A Cdo–Bnip-2–Cdc42 signaling pathway regulates p38α/β MAPK activity and myogenic differentiation

The p38α/β mitogen-activated protein kinase (MAPK) pathway promotes skeletal myogenesis, but the mechanisms by which it is activated during this process are unclear. During myoblast differentiation, the promyogenic cell surface receptor Cdo binds to the p38α/β pathway scaffold protein JLP and, via JLP, p38α/β itself. We report that Cdo also interacts with Bnip-2, a protein that binds the small guanosine triphosphatase (GTPase) Cdc42 and a negative regulator of Cdc42, Cdc42 GTPase-activating protein (GAP). Moreover, Bnip-2 and JLP are brought together through mutual interaction with Cdo. Gain- and loss-of-function experiments with myoblasts indicate that the Cdo–Bnip-2 interaction stimulates Cdc42 activity, which in turn promotes p38α/β activity and cell differentiation. These results reveal a previously unknown linkage between a cell surface receptor and downstream modulation of Cdc42 activity. Furthermore, interaction with multiple scaffold-type proteins is a distinctive mode of cell surface receptor signaling and provides one mechanism for specificity of p38α/β activation during cell differentiation.

RD rhabdomyosarcoma cells (cancer cells of the muscle lineage that have low differentiation capability) are defi cient in p38 ␣ / ␤ activity in response to differentiation-inducing culture conditions, and enforced p38 ␣ / ␤ activation in these cells by expression of activated MKK6 (an immediate upstream activating T he p38 ␣ / ␤ mitogen-activated protein kinase (MAPK) pathway promotes skeletal myogenesis, but the mechanisms by which it is activated during this process are unclear. During myoblast differentiation, the promyogenic cell surface receptor Cdo binds to the p38 ␣ / ␤ pathway scaffold protein JLP and, via JLP, p38 ␣ / ␤ itself. We report that Cdo also interacts with Bnip-2, a protein that binds the small guanosine triphosphatase (GTPase) Cdc42 and a negative regulator of Cdc42, Cdc42 GTPase-activating protein (GAP). Moreover, Bnip-2 and JLP are brought together through mutual interaction with Cdo. Gain-and loss-of-function experiments with myoblasts indicate that the Cdo -Bnip-2 interaction stimulates Cdc42 activity, which in turn promotes p38 ␣ / ␤ activity and cell differentiation. These results reveal a previously unknown linkage between a cell surface receptor and downstream modulation of Cdc42 activity. Furthermore, interaction with multiple scaffold-type proteins is a distinctive mode of cell surface receptor signaling and provides one mechanism for specifi city of p38 ␣ / ␤ activation during cell differentiation.
Cdc42 were expressed in different myoblast cell systems have produced contradictory results but, in general, both mutants interfered with myogenesis ( Takano et al., 1998 ;Gallo et al., 1999 ;Meriane et al., 2000 ), suggesting that major perturbations in Cdc42 activity are not tolerated.
Like other small GTPases, Cdc42 cycles between an inactive GDP-bound state and an active GTP-bound state. The activity cycle is directly regulated by its interaction with stimulatory guanine nucleotide exchange factors and inhibitory GTPaseactivating proteins (GAPs) and by interaction with additional proteins ( Van Aelst and D ' Souza-Schorey, 1997 ;Jaffe and Hall, 2005 ). A candidate regulator of Cdc42 is Bnip-2 ( Low et al., 1999( Low et al., , 2000a. Bnip-2 is a 314-aa protein that harbors a single recognizable motif, a BCH domain that spans its C-terminal half. Cdc42GAP (also known as p50RhoGAP/ARHGAP1) contains a BCH domain in its noncatalytic N-terminal region, and Bnip-2 and Cdc42GAP interact via their respective BCH domains ( Low et al., 1999( Low et al., , 2000b. The Bnip-2 BCH domain also binds Cdc42 itself ( Low et al., 2000a ). Transient expression of Bnip-2 in several cell types induces cellular elongation and membrane protrusions in a manner dependent on its ability to bind Cdc42 and on cellular Cdc42 activity ( Zhou et al., 2005 ). That Bnip-2 binds Cdc42GAP, a negative regulator of Cdc42, yet has the ability to induce morphological alterations that require Cdc42 binding and Cdc42 activity, suggests that Bnip-2 might function as a scaffold for dynamic regulation of Cdc42 signaling. However, modulation of cellular Cdc42 activity by Bnip-2 has not been demonstrated.
Cdo is a cell surface receptor of the Ig superfamily that promotes myogenesis in vivo and in vitro ( Kang et al., 1998( Kang et al., , 2003Cole et al., 2004 ). Primary myoblasts from Cdo Ϫ / Ϫ mice and C2C12 myoblasts that express Cdo siRNA differentiate defectively in culture, producing reduced levels of muscle-specifi c proteins and fusing into myotubes ineffi ciently Takaesu et al., 2006 ). During myogenesis, the Cdo intracellular region binds to JLP, a scaffold protein for the p38 ␣ / ␤ MAPK pathway and, via JLP, p38 ␣ / ␤ ( Takaesu et al., 2006 ). Cdo Ϫ / Ϫ myoblasts are defi cient in differentiation-associated p38 ␣ / ␤ activity, and their differentiation defect is specifi cally rescued by expression of activated MKK6 ( Takaesu et al., 2006 ); however, how association of JLP/p38 ␣ / ␤ with Cdo leads to activation of p38 ␣ / ␤ is unknown. We report in this paper that the Cdo intracellular region binds Bnip-2 and that this interaction regulates Cdc42 activity, thus identifying a novel linkage between a cell surface receptor and regulation of Cdc42. Bnip-2 and JLP do not directly associate but are brought together through mutual interaction with Cdo, and Bnip-2 and Cdc42 stimulate p38 ␣ / ␤ activation and myogenic differentiation. Interaction with multiple scaffold-type proteins is an unusual mode of cell surface receptor signaling and provides a mechanism by which p38 ␣ / ␤ can be specifi cally activated to promote cell differentiation.

Cdo interacts with Bnip-2
A yeast two-hybrid screen that used the Cdo intracellular region as bait previously identifi ed JLP as a Cdo-interacting protein ( Takaesu et al., 2006 ). This screen also identifi ed positive clones kinase for p38) rescued myogenesis . Cytokines and various types of cellular stress are activators of the p38 ␣ / ␤ and other pathways ( Zarubin and Han, 2005 ); however, treatment of RD cells with TNF ␣ , sorbitol or UV light failed to rescue differentiation despite activation of p38 ␣ / ␤ , revealing that differentiation-and stress-induced programs are distinct . Furthermore, the p38 ␣ / ␤ pathway functions as a switch in muscle satellite cells, required initially to phosphorylate unidentifi ed substrates that activate cell cycle entry and subsequently targeting substrates named in the previous paragraph to promote cell differentiation ( Jones et al., 2005 ). Presumably, these distinct roles of p38 ␣ / ␤ signaling require appropriate concentrations of p38 ␣ / ␤ to become activated at specifi c times and subcellular locations. However, the spatiotemporal regulatory mechanisms by which reiteratively used signaling pathways (like p38 ␣ / ␤ ) achieve such specifi city are, in most cases, unclear.
The Rho family of small GTPases regulates many biological processes, including cytoskeletal dynamics, cell polarity, signal transduction, and transcription ( Van Aelst and D ' Souza-Schorey, 1997 ;Jaffe and Hall, 2005 ). They are therefore well positioned to coordinate the changes in both gene expression and cell morphology that characterize cell differentiation. The role in vertebrate myogenesis of one such GTPase, Cdc42, is controversial. The concentration of active GTP-bound Cdc42 is relatively low in proliferating myoblasts and increases severalfold in differentiating cells ( Travaglione et al., 2005 ). Studies in which constitutively active and dominant-negative mutants of  Low et al., 2000b ;Zhou et al., 2005 ), was tested in an analogous manner. The deletion constructs ⌬ 261 -269, ⌬ 264 -284, and ⌬ 285 -292 each strongly diminished, but did not fully prevent, Cdo binding ( Fig. 2 C ). In contrast, ⌬ 217 -221, ⌬ 235 -239, and ⌬ 251 -263 bound Cdo normally, suggesting that a 31-aa region (261 -292) near the C terminus of Bnip-2 encompasses the site of Cdo binding.

Bnip-2 positively regulates myogenic differentiation
To explore a role for Bnip-2 in myogenesis, C2C12 cells with enhanced or diminished Bnip-2 levels were constructed by stable expression of a Bnip-2 cDNA or Bnip-2 siRNAs, respectively. Forced expression of a Bnip-2 cDNA generally increased overall immunoreactivity of Bnip-2 bands about fourfold ( Fig.  3 A ). Overexpression of Bnip-2 did not signifi cantly alter the morphology or proliferation of C2C12 cells cultured in GM (unpublished data). However, when triggered to differentiate, cells that overexpressed Bnip-2 (C2C12/Bnip-2 cells) displayed a greater fraction of cell nuclei in myosin heavy chain -positive (MHC + ) myotubes than did vector control cells ( Fig. 3, B and C ). Furthermore, some of the myotubes formed by C2C12/Bnip-2 cells had much larger numbers of nuclei than were observed with the vector control cells ( Fig. 3 C , arrow). When analyzed for DM-induced expression of muscle-specifi c proteins, C2C12/ Bnip-2 cells showed accelerated and enhanced levels of the differentiation markers myogenin, MHC, and troponin T relative to the control cultures ( Fig. 3 A ).
To assess the effects of diminished Bnip-2 levels on differentiation, three independent Bnip-2 sequences placed into the pSilencer siRNA vector were individually and stably expressed in C2C12 cells. Each sequence substantially reduced Bnip-2 protein levels ( Fig. 4 A ). Note that the most slowly migrating band was more variably affected than the faster migrating forms in Fig. 4 A and in later fi gures. This could represent a more encoding Bnip-2 ( Fig. 1 A ). The intracellular region of another Ig protein, Necl-2, did not interact as effi ciently with Bnip-2, nor did Cdo interact as effi ciently with a Necl-2 binding protein, Pals-2 ( Shingai et al., 2003 ;Takaesu et al., 2006 ). Consistent with their interaction in yeast, Cdo and fl ag epitope -tagged Bnip-2 coimmunoprecipitated when transiently expressed in 293T cells ( Fig. 1 A ). Like Cdo, Bnip-2 is expressed endogenously in C2C12 myoblasts, and Bnip-2 levels increased during a 4-d time course of differentiation ( Fig. 1 C ). On SDS-PAGE of C2C12 cell lysates, Bnip-2 migrated as a series of bands ( Fig.  1, C and D ) that likely represent alternatively spliced variously phosphorylated isoforms ( Low et al., 1999 ;unpublished data). To assess whether endogenous Cdo and Bnip-2 interact, C2C12 cells were harvested while proliferating in growth medium (GM) or over a 3-d time course after transfer to differentiation medium (DM). Cell lysates were immunoprecipitated with Bnip-2 antibodies and probed with Cdo antibodies ( Fig. 1 D ). Cdo coprecipitated with Bnip-2, with maximal interaction occurring over the fi rst 2 d in DM, which is similar to Cdo ' s interaction with JLP ( Takaesu et al., 2006 ).
To identify the Cdo binding site in Bnip-2, a series of previously characterized fl ag-tagged Bnip-2 deletion mutants ( Fig. 2 A ; Low et al., 2000b ;Zhou et al., 2005 ) were tested for their ability to coimmunoprecipitate Cdo in transiently transfected COS7 cells ( Fig. 2 B ). A construct comprising only the Bnip-2 BCH domain (aa 167 -314) was as effective as full-length Bnip-2 in coprecipitating Cdo, but a construct lacking this domain failed to do so. Therefore, Bnip-2 binds to Cdo via its BCH domain. Boc is a protein that forms cis complexes with Cdo. Their respective ectodomains have strong sequence similarity but their intracellular regions are unrelated ( Kang et al., 2002 ). Boc also coimmunoprecipitated with Bnip-2, but the interaction appeared less effi cient and was only observed with full-length Bnip-2 ( Fig. 2 B ). A series of Bnip-2 mutants with small deletions in the BCH domain, which disrupt specifi c protein -protein inter-  Low et al., 2000b ;Zhou et al., 2005 ). Note that ⌬ 217 -221 and ⌬ 235 -239 are also known as ⌬ M and ⌬ R, respectively ( Low et al., 2000b ;Zhou et al., 2005 ). (B) Lysates of COS7 cells transiently transfected with fl ag-tagged Bnip-2, fl ag-tagged BCH, fl ag-tagged ⌬ BCH, Cdo, myctagged Boc, or control ( Ϫ ) expression vectors as indicated were immunoprecipitated (IP) with fl ag epitope antibodies and then Western blotted with fl ag epitope, Cdo, or myc epitope antibodies. (C) Lysates of COS7 cells transiently transfected with fl ag-tagged Bnip-2, fl ag-tagged Bnip-2 deletion mutants, Cdo, or control ( Ϫ ) expression vectors as indicated were immunoprecipitated (IP) with fl ag epitope antibodies and then Western blotted with fl ag epitope or Cdo antibodies. For unknown reasons, a faster migrating Cdo degradation band is reproducibly seen when Cdo is coexpressed with the ⌬ 217 -221 mutant. Lower levels of this band are also seen when Cdo is coexpressed with the ⌬ 251 -263 and ⌬ 261 -269 mutants.
Transfection effi ciencies of ‫ف‬ 10% were used to minimize fusion of independent ␤ -gal + transfectants. 48 h after transfection, the cultures were transferred to DM, and 72 h later the cells were double-stained for ␤ -gal activity and for MHC expression. Note that when transfectants fused with nontransfected cells, many (often most) of the nuclei in the myotube became positive for ␤ -gal activity because the cytoplasmically translated protein diffuses within the myotube Takaesu et al., 2006 ). Expression of wild-type Bnip-2 stimulated MHC expression and production of multinucleated myotubes by ␤ -gal + transfectants ( Fig. 5, A and B ). The ⌬ 217 -221 mutant functioned similarly to wild-type Bnip-2, indicating that interaction with Cdc42GAP is not required for Bnip-2 ' s promyogenic activity. In contrast, the ⌬ 264 -284 and ⌬ 285 -292 mutants lost the ability to promote myogenesis, suggesting that interaction with Cdc42 and/ or Cdo are required for this activity ( Fig. 5, A and B ).

Cdo and Bnip-2 promote Cdc42 activity, and Cdc42 promotes myogenesis
The concentration of GTP-bound (active) Cdc42 rises, and is maintained, when C2C12 cells are transferred into DM ( Fig. 6 A ; Travaglione et al., 2005 ). We therefore tested whether modulation of Bnip-2 levels altered the amount of active Cdc42 in these cells. C2C12/Bnip-2 cells displayed elevated levels of GTPbound Cdc42 relative to vector control cells in both GM and DM ( Fig. 6 B ), whereas stable expression of Bnip-2 siRNA largely prevented the DM-induced increase in GTP-bound Cdc42 ( Fig. 6 C ). The concentration of active Cdc42 therefore correlates with Bnip-2 protein levels.
Because Cdo binds Bnip-2 and Bnip-2 binds Cdc42, we asked whether Cdc42 was present in Cdo immunoprecipitates from differentiating C2C12 cell lysates. Indeed, Cdc42 coprecipitated with Cdo with a maximal interaction 1 d after transfer of cultures to DM ( Fig. 6 D ). This is similar to Cdo ' s interaction with Bnip-2 and suggests that formation of a Cdo -Bnip-2 -Cdc42 complex promotes Cdc42 activation. To test this possibility further, C2C12 cells that express Cdo siRNA and cultured Cdo Ϫ / Ϫ myoblasts were analyzed for Cdc42 activity. In C2C12 cells that express Cdo siRNA, the DM-associated elevation in GTP-bound Cdc42 levels seen in control transfectants was almost completely blunted ( Fig. 6 E ). Primary myoblasts must be cultured in GM plus basic FGF to remain in a proliferative nondifferentiated state, and both Cdo +/+ and Cdo Ϫ / Ϫ myoblasts had abundant GTP-bound Cdc42 in this medium, likely because of the added growth factor; however, when transferred to DM, the Cdo Ϫ / Ϫ cells had a lower concentration of active Cdc42 than did the Cdo +/+ cells ( Fig. 6 F ).
Both Cdo and Bnip-2 positively regulate myogenic differentiation and promote activation of Cdc42, suggesting that Cdc42 is itself promyogenic. However, previous studies in which constitutively active and dominant-negative mutants of Cdc42 were expressed in myoblasts failed to provide evidence for this notion, as both mutants blocked differentiation ( Meriane et al., 2000 ). Expression of siRNA against Cdc42 in C2C12 cells also inhibited differentiation, but these cells acquired a strongly altered morphology (unpublished data). Therefore, to gain information on the role of Cdc42 in myogenesis, we sought to alter Cdc42 activity in a more subtle manner. It was reasoned stable species of Bnip-2 that is relatively resistant to RNAi-mediated knockdown or revelation of a protein nonspecifi cally recognized by the Bnip-2 antibody that comigrates with the top Bnip-2 band. C2C12 cells that expressed each siRNA sequence displayed a smaller percentage of cell nuclei in MHC + myotubes, and the myotubes that formed were shorter and thinner, than control cells that expressed an irrelevant siRNA sequence ( Fig. 4, B and C ). Expression of MHC was delayed and reduced in Bnip-2 siRNA-expressing cells, whereas expression of myogenin was not substantially altered, perhaps because of incomplete knockdown of Bnip-2 ( Fig. 4 D ). These latter results suggest that myotube formation may be more sensitive to partial loss of Bnip-2 than is expression of muscle-specifi c proteins.
Bnip-2 binds to Cdc42, Cdc42GAP, and, as is shown here, Cdo. The Bnip-2 deletion mutant ⌬ 217 -221 is selectively defective in Cdc42GAP binding. The ⌬ 285 -292 mutant does not bind to Cdc42 and is ineffi cient at binding Cdo but binds Cdc-42GAP. The ⌬ 264 -284 mutant is ineffi cient at binding Cdo but interacts normally with Cdc42 and Cdc42GAP ( Fig. 2, A and C ; Low et al., 2000b ;Zhou et al., 2005 ). The ability of these mutants to promote myotube formation was examined in a transient myogenesis assay that scores expression of MHC and myotube formation by transfectants Takaesu et al., 2006 ). C2C12 cells were cotransfected with control or Bnip-2 expression vectors plus, to mark transfectants, a vector that drives expression of nuclear-localized ␤ -galactosidase ( ␤ -gal). (A) Lysates of C2C12 cells stably transfected with a control expression vector lacking a cDNA ( Ϫ ) or with an expression vector harboring a Bnip-2 cDNA (+) were Western blotted with antibodies to Bnip-2, the indicated muscle-specifi c proteins, or Cdo and, as a control, with pan-cadherin antibody. (B) Photomicrographs of C2C12/Bnip-2 and vector control cells that were cultured in DM, fi xed, and stained with an antibody to MHC. The arrow indicates a C2C12/Bnip-2 cell myotube with many more nuclei than are seen with the control cells under these conditions. Bar, 0.1 mm. (C) Quantifi cation of myotube formation. Values represent means of triplicate determinations ± 1 SD. The experiment was repeated three times with similar results. Asterisks indicate difference from control, P < 0.01. A level of myotube formation by control cells ( ‫ف‬ 35% nuclei in MHC + cells) was selected so as to permit visualization of enhanced differentiation by Bnip-2.
To enhance Cdc42 activity, three independent siRNA sequences against Cdc42GAP were stably expressed in C2C12 cells. Two of these sequences effectively diminished Cdc42GAP protein levels, and this was associated with an increase in the amount of GTP-bound Cdc42 in these cells ( Fig. 8, A and B ). When cultured in DM, cells with diminished levels of Cdc42GAP produced larger myotubes ( Fig. 8 C ), had a higher percentage of nuclei present in MHC + cells ( Fig. 8 D ), and expressed more MHC protein than did control transfectants ( Fig. 8 E ). Collectively, the results indicate that Cdc42 activity promotes myogenesis.
Cdo brings JLP and Bnip-2 together to regulate p38 ␣ / ␤ activity Cdc42 signaling activates p38 ␣ / ␤ MAPK in several cell systems ( Coso et al., 1995 ;Minden et al., 1995 ;Moln á r et al., that the concentration of GTP-bound Cdc42 and, perhaps more importantly, the amount of time Cdc42 proteins spend in the active state could be altered by modulating the amount of Cdc-42GAP protein expressed by C2C12 cells. Two GAP proteins for Cdc42, Cdc42GAP and BPGAP ( Shang et al., 2003 ), were each stably overexpressed in C2C12 cells ( Fig. 7 A ). In both cases, this resulted in lower steady-state levels of GTP-bound Cdc42 in both GM and DM, relative to control vector transfectants ( Fig. 7 B ). When cultured in DM, C2C12/Cdc42GAP cells and C2C12/BPGAP cells each displayed a similar phenotype: relative to control cells, a smaller percentage of cell nuclei were found in MHC + myotubes, and the myotubes that formed were shorter and thinner ( Fig. 7, C and D ). Furthermore, overexpression of Cdc42GAP or BPGAP resulted in delayed induction of the differentiation markers myogenin and MHC ( Fig. 7 E ).  between two and fi ve nuclei ( ‫ف‬ 45%), and those that had greater than fi ve nuclei ( < 5%; Fig. 9, G and H ). The expression of Bnip-2 siRNA decreased the percentage of MHC + cells to ‫ف‬ 66%, with multinucleated cells representing only ‫ف‬ 16%, none of which were in the greater-than-fi ve-nuclei category. Therefore, transient expression of Bnip-2 siRNA reduced myogenesis, which is similar to stable knockdown of Bnip-2. Expression of MKK6(EE) alone enhanced myogenesis relative to control transfectants, resulting in ‫ف‬ 94% MHC + cells, with ‫ف‬ 30% of the total having greater than fi ve nuclei. Cells that coexpressed MKK6(EE) and Bnip-2 siRNA underwent robust myogenesis, though not quite to the level observed with expression of MKK6(EE) alone ( ‫ف‬ 95% MHC + cells, but only ‫ف‬ 15% with greater than fi ve nuclei). These results are consistent with the notion that Bnip-2 ' s promyogenic function is exerted mainly, though perhaps not exclusively, via activation of p38 ␣ / ␤ .
We also assessed the effects of MKK6(EE) expression on cells that coexpressed the Bnip-2 deletion mutants ⌬ 264 -284 and ⌬ 285 -292. Similar to the results shown in Fig. 5 , expression of either of these two mutants did not dramatically alter myogenesis relative to control transfectants, suggesting a loss of function ( Fig. 9, G and H ). Coexpression of MKK6(EE) enhanced differentiation of cells expressing these mutants, but the effect was seen more in the production of multinucleated MHC + cells, with a smaller effect on the percentage of MHC + versus MHC Ϫ cells. The results with the deletion mutants plus or minus MKK6(EE) are somewhat distinct from the effects seen with Bnip-2 siRNA plus or minus MKK6(EE) and suggest that these mutants may have a subtle inhibitory effect made apparent by coexpression of MKK6(EE).  Bourdoulous et al., 1998 ). The ability of Cdo to bind Bnip-2 and JLP, which in turn bind Cdc42 and p38 ␣ / ␤ , respectively, suggests that Cdo might coordinate Cdc42 → p38 ␣ / ␤ signaling. The possibility that Bnip-2 and JLP bind the same Cdo complexes was tested initially. COS7 cells were transfected with expression vectors encoding S epitope -tagged JLP (JLP-S), JLP-S and Bnip-2, or JLP-S, Bnip-2, and Cdo. Cell lysates were then precipitated with anti -S agarose and blotted with antibodies to each protein ( Fig. 9 A ). JLP coprecipitated Bnip-2 in the presence, but not the absence, of coexpressed Cdo. Therefore, JLP and Bnip-2 did not directly interact but were brought together by a mutual interaction with Cdo. To assess whether such complexes form endogenously, lysates of Cdo +/+ and Cdo Ϫ / Ϫ myoblasts were immunoprecipitated with antibodies to Cdo or to Bnip-2 and then Western blotted with antibodies to Cdo, Bnip-2, and JLP. As expected, Bnip-2 and JLP both coprecipitated with Cdo ( Fig. 9 B ; Takaesu et al., 2006 ). Furthermore, JLP coprecipitated with Bnip-2 from Cdo +/+ cell lysates but not Cdo Ϫ / Ϫ cell lysates, indicating that Bnip-2 and JLP associate in myoblasts in a Cdo-dependent manner ( Fig. 9 C ). C2C12/Bnip-2 cells, which had elevated Cdc42 activity in GM and DM ( Fig. 6 ), also had elevated levels of the dually phosphorylated (active) form of p38 ␣ / ␤ (pp38 ␣ / ␤ ) in GM and DM ( Fig. 9 D ). Conversely, C2C12 cells that stably express Bnip-2 siRNA, which poorly activated Cdc42 in response to DM ( Fig. 6 ), were also defi cient in DM-induced pp38 ␣ / ␤ production ( Fig. 9 E ). Furthermore, expression of Cdc42GAP siRNA increased pp38 ␣ / ␤ levels in C2C12 cells in both GM and DM ( Fig. 9 F ). Collectively, these results are consistent with a model in which Cdo brings Bnip-2 and JLP together to coordinate Cdc42 → p38 ␣ / ␤ signaling.
To assess whether p38 ␣ / ␤ is functionally downstream of Bnip-2, we asked whether coexpression of the p38 ␣ / ␤ activator MKK6(EE) could rescue the differentiation defect associated with expression of Bnip-2 siRNA. The transient C2C12 cell myogenesis assay described earlier ( Fig. 5 ) was used in this case. Approximately ‫ف‬ 83% of double control-vector transfectants were MHC + , and these cells were categorized as those that were mononucleated ( ‫ف‬ 35% of total cell nuclei), those that had express no Cdo protein, are also MHC + ( Takaesu et al., 2006 ). These results suggest that other pathways that activate p38 ␣ / ␤ are functional in Cdo-and Bnip-2 -depleted myoblasts.
To assess directly whether additional stimuli can activate p38 ␣ / ␤ in cells depleted of Cdo or Bnip-2, Cdo Ϫ / Ϫ myoblasts and C2C12 cells expressing Cdo or Bnip-2 siRNA were treated with TNF ␣ or with hyperosmotic stress and analyzed for production of pp38 ␣ / ␤ . Such cells produced pp38 ␣ / ␤ at levels similar to those of control cells in response to these stimuli ( Fig. 10, D -F ). Collectively, the results suggest that Cdo/Bnip-2 signaling is neither the sole mechanism by which p38 ␣ / ␤ becomes activated during myogenesis nor a general requirement for full p38 ␣ / ␤ activation but is likely to be specifi c for differentiation-mediated signaling.

Discussion
Many ubiquitous signaling pathways perform cell type -specifi c functions. Furthermore, such pathways can regulate disparate functions in the same cell type. Clearly, this requires that the myoblasts cultured in the presence of the p38 ␣ / ␤ inhibitor SB203580 have a more dramatic blockade to differentiation ( Cuenda and Cohen, 1999 ;Zetser et al., 1999 ;Wu et al., 2000 ). It seems likely, therefore, that Cdo/Bnip-2 -independent pathways also contribute to p38 ␣ / ␤ activation during myogenesis. To examine this point more closely, C2C12 cells that stably express Bnip-2 or Cdo siRNA were treated with SB203580 and assessed for myotube formation and expression of MHC ( Fig. 10, A -C ). The percentages of control cell nuclei that were present in MHC + versus MHC Ϫ cells was ‫ف‬ 80:20, whereas this MHC + /MHC Ϫ ratio was ‫ف‬ 10:90 in SB203580-treated control cells. The MHC + /MHC Ϫ ratio of C2C12 cells expressing either Cdo or Bnip-2 siRNA was ‫ف‬ 40:60, which is consistent with incomplete inhibition of differentiation. Treatment of such cells with SB203580 further reduced the ratio to ‫ف‬ 10/90, which is similar to that of vector control cells. Therefore, the cells with diminished levels of Cdo or Bnip-2 that express MHC remain dependent on residual p38 ␣ / ␤ activity to do so. Although the extent of siRNA-mediated depletion is not 100%, ‫ف‬ 40% of Cdo Ϫ / Ϫ myoblasts, which  (B) Levels of GTP-bound and total Cdc42 in C2C12 cells that express Cdc-42GAP siRNA (+) or an irrelevant sequence ( Ϫ ) in DM were assessed as described in Fig. 6 A . (C) Photomicrographs of C2C12 cells that express Cdc42GAP siRNA sequences or an irrelevant (pSilencer) sequence as indicated, cultured in DM, fi xed, and stained with an antibody to MHC. Bar, 0.1 mm. (D) Quantifi cation of myotube formation. Values represent means of triplicate determinations ± 1 SD. The experiment was repeated three times with similar results. Asterisks indicate difference from pSilencer control, P < 0.02. A level of myotube formation by control cells ( ‫ف‬ 45% nuclei in MHC + cells) was selected so as to permit visualization of enhanced differentiation by Cdc42GAP siRNA. (E) Western blot analysis of MHC expression by C2C12 cells that express Cdc42GAP siRNA sequences numbers 1 or 3 or an irrelevant sequence ( Ϫ ), cultured in DM for 48 h. Fig. 10 G ). JLP and Bnip-2 associate in a Cdo-dependent manner, implying that Cdc42 bound to Cdo via Bnip-2 activates p38 ␣ / ␤ bound to Cdo via JLP and that this represents a pool of p38 ␣ / ␤ specifi cally activated during differentiation. We speculate that binding of Bnip-2 -Cdc42 to Cdo allows Cdc42 to interact with a specifi c guanine nucleotide exchange factor, promoting nucleotide exchange on Cdc42 and subsequent binding to effector proteins that initiate a kinase cascade resulting in activation of p38 ␣ / ␤ . Alternatively, Cdc42 may be activated before association with Cdo, and subsequent interaction between Cdo, Bnip-2, and Cdc42 stabilizes Cdc42 in the active state and brings it into proximity with components of the p38 ␣ / ␤ pathway.
All the described components of this complex are ubiquitously expressed except Cdo, which, though not muscle specifi c, is highly enriched in muscle precursor cells and differentiating muscle ( Kang et al., 1998 ;Mulieri et al., 2000 ), suggesting that Cdo itself may provide some level of cell type specifi city to this signaling pathway. Cdo is also expressed at high levels in neuronal precursors, and similar results to those reported here have been obtained in experiments on neuronal differentiation of a activation of these pathways occurs at the correct time and subcellular location and with the appropriate magnitude and duration. The mechanisms by which such specifi city is achieved are not well understood. We previously reported that during myogenic differentiation, the cell surface receptor Cdo binds the p38 ␣ / ␤ pathway scaffold protein JLP and, via JLP, p38 ␣ / ␤ itself ( Takaesu et al., 2006 ). Cdo Ϫ / Ϫ myoblasts are defi cient in both p38 ␣ / ␤ activity and differentiation capability, and their ability to differentiate is rescued by expression of the p38 ␣ / ␤ activator MKK6(EE) ( Takaesu et al., 2006 ); however, these results did not explain how association of JLP/p38 ␣ / ␤ with Cdo leads to activation of p38 ␣ / ␤ .
In this paper, it is demonstrated that Cdo also associates with the Cdc42 binding protein Bnip-2, identifying a novel linkage between a cell surface receptor and downstream modulation of Cdc42 activity. This interaction stimulates Cdc42 and p38 ␣ / ␤ activities and regulates myogenic differentiation. Cdo, therefore, appears to promote activation of p38 ␣ / ␤ by assembly of multiprotein signaling modules for Cdc42 and p38 ␣ / ␤ via direct binding to scaffold proteins for each (i.e., Bnip-2 and JLP, respectively; Figure 9. Bnip-2 and JLP bind the same Cdo complexes, and Bnip-2 regulates p38 ␣ / ␤ activity to promote myogenesis. (A) Lysates of COS7 cells transiently transfected with JLP-S, Bnip-2, Cdo, or control ( Ϫ ) expression vectors as indicated were immunoprecipitated (IP) with anti -S agarose and then Western blotted with JLP, Bnip-2, or Cdo antibodies. (B) Lysates of myoblasts of the indicated Cdo genotype cultured in DM for 48 h were immunoprecipitated with Cdo antibodies and then Western blotted with Cdo, Bnip-2, or JLP antibodies. Total cell lysates were also Western blotted with Bnip-2 or JLP antibodies. (C) Lysates of myoblasts of the indicated Cdo genotype cultured in DM for 48 h were immunoprecipitated with Bnip-2 antibodies and then Western blotted with Cdo, Bnip-2, or JLP antibodies. Total cell lysates were also Western blotted with Cdo or JLP antibodies. The Cdo band below the one indicated by the arrow is a partial degradation product. (D) Lysates of C2C12/Bnip-2 (+) or vector control ( Ϫ ) cells in GM or cultured for 48 h in DM were Western blotted with anti -phospho p38 ␣ / ␤ (pp38) or p38 ␣ / ␤ (p38) antibodies. (E) Lysates of C2C12 cells stably expressing a Bnip-2 siRNA sequence (+) or an irrelevant sequence ( Ϫ ) in GM or cultured for 48 h in DM were Western blotted with pp38 or p38 antibodies. (F) Lysates of C2C12 cells stably expressing a Cdc42Gap siRNA sequence (+) or an irrelevant sequence ( Ϫ ) in GM or cultured for 48 h in DM were Western blotted with pp38 or p38 antibodies. As the lanes were somewhat unevenly loaded in F, the pp38 and p38 signals were quantifi ed by densitometry, and the pp38/p38 ratio reported under each lane in arbitrary units with the control transfectants in GM set to 1. (G) C2C12 cells were cotransfected with control, Bnip-2 siRNA, or Bnip-2 deletion mutant expression vectors, control or MKK6(EE) expression vectors, and pQ-lacZ (a vector that drives expression of nuclear-localized ␤ -gal to mark transfectants). Differentiated cultures were double-stained for ␤ -gal activity (blue) and for MHC expression (brown). Bar, 0.1 mm. (H) Quantifi cation of C2C12 cell differentiation shown in G. Cultures were scored as MHC Ϫ or MHC + , with MHC + cells further scored as having a single (1) nucleus, two to fi ve nuclei, or greater than fi ve nuclei. Values represent means of triplicate determinations ± 1 SD. The experiment was repeated three times with similar results.
Active cycling of Cdc42 may be required for effi cient myogenesis, as the results presented in this paper demonstrate that Cdc42 activity is important for this process, but expression of constitutively active or dominant-negative mutants of Cdc42 each block differentiation ( Meriane et al., 2000 ). The ability of Cdc42 to trigger p38 ␣ / ␤ activation fi ts well with the known importance of p38 ␣ / ␤ in myogenic differentiation ( Lluis et al., 2006 ). However, Cdc42 regulates additional processes that are also likely to be relevant to differentiation, such as the formation of fi lopodia, which may be required for cell -cell interactions in preparation for fusion, and regulation of cell polarity ( Jaffe and Hall, 2005 ). That forced expression of the p38 ␣ / ␤ activator MKK6(EE) has a somewhat greater effect on differentiation of control myoblasts than on myoblasts depleted of Bnip-2 is consistent with the notion that promyogenic pathways other than p38 ␣ / ␤ lie downstream of Cdc42, but additional work will be required to establish that this is the case. neural precursor line (unpublished observations). It is also clear, however, that not all p38 ␣ / ␤ pathway activity during myoblast differentiation is Cdo-or Bnip-2 -dependent, as residual pp38 ␣ / ␤ is detected in cells depleted for Cdo or Bnip-2, and treatment of such cells with the p38 ␣ / ␤ inhibitor SB203580 further inhibits their differentiation. Potential additional mechanisms for activation of p38 ␣ / ␤ in differentiating myoblasts include low-level autocrine TNF ␣ signaling and signaling by semaphorin 4C ( Ko et al., 2005 ;Chen et al., 2007 ;Riuzzi et al., 2007 ;Wu et al., 2007 ).
In addition to binding Cdc42, Bnip-2 binds its negative regulator, Cdc42GAP ( Low et al., 1999 ). However, loss-and gain-of-function experiments in myoblasts indicate that Bnip-2 stimulates Cdc42 activity. These data suggest that Bnip-2 may function as a scaffold for dynamic signaling through Cdc42, modulating the balance or kinetics of its activity cycle. This is the fi rst paper to fi nd an endogenous function for Bnip-2, and we are unaware of another protein with this type of activity for Cdc42. Figure 10. Other mechanisms of p38 ␣ / ␤ activation occur in the absence Cdo/Bnip-2 signaling. (A) Photomicrographs of C2C12 cells stably transfected with control vector or vectors expressing Cdo or Bnip-2 siRNA that were cultured in DM plus SB203580 (+SB) or DMSO vehicle ( Ϫ SB), fi xed, and stained with an antibody to MHC. Bar, 0.1 mm. (B) Quantifi cation of the percentage of nuclei in cultures shown in A that were in MHC + or MHC Ϫ cells. Control vector cells are indicated by the negative sign for both Cdo and Bnip-2 siRNA. Values represent means of triplicate determinations ± 1 SD. The experiment was repeated three times with similar results. Asterisks indicate difference from vector control, P < 0.01. (C) Western blot analysis of cultures shown in A. Extracts were probed with antibodies to MHC or total p38 ␣ / ␤ (p38). (D) Myoblasts of the indicated Cdo genotype were treated with 0.9 M NaCl or 10 ng/ml TNF ␣ for 15 min and analyzed for pp38 ␣ / ␤ and p38 ␣ / ␤ . (E) C2C12 cells stably expressing siRNA against Cdo were treated with NaCl or TNF ␣ and analyzed as in D. (F) C2C12 cells stably expressing siRNA against Bnip-2 were treated with NaCl or TNF ␣ and analyzed as in D. (G) Model of Cdo-mediated p38 ␣ / ␤ activation during myogenic differentiation. Cdo binds directly to JLP and, via JLP, to p38 ␣ / ␤ . Cdo also binds to Bnip-2 and, via Bnip-2, Cdc42. Formation of a Cdo -Bnip-2 -Cdc42 complex promotes or stabilizes activation of Cdc42, which in turn triggers signals culminating in phosphorylation and activation of p38 ␣ / ␤ bound to Cdo via JLP. Cdo interacts with itself ( Kang et al., 2003 ) and is shown as a dimer. JLP and Bnip-2 are shown as binding to different Cdo proteins of the dimer for convenience and does not preclude the possibility that JLP and Bnip-2 bind the same Cdo protein simultaneously. See text for additional details.
2.1-U6 hygro vector harboring an irrelevant sequence (Ambion) was used as a control. These vectors were transfected into C2C12 cells with Fu-Gene6 and cultures were selected in hygromycin-containing medium. Drug-resistant cells were pooled and analyzed. Multiple such pools were studied in each case.
For Cdo -Bnip-2 interaction studies, expression vectors encoding Cdo, myc-tagged Boc, Bnip-2, and Bnip-2 deletion mutants ( Low et al., 2000b ;Kang et al., 2002 ;Zhou et al., 2005 ) were transiently transfected into COS7 or 293T cells with FuGene6 or calcium phosphate, respectively. 48 h later, cells were harvested for Western blot and immunoprecipitation analyses as described in the next section.
Collectively, these results reveal a distinctive mechanism of signaling: the Cdo intracellular region binds to scaffold proteins that in turn bind multiple components of specifi c pathways. This is different from other Ig/FNIII repeat receptors related to Cdo, such as the Robo proteins, which bind to the nonreceptor tyrosine kinase, c-Abl, the Rho family GAPs srGAP and Vilse, the SH2/SH3 adaptor Nck, and the actin regulator Ena/ Vasp ( Dickson and Gilestro, 2006 ). Furthermore, Cdo functions in multiple contexts at the extracellular face of the cell surface. In myoblasts, it forms cis complexes with N-cadherin and neogenin, the latter a receptor for the netrin and RGM families of ligands ( Kang et al., 2003Cole et al., 2007 ). Additionally, Cdo binds Sonic hedgehog, possibly as a coreceptor with Patched1 ( Tenzen et al., 2006 ;Yao et al., 2006 ;Martinelli and Fan, 2007 ). The mechanism by which binding of JLP and Bnip-2 to Cdo is induced during myogenesis is not clear, but a connection with N-cadherin is likely. Expression in C2C12 cells of a Cdo deletion mutant that is specifi cally defi cient in its ability to bind N-cadherin blocks differentiation ( Kang et al., 2003 ). N-cadherin -based adhesion enhances p38 ␣ / ␤ activity in C2 myoblasts ( Lovett et al., 2006 ), and preliminary results suggest that Cdo, JLP, and Bnip-2 can be recruited to sites of N-cadherin ligation (unpublished data). Cdo may function to link cadherinbased adhesion to the p38 ␣ / ␤ pathway, which promotes the muscle-specifi c transcriptional program. That Cdc42, a known regulator of actin dynamics, is directly involved in this process provides a mechanism by which such changes in gene expression can be coordinated with the alterations in cell morphology that are also required for cell differentiation.

Materials and methods
Yeast two-hybrid screen Bnip-2 was identifi ed in a previously reported screen in which the transmembrane plus intracellular region of mouse Cdo was used as bait ( Takaesu et al., 2006 ). Two independent clones were isolated a total of eight times. Both clones started in the 5 Ј UTR, were in frame, and encoded the entire Bnip-2 ORF.
Cell culture C2C12, 293T, COS7, and myoblasts derived from Cdo +/+ and Cdo Ϫ / Ϫ mice were cultured as previously described ( Kang et al., 1998Cole et al., 2004 ). To induce differentiation of C2C12 cells, cultures were transferred from DME containing 15% FBS (GM) to DME containing 2% horse serum (DM). Myotube formation in stable and transient assays was performed and quantifi ed as previously described . Statistical analysis of the number of nuclei in MHC + versus MHC Ϫ cells was done with Student ' s t test.
For stable overexpression studies in myoblasts, pXJ40 vectors encoding fl ag-tagged forms of Bnip-2, Cdc42GAP, or BPGAP ( Low et al., 1999( Low et al., , 2000a were cotransfected with pBabePuro ( Morgenstern and Land, 1990 ) into C2C12 cells with FuGene6 (Roche), and cultures were selected in puromycin-containing medium. Drug-resistant cells were pooled and analyzed. Multiple such pools were studied in each case.