0.1-1.0-m z-steps and projected onto a single plane or used to obtain a z-plane image. Images were collected at a resolution of 1024 ϫ 1024 pixels (GFP-csp) or 2048 ϫ 2048 pixels (GFP-syntaxin 1a).
Immunoprecipitation-Oocytes were disrupted in extraction buffer (1% Triton X-100 in phosphate-buffered saline with 2 mM ADP) and centrifuged at 15,000 ϫ g for 1 min. The supernatant was precleared for 15 min with 0.1 volume of protein A/G-agarose (Santa Cruz). Cleared extract from 2-3 oocytes was incubated with or without affinity-purified csp antibody (1-2 g), and as a separate control, csp antibody was added to extraction buffer alone. After 1 h at 4°C, protein A/G-agarose (10 l) was added, and samples were gently mixed by rotation. After 30 -40 min, the agarose beads were collected by centrifugation and washed three times with extraction buffer before dispersion in sample buffer. Recovered protein was resolved electrophoretically for immunoblot analysis using Hsp/Hsc 70 antibody or csp antibody, as above. Blots were also probed for two abundant oocyte proteins, actin and ␤-tubulin.
Assay of Constitutive Exocytosis-Plasma membrane proteins are efficiently delivered to the surface of the oocyte via a constitutive exocytotic pathway involving carrier vesicles that are ϳ100 nm in diameter (30). We developed a biochemical assay of this process by expressing GFP-syntaxin 1a in oocytes and monitoring the appearance of GFPsyntaxin 1a immunoreactivity in the low speed pellet fraction of oocytes (generated as in Ref. 7; results from confocal fluorescence microscopy confirm that GFP-syntaxin 1a reaches the plasma membrane of oocytes). To verify that the presence of GFP-syntaxin 1a in the low speed pellet requires intact protein trafficking via the Golgi apparatus, some oocytes were treated with the Golgi-dismantling reagent, brefeldin A (50 M) subsequent to the injection of RNA for GFP-syntaxin 1a.
To assess the impact of csp constructs on the trafficking of GFP-syntaxin, oocytes were injected with csp RNA 2 days prior to injection with GFP-syntaxin 1a RNA.

RESULTS
Functional Consequence of csp Overexpression-Xenopus oocytes typically secrete 1-1.5 g of cortical granule lectin in response to PMA csp/Hsc 70 Involvement in Exocytosis (2,7,26). This is illustrated in Fig. 1A, which shows the broad band of highly glycosylated lectin that is collected from three individual control oocytes exposed to 50 nM PMA. In contrast, secretion of this lectin is greatly diminished (or absent) for cells that overexpress full-length csp (Fig. 1A). Quantification of this inhibitory effect reveals that the mean amount of secreted cortical granule lectin drops to 20 Ϯ 13% of control (for 29 control cells and 26 cells overexpressing csp) in oocytes challenged with PMA 3-4 days after injection of csp RNA (this difference is significant at p Ͻ 0.01).
Immunoblot analysis revealed that oocytes injected 3-4 days earlier with 30 -50 ng of Xcsp RNA expressed 20 Ϯ 15 times more csp than control oocytes (Fig. 1B). The overexpressed csp was present as two distinct species in the RNA-injected oocytes. In addition to csp immunoreactivity at the same mobility of csp in control oocytes (32-33 kDa, see Fig. 1B), there was an additional band at ϳ27 kDa (Fig. 1B). Because this latter species exhibited mobility that is consistent with the mass of csp prior to fatty acylation (or after deacylation; see Refs. 18, 31, and 32), it is very likely that this form of overexpressed csp lacks lipid modification (indeed, when extracts of csp overexpressing oocytes were treated with the deacylating reagent, hydroxylamine (1 M (pH 7.0), followed by reduction and alkylation) all of the 32-33 kDa csp was converted to the 27-kDa species, data not shown). Together with the data of Fig. 1A these results indicate that csp is overexpressed in oocytes, and this overexpression interferes with the normal pathway by which PMA elicits the secretion of cortical granule lectin. It is also important to note that with less robust overexpression (Ͻ10-fold) of csp there is proportionately less inhibition of cortical granule exocytosis (data not shown).
The results of Fig. 1A could be explained by an effect of overexpressed csp that either impairs the exocytotic pathway or that alters the pool of cortical granule lectin available for secretion (for instance, by promoting degradation of cortical granules or by prematurely evoking secretion). We excluded an effect of overexpressed csp on the available pool of cortical granules via three separate avenues. First, individual oocytes were transferred to 12 l of Barths solution 2 h after injection of csp RNA and after 2 days the fluid was collected and assayed for secreted cortical granule lectin. No lectin was detected (data not shown), indicating that csp overexpression does not, by itself, trigger secretion from cortical granules (in contrast, overexpression of protein kinase C does trigger cortical granule exocytosis; see Ref. 31). Second, we exposed oocytes (control or csp overexpressing) to PMA (50 nM for 30 min) and verified that secretion was significantly diminished in oocytes overexpressing csp (Fig. 1C). Then, the same oocytes were incubated in a solution containing 10 M ionomycin plus 20 mM added Ca 2ϩ (this procedure promotes secretion via an apparently protein-independent effect on oocyte membranes; see "Materials and Methods"). This second incubation culminated in the discharge of very little cortical granule lectin from the control oocytes, because these cells had discharged most of the available lectin in response to PMA (Fig. 1C). However, the csp overexpressing oocytes released essentially as much cortical granule lectin during the high Ca-ionomycin incubation as control oocytes had released in response to PMA (Fig. 1C). These data indicate that csp overexpressing oocytes retain cortical granule lectin in a pool that is accessible to the nonspecific secretory effect of high Ca 2ϩ plus ionomycin. For a final control, we evaluated oocyte extracts for cortical granule lectin immunoreactivity after 30 min in 50 nM PMA. Under these conditions, control oocytes typically secrete ϳ80% of their cortical granule lectin (data not shown; see Ref. 31). In contrast, csp overexpressing oocytes retained a level of cortical granule lectin immunoreactivity that was indistinguishable from cells that had not been treated with PMA (data not shown; however, images in Fig. 3B reveal that cortical granules are present in oocytes expressing high levels of GFP-csp). Taken together, these experiments indicate that cortical granules persist in oocytes that overexpress csp, but there is a block of the pathway by which PMA elicits cortical granule exocytosis.
Distribution of Overexpressed csp in Oocytes-To assess further the basis of the secretory inhibition in oocytes overexpressing csp, we evaluated the subcellular distribution of the newly produced csp using biochemical and cell biological approaches. As reported previously (26), when control oocytes are gently disrupted and subjected to low speed centrifugation, 80 -90% of the endogenous csp immunoreactivity (and Ͼ90% of the cortical granule lectin immunoreactivity; Ref. 26) sediments with the low speed pellet (Fig. 2). (The utility of this fractionation procedure for discriminating oocyte compartments is further evidenced by the fact that ␤-tubulin immunoreactivity is detected only in the low speed supernatant of control and csp overexpressing oocytes (Fig. 2). Conversely, when oocytes that had been incubated with a membraneimpermeant alkylating agent are fractionated via this protocol, the labeled proteins, exemplified by the 35-kDa species in Fig. 2, are detected only in the pellet). However, fractionation of oocytes that overexpress csp yielded a pattern that was qualitatively and quantitatively different from control oocytes. In addition to there being abundant csp in the low speed supernatant (overall, csp in the low speed supernatant  Single control or csp overexpressing oocytes were fractionated to yield a supernatant (sup) and low speed pellet (pel) and analyzed by immunoblot for csp and tubulin (tubulin signal in the pellet was undetectable). In addition, a 35-kDa protein (labeled using a membraneimpermeant alkylating agent) partitions almost exclusively in the pellet. SEPTEMBER 23, 2005 • VOLUME 280 • NUMBER 38 of csp overexpressing cells was Ͼ20-fold higher than csp in the supernatant of control oocytes), the 27-kDa form of csp (that lacks lipid modification) was distributed approximately equally between the low speed supernatant and pellet (Fig. 2). Although these data indicate that csp is differentially distributed in oocytes that overexpress this protein, the results do not distinguish where in the cell csp is localized. Because csp is predominantly associated with cortical granules in control oocytes (26), a functionally important issue is whether any of the overexpressed csp is targeted to these granules.

csp/Hsc 70 Involvement in Exocytosis
To evaluate whether overexpressed csp associates with cortical granules, we expressed GFP-tagged csp in oocytes and assessed its subcellular distribution biochemically (Fig. 3A). Of particular interest was the fact that nearly 50% of the GFP-csp partitioned in the low speed pellet (Fig. 3A). Because this pellet includes most of the cortical granules, as well as the plasma membrane, this result indicates that GFP-csp associates with one or both of these structures. To resolve this issue, we used confocal fluorescence microscopy to examine isolated cortical fragments of oocytes expressing GFP-csp (Fig. 3B). As the image stack of Fig.  3B indicates, GFP-csp yielded a "Swiss cheese" pattern of fluorescence, where the "holes" had the dimensions of cortical granules (the diameter of these holes was between 1.2 and 2.9 m for n ϭ 100; a range typical for cortical granules; Ref. 1). For comparison, we also examined oocytes expressing GFP-syntaxin 1a (syntaxin 1a is a t-SNARE that is prominently associated with the plasma membrane of a wide variety of regulated secretory cells; Ref 33). The pattern of GFP-syntaxin 1a fluorescence was essentially indistinguishable from the pattern of GFP-csp (Fig. 3B). Oblate structures (whose diameter ranged from 1.1 to 2.7 m for n ϭ 100) were devoid of GFP-syntaxin 1a fluorescence and surrounded by interstitial zones of strong fluorescence (Fig. 3B). Similar patterns of GFP-csp and GFP-syntaxin 1a fluorescence were also apparent in the z-plane (Fig. 3B). Because GFP-csp is distributed similarly to GFP-syntaxin 1a (which should traffic exclusively to the plasma membrane), these data indicate that the csp in the low speed pellet fraction of oocytes is predominantly associated with the plasma membrane and is not detectably present on the cortical granules.
Overexpression of the J-domain of csp Inhibits Cortical Granule Exocytosis-The preceding results raise the possibility that csp overexpression inhibits cortical granule exocytosis, because mislocalized csp interacts non-productively with one or more proteins that are necessary for the secretory cascade. Although csp has been reported to interact in vitro with a large number of other proteins (9, 10), its affiliation with members of the Hsp 70 and Hsc 70 family is indisputable (34 -38,40).
Thus, we tested whether expression of the J-domain of csp, the region that binds Hsp/Hsc 70 proteins (9, 10, 39), suffices to inhibit cortical granule exocytosis. The results (Fig. 4A) indicate that J-domain overexpression inhibits cortical granule exocytosis. Across a series of trials (18 control and 20 J-domain-expressing cells), secretion fell significantly (p Ͻ 0.01) to 30 Ϯ 17% of control (Fig. 4A). Concomitantly, using antibodies that target only the NH 2 -terminal region of csp (N-end antibody), we observed a dramatic overexpression of the csp J-domain in the cells that exhibit the secretory inhibition (Fig. 4B). Densitometry reveals that the average signal for the overexpressed J-domain exceeds that of the endogenous csp by 36 Ϯ 16-fold. Thus, by itself, the Hsc 70-interacting domain of csp is capable of blunting cortical granule exocytosis.
Based on the preceding results, we predicted that specific mutations of the J-domain (which have been shown in other J-domain proteins (39) and in csp (35,40) to interfere with binding to Hsp/Hsc 70 isoforms) should largely eliminate the secretory inhibition associated with J-domain overexpression. As is evident in Fig. 4A, despite substantial overexpression (relative to csp), the H43Q-or D45A-mutated versions of the J-domain had little impact on cortical granule exocytosis. For cumulative evaluation of these data, we analyzed secretory results from oocytes that exhibited at least a 20-fold overexpression of the H43Q or D45A construct (this threshold was chosen, because Ͼ20-fold overexpression of the wild-type J-domain was necessary to inhibit cortical granule exocytosis by ϳ70%; Fig. 4B). Thus, for 8 oocytes (which overexpressed the D45A mutant construct by a mean of 34-fold) secretion was 81 Ϯ 12% of control. Because of variability among control oocytes, this apparent reduction of secretion was not significant (p Ͼ 0.10) indicating that the construct with the D45A mutation negligibly affects cortical granule exocytosis. The more important comparison is between the mutant and wild-type J-domain, where the small reduction of secretion with the D45A mutant construct was significantly less (p Ͻ 0.001) than the 70% decline observed with native J-domain. Extending this analysis to the H43Q mutation of the J-domain (where 17 oocytes reached the criterion of Ͼ20-fold overexpression of this construct), we found that secretion was 99 Ϯ 9% of control. This represents a significant (p Ͻ 0.001) loss of inhibitory effect relative to wild-type J-domain.
Csp Overexpression Alters the Subcellular Distribution of Hsc 70 in Oocytes- In Fig. 5A, the immunoblot shows the pattern of Hsp/Hsc 70 immunoreactivity obtained for an extract of a single oocyte using the

csp/Hsc 70 Involvement in Exocytosis
broad spectrum (in terms of species recognition) Hsp 70 monoclonal antibody BRM-22. In addition to a pair of very closely overlapping bands at 70 kDa (which were also detected using Hsp/Hsc 70 monoclonal antibody N27F3-4 from Stressgen; results not shown), there was a lower mass species at ϳ67 kDa (the abundance of this 67-kDa species varied considerably among batches of oocytes; data not shown). Although prior work (40,41) has documented the presence in Xenopus oocytes of two Hsc 70 isoforms, we have not resolved further the identity of the immunoreactive species in Fig. 5A. However, as expected for soluble proteins, when oocytes were fractionated (as in Fig. 2), most (Ͼ90%) of the immunoreactive Hsp/Hsc 70 remained in the low speed supernatant (Fig. 5B). Interestingly, in csp overexpressing oocytes, appreciably more Hsc 70 (but, not the 67-kDa species) was detected in the low speed pellet (Fig. 5B, csp). Quantifying this effect in three separate experiments, we observed that the level of Hsp/Hsc 70 immunoreactivity in the pellet of csp overexpressing cells was Ͼ5-fold the level in control cells (this difference was significant at p Ͻ 0.01), arguing that the abundant csp in the pellet of csp overexpressing cells (Fig. 2) contributes to a redistribution of Hsp/Hsc 70.
Co-immunoprecipitation of csp and Hsp/Hsc 70-As independent evidence that csp and Hsp/Hsc 70 interact in uninjected control oocytes, we immunoprecipitated csp and tested for co-immunoprecipitation of Hsp/Hsc 70. Results in Fig. 6 indicate that Hsp/Hsc 70 coimmunoprecipitates with csp. (Controls for this experiment included samples of csp antibody alone or extract alone to which were added protein A/G-agarose; no csp or Hsp/Hsc 70 signal was detected in these samples; independently, we found that neither actin nor tubulin coimmunoprecipitated with csp; data not shown). However, perhaps because of limited access of the Hsp/Hsc 70 monoclonal antibody to the antigen, we observed a very low efficiency (Ͻ5%) of immunoprecipitation of Hsp/Hsc 70 from oocyte extracts and no detectable co-immunoprecipitation of csp (data not shown).
Effect of csp Overexpression on Constitutive Exocytosis-Oocytes exhibit robust trafficking of plasma membrane components, and the entire plasma membrane apparently turns over within 24 h (30). To ascertain whether csp overexpression interferes with this process, we fractionated oocytes to monitor the subcellular distribution of co-expressed GFP-syntaxin 1a. In control oocytes, ϳ60% of the GFP-syntaxin 1a immunoreactivity was associated with the low speed pellet of oocytes, and this percentage was unchanged in oocytes overexpressing csp (Fig. 7, the GFP-syntaxin 1a that remained in the low speed supernatant was presumably associated with Golgi apparatus and endoplas-mic reticulum, as it sedimented after 1 h at 100,000 ϫ g; data not shown). Cumulatively, in two experiments involving 12 control and 12 csp overexpressing oocytes, the percentage of GFP-syntaxin 1a in the pellet was 57 Ϯ 5% for control and 60 Ϯ 3% for csp overexpressing cells (this difference was not significant; in addition, the J-domain of csp did not affect GFP-syntaxin 1a distribution; data not shown). However, if oocytes were maintained in brefeldin A (50 M), a reagent that disrupts the Golgi apparatus (43), then no GFP-syntaxin 1a appeared in the low speed pellet (Fig. 7). Collectively, these results indicate that Golgi-dependent membrane trafficking (including constitutive exocytosis) is necessary for the normal distribution of GFP-syntaxin 1a in oocytes and that csp overexpression does not affect this process.

DISCUSSION
This study was initiated with the goal of evaluating the role of csp in a regulated exocytotic pathway that is independent of Ca 2ϩ . This is an important issue, because it has been argued, based on studies of csp null mutant Drosophila, that csp plays a vital role in controlling the Ca 2ϩ sensitivity of the regulated secretory machinery (25). Although the current data do not exclude the possibility that csp displays additional functions at nerve terminals (where Ca 2ϩ sensing is important for the stimulus-evoked release of transmitter), our results indicate that csp participates in the regulated exocytotic cascade at a step that is independent of Ca 2ϩ . This conclusion is based on the fact that, as reviewed in the Introduction, PMA-triggered cortical granule exocytosis in Xenopus oocytes does not involve changes of cytosolic Ca 2ϩ and proceeds normally even when cytosolic Ca 2ϩ is buffered (5)(6)(7)31). However, cortical granule exocytosis is significantly disrupted by the overexpression of full-length csp, and the remainder of this investigation was aimed at clarifying the basis of this secretory blockade.
It was our original hypothesis that csp overexpression in frog oocytes might augment cortical granule exocytosis. This expectation was based on results from several other investigations. For instance, absence of csp in csp null mutant Drosophila leads to a decline of stimulus-dependent transmitter release (44), and csp antibody causes a reduction of evoked transmitter release at embryonic frog motor nerve terminals (16).  Fig. 2) to yield low speed supernatants (sup) and pellets (pel), which were subjected to immunoblot analysis for Hsp/Hsc 70. FIGURE 6. Hsp/Hsc 70 co-immunoprecipitates with csp. Oocyte extract was subjected to immunoprecipitation (IP) using csp antibody and protein A/G-agarose to capture immune complexes. Samples were resolved electrophoretically for sequential immunoblot analysis using Hsp/Hsc 70 antibody followed by csp antibody. Immunoblot signals in the lane labeled extract correspond to 25% of the oocyte extract used for immunoprecipitation. FIGURE 7. Brefeldin A affects the subcellular distribution of GFP-syntaxin 1a, whereas overexpression of csp does not. Control oocytes, oocytes overexpressing csp, or oocytes maintained in brefeldin A (50 M) were injected with RNA encoding GFP-syntaxin 1a. After 2 days (to permit expression of GFP-syntaxin 1a), single oocytes were fractionated to yield low speed supernatants (S) and pellets (P), and the extracts were subjected to immunoblot analysis to detect GFP-syntaxin 1a (which migrates at 60 kDa). Blots were reprobed to verify csp overexpression (only acylated csp is shown).
Together, these results imply that loss of csp function correlates with an impaired secretory response. Conversely, stable overexpression of csp in PC12 cells culminates in enhanced secretion of dopamine in response to Ca 2ϩ or GTP␥S (22), and Li ϩ -induced up-regulation of csp is also associated with elevated dopamine secretion from PC12 cells (46). However, contrary to our expectation, csp overexpression in oocytes strongly inhibited cortical granule exocytosis (note that inhibitory effects of transient csp overexpression have independently been reported in several other systems, Refs. 20, 23, 24, and 47). The inhibition of secretion in oocytes also relied on the extent of csp overexpression. Thus, little or no impact on cortical granule exocytosis was observed when csp overexpression was Ͻ10 ϫ the level of csp in control oocytes (data not shown), but profound inhibition was seen when csp exceeded the control level by Ͼ20-fold.
In seeking to explain the inhibition of cortical granule exocytosis in oocytes overexpressing csp, we initially evaluated the possibility that overexpressed csp failed to reach its normal destination, the cortical granules. This would presumably leave a large pool of mislocalized csp to interfere with exocytosis. Subcellular fractionation of csp overexpressing oocytes (Fig. 2) revealed a substantial increase of csp in the low speed supernatant, a fraction that typically contains very little csp and is essentially devoid of intact cortical granules (26,31). Concomitantly, results obtained by confocal fluorescence microscopy showed that GFPcsp fluorescence was effectively excluded from cortical granules. Instead, GFP-csp was clearly associated with the plasma membrane of the oocyte, exhibiting fluorescence that was indistinguishable from the pattern of GFP-syntaxin 1a, a plasma membrane protein (33). Thus, in contrast to control oocytes, where most of the csp is associated with cortical granules (26), oocytes overexpressing csp appear to accumulate this protein at sites other than cortical granules. In retrospect, this result is consistent with what is known about cortical granule biogenesis in oocytes. Balinsky and Devis (48) reported that production of cortical granules by the Golgi apparatus ceases once oocytes reach 0.5-0.6 mm in diameter. Similarly, Dumont (49) observed that a few small cortical granules remained in the cytoplasm of stage IV (0.6 -1-mm diameter) oocytes, whereas by stage V-VI, the granules uniformly assumed a position immediately beneath the plasma membrane. These results (48,49) suggest that the turnover of cortical granules is negligible in stage V-VI oocytes (supporting this conclusion, we have detected no decline over 3 days in the amount of cortical granule lectin in oocytes incubated with cycloheximide at a level (100 g/ml) that blocks ϳ98% of protein synthesis; data not shown), and would explain why very little of the overexpressed csp is found in association with these relatively quiescent structures. Nevertheless, because our prior work on oocytes documented that soluble csp becomes membrane-associated as a consequence of fatty acylation (18), the current data imply that cortical granule membranes lack the capability to bind and fatty acylate newly produced csp.
Because most of the overexpressed csp in oocytes appears to be mislocalized (being present in the cytosol or associated with membranous structures other than cortical granules), it was reasonable to postulate that the mislocalized csp was sequestering one or more proteins that are necessary for cortical granule exocytosis. Abundant work has shown that csp interacts via its J-domain with members of the Hsp 70 family of molecular chaperones (34 -38, 40). In addition, oocytes constitutively express at least two Hsp/Hsc 70 isoforms (Refs. 41 and 42 and Fig. 5; independently, cDNAs encoding two closely related Hsc 70 isoforms have been reported for Xenopus; Refs. 45 and 50), and we found that oocyte Hsp/Hsc 70 co-immunoprecipitates with csp. Moreover, subcellular fractionation of oocytes reveals a significant redistribution of Hsp/ Hsc 70 to the fraction that contains the newly synthesized csp (Fig. 5B).
Thus, it was reasonable to consider the possibility that overexpressed csp interferes with an interaction between Hsp/Hsc 70 and granuleassociated csp that is important for regulated exocytosis. Two results support this conclusion. First, by itself, overexpression of the J-domain of csp significantly blunts cortical granule exocytosis. Because the J-domain underlies the interaction of csp with members of the Hsp/Hsc 70 family of proteins (34 -38), this result supports a role for csp-Hsp/Hsc 70 interaction in cortical granule exocytosis. Second, mutation of the J-domain of csp (to produce constructs that have been shown not to associate with Hsp/Hsc 70; see Refs. 35, 40, and 41) significantly reduces the inhibitory effect of these constructs on cortical granule exocytosis. Collectively, these results suggest that the normal pathway for cortical granule exocytosis involves an interaction between granule-associated csp and Hsp/Hsc 70. However, additional work will be necessary to obtain a more detailed understanding of the role of this interaction in the secretory cascade. As a final comment on this issue, recent work (47) concluded that the J-domain of csp was not intrinsically vital for insulin secretion from insulinoma cells. A possible explanation for the apparent discrepancy between this finding and our results is that overexpression of the J-domain in the insulin-secreting cells may have been insufficient to produce the inhibition seen in oocytes.
Although csp overexpression inhibits cortical granule exocytosis, the constitutive exocytotic pathway for the delivery of proteins to the plasma membrane remains intact. This conclusion is supported by the observation that syntaxin 1a trafficking to the plasma membrane is unaltered by csp overexpression. Interestingly, the finding that GFP-csp is also targeted to the plasma membrane of oocytes (Fig. 3) suggests that this pool of csp may also reach the plasma membrane via ongoing constitutive exocytosis. Several prior investigations have noted that perturbation of csp function in the regulated pathway does not affect spontaneous neurotransmitter release at nerve endings (16,44) or the basal secretory process in adrenal or pancreatic cells (22)(23)(24)47). These results support the hypothesis that csps evolved to perform a function important for the regulated secretory pathway. Concomitantly, the current data indicate that the role of csp in regulated exocytosis depends on its interaction with Hsc 70.