MST3 Kinase Phosphorylates TAO1/2 to Enable Myosin Va Function in Promoting Spine Synapse Development

Summary Mammalian Sterile 20 (Ste20)-like kinase 3 (MST3) is a ubiquitously expressed kinase capable of enhancing axon outgrowth. Whether and how MST3 kinase signaling might regulate development of dendritic filopodia and spine synapses is unknown. Through shRNA-mediated depletion of MST3 and kinase-dead MST3 expression in developing hippocampal cultures, we found that MST3 is necessary for proper filopodia, dendritic spine, and excitatory synapse development. Knockdown of MST3 in layer 2/3 pyramidal neurons via in utero electroporation also reduced spine density in vivo. Using chemical genetics, we discovered thirteen candidate MST3 substrates and identified the phosphorylation sites. Among the identified MST3 substrates, TAO kinases regulate dendritic filopodia and spine development, similar to MST3. Furthermore, using stable isotope labeling by amino acids in culture (SILAC), we show that phosphorylated TAO1/2 associates with Myosin Va and is necessary for its dendritic localization, thus revealing a mechanism for excitatory synapse development in the mammalian CNS.

Empty vector or MST3 shRNA expressing vector transfected neurons are stained with a mouse monoclonal MST3 antibody. The staining in the cell body is significantly reduced (arrows and dotted line around the soma) (p<0.001) (n = 5 neurons each). Scale bar = 25 µm. C. Average filopodium length is also increased upon loss of function of MST3. D. Total filopodia length per 50 µm is not altered in MST3* (wild type MST3 that is shRNA resistant) expressing neurons (n = 6 for GFP and 5 for MST3*). E. Control shRNA does not alter length filopodia (n = 6 for GFP and 7 for control shRNA). F. Lentivirus was generated for pLentiLox 3.7 vector alone or vector expressing control shRNA (Ctrl sh), MST3 sh1 and MST3 sh2. Neurons were infected at DIV6 and protein lysates were obtained at 16 DIV. Endogeneous MST3 expression levels are reduced confirming knock-down (using rabbit polyclonal MST3 antibody). Tubulin antibody is used as a control for total protein levels. MST3 migrates at 60 kDa. Also see Figure S4F. G. Expression of HA tagged MST3-KD and HA-MST3* is detected by anti-HA antibodies for all data acquisition. Scale bar = 25 µm. H. MST3 shRNA #2 significantly increases dendrite total filopodia length per dendrite length, comparable to the first MST3 shRNA used (n = 7 neurons each). All error bars reflect standard errors.   shRNA and TAO1A increases these measurements significantly (n = 21, 9, 14, 12 and 12 for GFP, MST3 shRNA, TAO1A, TAO1D and TAO1WT, respectively). C. TAO1A expression increases dendritic filopodia length when compared to GFP alone and TAO1D in hippocampal neurons transfected at DIV4 and imaged at DIV8 (n = 7, 7 and 10 for GFP, TAO1A, TAO1D, respectively).
D. Representative images of dendrites expressing three separate TAO1 shRNAs (which do not target TAO2) are shown. Scale bar is 7.5 µm. E. None of the three TAO1 shRNAs has any significant effect on filopodia density when expressed from 3DIV to 7DIV (n = 5, 6, 6, 5 and 6 neurons for GFP, TAO1sh1, TAO1sh2, TAO1sh3 and TAO1/2shRNA, respectively). TAO1/2 significantly increases filopodia density as shown earlier. F. Lentivirus expressing shRNA as labelled are used to infect cultured neurons at DIV6 and neuronal lysates were collected at DIV11. MST3 shRNAs are highly effective as shown earlier, TAO1/2 shRNA can effectively knock down TAO2 but also reduces TAO1 levels. Proteins migrate at, TAO1 120-130 kDa, TAO2 140-150 kDa, MST3 60 kDa. G. Filopodia density is also increased in TAO1/2 shRNA and TAO2A expressing neurons (n = 14, 14 and 6 neurons for GFP, TAO1/2 shRNA and TAO2A). H. PSD95-GFP and Td-tomato was expressed with TAO1/2 shRNA in hippocampal neurons. Scale bar = 5 µm. I. PSD95-GFP containing dendritic protrusion density is reduced in TAO1/2 shRNA expressing neurons (analysis also shown in Figure   2C & D). All error bars reflect standard errors.  MyosinVa-GTD tagged with HA is co-expressed with a vector expressing GFP alone or GFP with TAO1/2 shRNA in dissociated cortical neurons. GFP is shown in green, HA antibody staining is shown in red. Images are maximum z projections of image stacks obtained by confocal imaging. Myosin Va-GTD-HA is localized to dendrites in control neurons but is restricted to the cell body in TAO1/2 shRNA expressing neurons. Cortical neurons were transfected at DIV 13-14 and fixed in 3 to 4 days. B. Myosin Vb-GTD-HA (red) is expressed in neurons with GFP or GFP together with TAO1/2 shRNA. The Myosin Vb-GTD puncta shown in red (right) is seen as yellow due to colocalization with GFP inside the neurons (left). Scale bars are 10 µm. C. Quantification of Myosin Va-HA intensity per pixel along one primary dendrite in neurons expressing GFP alone or TAO1/2 shRNA and GFP. In TAO1/2 shRNA expressing neurons Myosin Va-GTD-HA is reduced (p < 0.01). D. TAO1/2 shRNA causes increase in Myosin Vb-GTD-HA puncta in dendrites. Number of Myosin Vb-GTD-HA puncta is increased in TAO1/2 shRNA expressing neurons (p < 0.001). E. MyosinVa-GTD-GFP expressed in dissociated cortical neurons and stained by anti-Rab11. MyosinVa-GTD-GFP (green) is colocalized with Rab11 (red). Scale bar = 10 µm. F. In a subset of TAO1/2 shRNA expressing neurons, it can be clearly observed that axon is the only process that contains Myosin Va-GTD-GFP (yellow arrow). Scale bar = 10 µm. All error bars reflect standard errors. Table S1: The enrichment of proteins in Experiment 1 is shown as -values and in Experiment 2 as positive values. The columns are ordered with ascending median log2 value for Experiment 2. The number of unique peptides identified from each protein is shown in #peptides columns. Proteins that are enriched in phosphopeptide pulldown are shown in bold at the bottom of the list.

In Utero Electroporation
Timed pregnant C57Bl6 mice were used for in utero electroporation for the first set of experiments with MST3 shRNA. Surgery was performed at E14.5-15.5, when mice were anesthetized with sodium pentobarbital at 50 mg/kg body weight. A midline incision was made and the uterus was exposed. DNA solution including the plasmid and 0.04% trypan blue was injected into the lateral ventricle with a glass micropipette. After injection, embryos were subjected to electrical pulses (32Vfor E14.5, 36V for E15.5) with 50 ms duration that were delivered five times at 950 ms interval using a square-pulse electroporator BTX830. Embryos were injected with either 1 μg/μl pCAG-GFP (control) or 0.4 μg/μl pCAG-GFP + 1 μg/μl MST3 shRNA #1 in pLenti-Lox 3.7. For a second set of experiments timed pregnant Parkes mice were used. In these experiments animals were anaesthetized with isofluorane during the surgery. Embryos were injected with either 1 μg/μl pCAG-GFP (control) or 0.4 μg/μl pCAG-GFP + 1 μg/μl MST3 shRNA #1 or 0.4 μg/μl pCAG-GFP + 1 μg/μl TAO1/2 shRNA in pLenti-Lox 3.7. After the procedure the abdominal wall and skin were sutured to allow the embryos to develop full term. Litters were inspected for GFP expression in the cortex by fluorescent goggles in first week (Nightsea). Mice were perfused at P18-P20 using 4% paraformaldehyde and 4% sucrose. Brains were post-fixed in the same fixative solution at 4°C. 100 μm thick coronal sections were obtained using cryostat sectioning. Sections were dried on glass slides, blocked by 10% Normal Donkey Serum and 0.5% Triton-X and immunostained by mouse GFP antibody (1:1000, Roche) in blocking buffer, followed by goat anti-mouse conjugated with Alexa-488 (1:500, Invitrogen).

Confocal Microscopy
Dendrites were imaged using an inverted Leica SP5 confocal microscope using a 63X (NA 1.4) objective at 6X zoom. Z sections were obtained across the dendrite depth at 0.5 μm z-intervals.
Spine density and categorization was done manually using Leica image analysis. Spine head diameter measurements were done using a custom plugin in ImageJ (Ultanir et al., 2007).

Quantification of PhosphoTAO1/2 Staining in Cultures
Cultured rat hippocampal neurons were transfected with either control scrambled shRNA or shRNA against MST3 at DIV13 using Lipofectamine2000, and then fixed using 4%PFA+4% sucrose at DIV16 for 10 min at room temperature. Neurons were then incubated with blocking solution (10% normal donkey serum+ 0.2M glycine+0.2% triton x-100 in PBS) for an hour followed by incubation with primary antibody against p-TAO1/2 (rabbit polyclonal) and GFP (monoclonal from Roche) at 4 degrees overnight. After 3 washes with PBS, neurons were incubated for 2 hours at room temperature with Alexa 488 and 568 secondary antibodies. Coverslips were mounted using fluoromont after six washes with PBS. Imaging was performed on a scanning disk confocal SP5 Leica system using 40X, 1.25N objective at 1.5 times zoom. Image stacks were acquired at z spacing of 0.4 microns.
Quantification of pTAO staining was performed using ImageJ Measure function. Average projection of Image stacks were acquired using ZProject. The fluorescence in the neuronal soma which was chosen as ROI was measured using Measure function in ImageJ. The fluorescence intensity of untransfected neighboring neuronal soma which was absent in GFP staining was used as internal control in each image. The normalized fluorescence intensity of neuronal soma from ten neurons per experiment was calculated and the average of three different experiments was then plotted. Two tailed-unequal variance Student t-test was used to calculate p-values. bis -tris gels (Invitrogen). MST3 autophosphorylation was detected by anti-thiophosphate ester antibody (1:10,000 -1:5,000, Epitomics) followed by HRP conjugated secondary antibody.

MST3 Substrate Labelling
Covalent capture method for kinase substrate identification was done as previously described (Ultanir et al., 2012). Briefly, MST3 kinase was purified from COS-7 or HEK293T cells on HAbeads as described above. Cells expressing MST3-MG were treated with 0.5 µM okadaic acid for 1 hour. Untransfected cells and cells expressing MST3-KD were used as negative controls. Cells were lysed with 20 mM Tris HCL, 150 mM NaCl, Protease inhibitor cocktail, phosphatase inhibitor I (Sigma), 10% glycerol and 1% NP40. Lysate was incubated on ice for 30 min, followed by centrifugation at 20,000g for 15 min. Supernatant was pre-cleared with IgG sepharose beads. 50 µL 1:1 slurry anti-HA resin was incubated with protein lysate for 1,5 -3 hours. P3 and P13 mouse brain lysates were obtained in 0.25% NP-40, 10 mM MgCl2, 100 Mm NaCl, 20 mM Tris pH=7.5, 0.5 mM DTT and 1X protease inhibitor cocktail. 100 µl brain lysate of 20 µg/µl protein concentration was used to be labeled by MST3-MG or MST3-KD containing beads, each. A lysate only sample was used in which no HA beads were added as a second negative control for each experiment. Total 4 experiments were conducted twice with P13 and twice with P3 brain lysates. Labelling reaction contained 1 µM cyclic AMPdependent protein kinase inhibitor, 10 μM protein kinase C inhibitor (Bisindolylmaleimide I -Calbiochem), 3 mM GTP, 100 µM ATP, 1 µM O.A and 0.5 mM Benzyl-ATP-γ-S.

Multiplex SILAC labelling of neuronal cultures
Cortical neuronal cultures were labelled using Multiplex SILAC as described (
TAO2 T475 peptide (C RNRDHFAT*IRTASLVSR) was synthesized with or without a phosphorylation at the indicated Threonine (Elim Biopharmaceuticals). Peptide was conjugated to iodoacetyl agarose beads (Sulfolink Resin, Thermo Scientific) as described in manufacturer's protocol. 30 µl resin conjugated to 22 nmol of peptide was used to for pull-down in 1.6 mg SILAC labeled protein lysate. Beads were incubated with lysate for 1.5 hours at room temperature. Beads were washed four times with lysis buffer and two additional times with lysis buffer without NP40 or protease inhibitors. Precipitated proteins were subjected to in-bead typsin digest.

Reversed-phase liquid chromatography-electrospray tandem mass spectrometry (LC-MS/MS) analysis
In-beads digestion. After incubation with the SILAC cell extracts, phospho and non phospho peptide conjugated beads were washed with the conjugation buffer twice, and then mixed. After a short spin  Peaklists were generated using PAVA in-house software (Guan et al., 2011), based on the RawExtract script from Xcalibur v2.4 (Thermo Fisher Scientific, San Jose, CA). The peak lists were searched in three separate searches against the rat subset of the UniProt database as of March 21, 2012, using inhouse ProteinProspector version 5.10.10 (a public version is available on line). A randomized version of all entries was concatenated to the database for estimation of false discovery rates in the searches.
Peptide tolerance in searches was 20 ppm for precursor and 20 ppm for product ions, respectively.
Peptides containing two miscleavages were allowed. Carbamidomethylation of cysteine was allowed as constant modification; acetylation of the N terminus of the protein, pyroglutamate formation from N terminal glutamine and oxidation of methionine, were allowed as variable modifications in initial searches. A second search was performed allowing also for 2H(4) labelling in lysine and 13C (6) labelling in arginine as constant modifications; and a third search allowing for 15N (2) 13C (6) labelling in lysine and 15N(4) 13C(6) labelling in arginine as constant modifications. In all cases, the number of modification was limited to two per peptide. The 3 searches merged into a single result file.
A minimal ProteinProspector protein score of 20, a peptide score of 15, a maximum expectation value of 0.05 and a minimal discriminant score threshold of 0.0 were used for initial identification criteria.
Protein hits were considered significant when at least two peptide sequences matched a protein entry and the Prospector score was above the significance level. For identifications based on one single peptide sequence with high scores, the MS/MS spectrum was reinterpreted manually by matching all the observed fragment ions to a theoretical fragmentation obtained using MS Product (Protein Prospector) (Clauser et al., 1999).
Quantification-SILAC quantification measurements were extracted from the raw data by Search Compare in Protein Prospector (http://prospector.ucsf.edu). Search Compare averaged together MS scans from -10 s to +30 s from the time at which the MS/MS spectrum was acquired in order to produce measurements averaged over the elution of the peptide. SILAC ratios (medium heavy/heavy) were calculated, and base 2 logarithms of these values were used. If quantitative data were available from isotopic envelopes identified as different charge states of the same peptide, the median of the log2 of the calculated SILAC ratios was used for that peptide. For proteins, the median of all the log2 ratios for peptides unique to that protein was calculated. Protein log2 ratios were plotted in a histogram with bin size 0.2, and log2 ratios were then corrected for differences in protein level between the heavy and medium heavy sample using the deviation from 0 of the mode of this distribution.

Co-immunoprecipitation in HEK293T cells
HEK293T cells were transfected with Myosin Va GTD-GFP or control GFP construct, HA tagged TAO1 wild type and HA tagged TAO1 phosphomutant T440A separately. Cell lysates were made in lysis buffer (20mM Hepes pH 7.4, 150mM NaCl, 1% Triton-X100, 0.5% Deoxycholate, 0.1% SDS, 2mM EDTA, 2mM DTT with protease inhibitor cocktail from Roche). Lysate from Myosin Va-GFP or control GFP transfected cells was incubated with Protein G beads prebound with monoclonal GFP antibody, and then lysates from either HA-TAO1-wildtype or HA-TAO1-A phosphomutant transfected cells were added and incubated for 3 hours at 4 degrees with rotation. Beads were then washed thrice with lysis buffer, twice with lysis buffer without detergent and then sample buffer was added. Samples were run after boiling on a 4-12% gradient Bis-Tris gel, transeferred on PVDF membranes and then blotted with antibodies against HA to detect presence of TAO1 and GFP to detect equal load of GFP or Myosin Va-GFP.