his cap1  (Cytiva Europe)

Journal: PLOS Genetics

Article Title: The RCAR12-CAP1-OST1 module controls ABA-mediated stomatal closure in Arabidopsis

doi: 10.1371/journal.pgen.1012092

Figure Lengend Snippet: (A) Interaction between OST1 and CAP1 validated by BiFC assays. The images are the representative of three independent experiments ( n = 3). OST1 K50R indicates the kinase-dead form of OST1. NLS-mCherry indicates nuclear localization. We repeated the experiment four times with similar results and at least 8 leaves were observed per time. Scale bars = 50 μm. (B) Co-IP assay was performed to test the interaction of CAP1 with OST1 and OST1 K50R , detected with anti-Flag and anti-MYC antibodies. GFP-2Flag was set as a negative control. The images are the representative of two independent experiments ( n = 2) with the same results. (C) In vitro kinase assay showing the phosphorylation of CAP1 by OST1. The phosphorylation of His-CAP1 was examined using an anti-thiophosphate ester (thioP). The loading amounts of His-CAP1 and GST-OST1 were detected using Coomassie Brilliant Blue (CBB) staining. (D) OST1 phosphorylates CAP1 in vitro . CAP1 from OE- CAP1 and ost1 :OE- CAP1 seedlings was immunoprecipitated and separated by a Pho-tag-PAGE gel, then analyzed with anti-Flag antibody. The IP input was analyzed by regular SDS-PAGE gel with anti-Flag antibody. (E) The phospho-sites of CAP1 identified from mass spectrometry analysis. (F) Phosphorylation levels of mutation of phosphosites (serine to alanine, S to A) by OST1, detected with anti-phosphoserine antibody. The loading amounts of His-CAP1 and GST-OST1 were detected by CBB staining. Values indicate mean ± SD ( n = 3 biological repeats). CAP1 (3 μM), ADF4 (3 μM), RCAR12 (1 μM), ABA (1 μM), F-actin (3 μM).

Article Snippet: GST-RCAR12 or GST (5 μg) were incubated with His-CAP1 (5 μg) and 25 μL glutathione S-Sepharose 4B (GS4B, GE Healthcare, PA, USA) resin in 500 μL binding buffer containing 25 mM Tris, pH 7.5, 150 mM NaCl, 1% Triton X-100 and ABA.

Techniques: Co-Immunoprecipitation Assay, Negative Control, In Vitro, Kinase Assay, Phospho-proteomics, Staining, Immunoprecipitation, SDS Page, Mass Spectrometry, Mutagenesis

Journal: PLOS Genetics

Article Title: The RCAR12-CAP1-OST1 module controls ABA-mediated stomatal closure in Arabidopsis

doi: 10.1371/journal.pgen.1012092

Figure Lengend Snippet: (A(A) GST pull-down assay was performed for the interaction of ADF4 with CAP1. In vitro phosphorylation assay was performed, followed by GST pull-down. OST1 K50R indicates the kinase-dead form of OST1. λ-PPase, Lambda Protein Phosphatase. The loading amounts of His-CAP1, GST-ADF4 and GST were detected by CBB staining.(B) Relative protein levels of His-CAP1 in (A). The intensity of His-band was measured using ImageJ software. Three independent experiments were provided for the data statistics. Values are means ± SD ( n = 3). Different letters indicate statistically significant differences by one-way ANOVA with Tukey’s test ( P < 0.05). (C) Analysis of the interaction of CAP1 using Co-IP assay before and after 50 μΜ ABA treatment for 3 h. CAP1-6Flag was immunoprecipitated using anti-Flag-M2 agrose, and immunoblotting assays were performed using anti-Flag and anti-MYC antibodies. (D) Relative protein levels of ADF4 in (C). The intensity was measured using ImageJ software. Four independent experiments were provided for the data statistics. Values are means ± SD (n = 4). Asterisk indicates statistically significant differences (*P < 0.05, Student’s t- test, two-sided). (E) The interactions of the phosphomimic variant of CAP1 S246/249/250/253-256D and CAP1 with ADF4 were analyzed using in vitro GST pull-down assay. The loading was detected by CBB staining. (F) The interactions of the phosphomimic variant of CAP1 S246/249/250/253-256D and CAP1 with ADF4 were analyzed using LCI (Luciferase Complementation Imaging) assay. The indicated constructs were transiently expressed in N. benthamiana leaves. Leaves were photographed 3-day after infiltration. (G) Representative confocal images of F-actin. F-actin was incubated with indicated proteins for 30 min before staining with Alexa488-Phalloidin. Scale bar = 5 μm. (H) Quantification of the length of F-actin in (G) by ImageJ. More than 100 filaments were measured for each treatment. Values are mean ± SEM. * P < 0.05, as determined by one-way ANOVA with Tukey’s test. (I) Depolymerization of F-actin (10% pyrene labeled). F-actin disassembly was monitored by tracking the decrease in pyrene fluorescence per minute. a.u., arbitrary units. Values indicate mean ± SD ( n = 3 biological repeats). CAP1 (3 μM), ADF4 (3 μM), OST1 (1 μM), F-actin (3 μM).

Article Snippet: GST-RCAR12 or GST (5 μg) were incubated with His-CAP1 (5 μg) and 25 μL glutathione S-Sepharose 4B (GS4B, GE Healthcare, PA, USA) resin in 500 μL binding buffer containing 25 mM Tris, pH 7.5, 150 mM NaCl, 1% Triton X-100 and ABA.

Techniques: Pull Down Assay, In Vitro, Phospho-proteomics, Staining, Software, Co-Immunoprecipitation Assay, Immunoprecipitation, Western Blot, Variant Assay, Luciferase, Imaging, Construct, Incubation, Labeling, Fluorescence

Journal: Molecular and Cellular Biology

Article Title: Dynamic Phosphorylation and Dephosphorylation of Cyclase-Associated Protein 1 by Antagonistic Signaling through Cyclin-Dependent Kinase 5 and cAMP Are Critical for the Protein Functions in Actin Filament Disassembly and Cell Adhesion

doi: 10.1128/MCB.00282-19

Figure Lengend Snippet: Phosphorylation mutants of CAP1 with point mutations at S307/S309 that resist transient phosphorylation had defects in rescuing the reduced actin filament disassembly in the CAP1 knockdown HeLa cells. (A) Both the phosphomimetic (DD) and nonphosphorylatable (AA) mutant had defects in rescuing the reduced actin filament disassembly in CAP1 knockdown HeLa cells driven by LA. CAP1 knockdown HeLa cells stably reexpressing WTCAP1 (R-WT) or the AA (R-AA) or DD mutant (R-DD) or harboring a control vector (R-Vec) were treated with 1 μM LA for 10 min and 30 min. The cells were stained with fluorescent phalloidin to visualize filamentous actin, and images were taken under fluorescence microscopy. HeLa cells harboring an empty shRNA vector (RNAi-Vec) were also included. (B) Fluorescence intensity per cell was measured from 25 cells using ImageJ and analyzed using Student’s t test by comparing to that in the R-Vec cells. The percentages shown reflect the integrity of the actin cytoskeleton at the specific time points of LA treatment compared to that of the same cell type without LA treatment. Both phosphorylation mutants show reduced capability in rescuing the reduced rate of actin filament disassembly compared to the rate in WTCAP1, especially at the 10-min time point. The error bars in the graph represent standard deviations of data from three independent experiments. (C) The phosphorylation mutants also had compromised capability in restoring the level of inhibitory phosphorylation of cofilin at Ser3 in CAP1 knockdown HeLa cells, as detected in Western blotting. Reexpression of the 6×His-/Xpress-tagged WTCAP1 or the AA or DD phosphorylation mutant was confirmed in Western blotting using an antibody against the 6×His tag. Results from three experiments were quantified through densitometry and analyzed using Student’s t test. The error bars in the graph represent standard deviations. *, P < 0.05; **, P < 0.01.

Article Snippet: Recombinant 6×His-tagged CAP1 (full length or truncated) purified from E. coli cells and conjugated to nickel beads (Qiagen, Hilden, Germany) was incubated with active CDK5 or CDK2 purchased from SignalChem (Richmond, Canada) in kinase assay buffer (5 mM MOPS [morpholinepropanesulfonic acid; pH 7.2], 2.5 mM β-glycerol-phosphate, 5 mM MgCl 2 , 1 mM EGTA, and 250 μm ATP) for 30 min at 30°C.

Techniques: Mutagenesis, Stable Transfection, Plasmid Preparation, Staining, Fluorescence, Microscopy, shRNA, Western Blot

Journal: Molecular and Cellular Biology

Article Title: Dynamic Phosphorylation and Dephosphorylation of Cyclase-Associated Protein 1 by Antagonistic Signaling through Cyclin-Dependent Kinase 5 and cAMP Are Critical for the Protein Functions in Actin Filament Disassembly and Cell Adhesion

doi: 10.1128/MCB.00282-19

Figure Lengend Snippet: The phosphorylation mutants of CAP1 also had defects in suppressing the elevated FAK activity and focal adhesion phenotypes in the CAP1 knockdown HeLa cells. (A) The reexpressed AA and DD mutants had defects in suppressing the elevated FAK activity in the CAP1 knockdown HeLa cells compared to the results for WTCAP1. HeLa cells that harbor the empty shRNA vector were also included as a control. Phosphorylation-specific antibody against Tyr397 was used to assess FAK activity in Western blotting. (B) Phosphorylation signals from three independent Western blot experiments as shown in panel A were measured through densitometry and statistically analyzed using Student’s t test. The phosphorylation mutants had significantly reduced capability in suppressing the elevated FAK activity. (C) The AA and DD mutants had defects in rescuing the focal adhesion phenotypes in the CAP1 knockdown HeLa cells compared to the results for WTCAP1. Cells were cultured on fibronectin (FN)-coated dishes with a glass bottom for 2.5 h, followed by fixation and staining with an antivinculin antibody. The focal adhesions (FAs) were visualized and images taken under a confocal microscope. (D) The sizes of FAs in 25 cells of each cell type in the experiment whose results are shown in panel C were measured using ImageJ and statistically analyzed using Student’s t test. The error bars in the graph represent standard deviations of data from three independent experiments. (E) The numbers of FAs per cell were scored from 25 cells using ImageJ, normalized to the cell area, and then statistically analyzed using Student’s t test. The error bars represent standard deviations of results from three independent experiments. **, P < 0.01.

Article Snippet: Recombinant 6×His-tagged CAP1 (full length or truncated) purified from E. coli cells and conjugated to nickel beads (Qiagen, Hilden, Germany) was incubated with active CDK5 or CDK2 purchased from SignalChem (Richmond, Canada) in kinase assay buffer (5 mM MOPS [morpholinepropanesulfonic acid; pH 7.2], 2.5 mM β-glycerol-phosphate, 5 mM MgCl 2 , 1 mM EGTA, and 250 μm ATP) for 30 min at 30°C.

Techniques: Activity Assay, shRNA, Plasmid Preparation, Western Blot, Cell Culture, Staining, Microscopy

Journal: Molecular and Cellular Biology

Article Title: Dynamic Phosphorylation and Dephosphorylation of Cyclase-Associated Protein 1 by Antagonistic Signaling through Cyclin-Dependent Kinase 5 and cAMP Are Critical for the Protein Functions in Actin Filament Disassembly and Cell Adhesion

doi: 10.1128/MCB.00282-19

Figure Lengend Snippet: Inhibitors that target both CDK5 and CDK2 reduced S307/S309 phosphorylation on CAP1 in cells. (A) In silico analyses reveal that the S307/S309 tandem site is conserved in mammalian CAP1 homologues from humans (S308/S310), mouse, and rat (Homo sapiens, Mus musculus, and Rattus norvegicus). The two residues form part of the recognition motif for CDKs. (B) Treatment with a pan-inhibitor of CDKs, PHA-793887, reduced CAP1 phosphorylation at S307/S309 remarkably in both HEK293T and HeLa cells. The phosphorylation signals were detected using a phosphorylation-specific antibody that recognizes signals on both S307 and S309 residues. (C) Roscovitine, an inhibitor specific for CDK2 and CDK5, also considerably reduced CAP1 phosphorylation at S307/S309 in HEK293T cells. (D) PD 033299, an inhibitor specific for both CDK4 and CDK6, did not show detectable effect in reducing S307/S309 phosphorylation on CAP1 in HEK293T and HeLa cells, after treatment of cells for 16 h at the indicated concentrations. The Western blot results shown are representative of those from three independent experiments. DMSO, dimethyl sulfoxide.

Article Snippet: Recombinant 6×His-tagged CAP1 (full length or truncated) purified from E. coli cells and conjugated to nickel beads (Qiagen, Hilden, Germany) was incubated with active CDK5 or CDK2 purchased from SignalChem (Richmond, Canada) in kinase assay buffer (5 mM MOPS [morpholinepropanesulfonic acid; pH 7.2], 2.5 mM β-glycerol-phosphate, 5 mM MgCl 2 , 1 mM EGTA, and 250 μm ATP) for 30 min at 30°C.

Techniques: In Silico, Western Blot

Journal: Molecular and Cellular Biology

Article Title: Dynamic Phosphorylation and Dephosphorylation of Cyclase-Associated Protein 1 by Antagonistic Signaling through Cyclin-Dependent Kinase 5 and cAMP Are Critical for the Protein Functions in Actin Filament Disassembly and Cell Adhesion

doi: 10.1128/MCB.00282-19

Figure Lengend Snippet: Results from in vitro kinase assays and manipulation of CDK5 activity in cells support the idea that CDK5 phosphorylates CAP1. (A) In vitro kinase assays show that CDK5 strongly phosphorylates S307/S309 on CAP1, while CDK2 exhibits a much more modest activity toward the tandem site. Recombinant and full-length proteins were incubated with the same amount of active CDK2 or CDK5 in the kinase buffer. The samples were then resolved on SDS-PAGE gels and blotted with a phosphorylation-specific antibody that detects phosphorylation signals on both S307 and S309. (B) Results from overexpression of CDK2 (HA-CDK2), its activating partner cyclin D1 (D1), or a dominant-negative (DN) HA-CDK2, alone or in combination, did not support a role for CDK2 in phosphorylating S307/S309 on CAP1 in the cell. HEK293T and HeLa cells were transiently transfected with either a control vector or plasmids that express the above-named proteins; cell lysates were prepared 24 h after transfection and analyzed in Western blotting for effects on S307/S309 phosphorylation. (C) Activation of CDK5 increased S307/S309 phosphorylation on CAP1 in cells. HEK293T and HeLa cells were transiently transfected with either a control vector or plasmids that express HA-CDK5, its activating partner p35, or a DN HA-CDK5, alone or in combination. Cell lysates were prepared 24 h after transfection and analyzed in Western blotting for effects on S307/S309 phosphorylation. (D) Stable silencing of CDK5 in HeLa cells with two independent lentivirus-based shRNA constructs consistently led to reduced CAP1 phosphorylation at S307/S309. CAP1 phosphorylation signals from three independent experiments were measured through densitometry and statistically analyzed using Student’s t test. The graph shows significantly reduced phosphorylation in the knockdown cells. The error bars represent standard deviations. **, P < 0.01.

Article Snippet: Recombinant 6×His-tagged CAP1 (full length or truncated) purified from E. coli cells and conjugated to nickel beads (Qiagen, Hilden, Germany) was incubated with active CDK5 or CDK2 purchased from SignalChem (Richmond, Canada) in kinase assay buffer (5 mM MOPS [morpholinepropanesulfonic acid; pH 7.2], 2.5 mM β-glycerol-phosphate, 5 mM MgCl 2 , 1 mM EGTA, and 250 μm ATP) for 30 min at 30°C.

Techniques: In Vitro, Activity Assay, Recombinant, Incubation, SDS Page, Over Expression, Dominant Negative Mutation, Transfection, Plasmid Preparation, Western Blot, Activation Assay, shRNA, Construct

Journal: Molecular and Cellular Biology

Article Title: Dynamic Phosphorylation and Dephosphorylation of Cyclase-Associated Protein 1 by Antagonistic Signaling through Cyclin-Dependent Kinase 5 and cAMP Are Critical for the Protein Functions in Actin Filament Disassembly and Cell Adhesion

doi: 10.1128/MCB.00282-19

Figure Lengend Snippet: CAP1 associates with CDK5/p35 in the cell, and both the S307 and S309 residue on CAP1 are phosphorylated by CDK5. (A) Recombinant GST-CAP1, but not GST alone, coprecipitated with CDK5 and p35 in pulldown assays of lysates of HEK293T cells transiently expressing p35 and HA-CDK5. Bottom, input of GST and GST-CAP1, where an asterisk indicates the correct band of GST-CAP1 on the Coomassie blue-stained gel. (B) Immunoprecipitation with an anti-p35 antibody, but not the control IgG, coprecipitated Xpress-CAP1 from the lysate of CAP1 knockdown HeLa cells that stably reexpress WTCAP1. (C) Immunoprecipitation of transiently expressed HA-CDK5 with the anti-HA antibody coprecipitated stably expressed Xpress-CAP1 in the CAP1 knockdown HeLa cells. (D) Endogenous CAP1 and CDK5 coprecipitated in the immunoprecipitation assays. HeLa cell lysate was incubated with a bead-conjugated mouse anti-CAP1 antibody, and the coprecipitated CDK5 was detected with a rabbit anti-CDK5 antibody in Western blotting. WCL, whole-cell lysate. (E) The N-terminal domain of CAP1 is responsible for mediating the interaction between CAP1 and CDK5. HEK293T cells were transiently transfected with plasmids that express Xpress-tagged full-length CAP1 (FL), the N terminus (NT), the middle domain (MD), or the C terminus (CT). Cell lysates were incubated with a bead-conjugated anti-CDK5 antibody, and the coprecipitated Xpress-tagged CAP1 (FL or truncated domains) was detected in Western blotting with the anti-Xpress antibody. Asterisks indicate the correct signal bands for FL CAP1 and the truncated domains. Note that the expression level of the middle domain was much lower and only detected with extended exposure in Western blotting (not shown). (F) Results from in vitro kinase assays suggest that CDK5 phosphorylates CAP1 at both Ser307 and Ser309. Truncated CAP1 fragments (aa 289 to 474, which covers the CT domain and the P2 region of the middle domain, as WT or harboring indicated single or double point mutations) purified from bacteria were incubated with CDK5 in kinase buffer. The samples were analyzed in Western blotting using phosphorylation-specific antibodies that recognize either signals on both Ser307 and Ser309 residues (top) or the signal on Ser309 alone (middle). The Coomassie-stained gel at the bottom shows the 6×His-tagged truncated CAP1 proteins used in the kinase assays. Asterisks indicate protein fragments that are either nonspecific or a product derived from partial cleavage of the truncated fragments. The schematic representation highlights the CT domain and the P2 region in the middle domain covered in the truncated substrate (shaded) used in the kinase assays. (G) GST-cofilin pulldown assay shows that activation of CDK5 in HEK293T cells through overexpression of p35 inhibited the binding between CAP1 and cofilin. The Coomassie blue-stained gel in the middle shows the input of GST and GST-cofilin proteins used in the pulldown assays. The graph at the bottom shows the quantitated and statistically analyzed results from three independent experiments. The error bars represent standard deviations. **, P < 0.01. (H) Activation of CDK5 did not alter the activity of GSK3. Lysates from HEK293T cells that transiently express the indicated proteins were used in Western blotting. The activity of GSK3 was assessed using an antibody that detects inhibitory phosphorylation on both GSK3 isoforms.

Article Snippet: Recombinant 6×His-tagged CAP1 (full length or truncated) purified from E. coli cells and conjugated to nickel beads (Qiagen, Hilden, Germany) was incubated with active CDK5 or CDK2 purchased from SignalChem (Richmond, Canada) in kinase assay buffer (5 mM MOPS [morpholinepropanesulfonic acid; pH 7.2], 2.5 mM β-glycerol-phosphate, 5 mM MgCl 2 , 1 mM EGTA, and 250 μm ATP) for 30 min at 30°C.

Techniques: Recombinant, Expressing, Staining, Immunoprecipitation, Stable Transfection, Incubation, Western Blot, Transfection, In Vitro, Purification, Derivative Assay, Activation Assay, Over Expression, Binding Assay, Activity Assay

Journal: Molecular and Cellular Biology

Article Title: Dynamic Phosphorylation and Dephosphorylation of Cyclase-Associated Protein 1 by Antagonistic Signaling through Cyclin-Dependent Kinase 5 and cAMP Are Critical for the Protein Functions in Actin Filament Disassembly and Cell Adhesion

doi: 10.1128/MCB.00282-19

Figure Lengend Snippet: Activation of cAMP signaling in the cell induced dephosphorylation of CAP1 at S307/S309, and both PKA and Epac1 are involved in mediating the cAMP signals. (A) Treatment of HEK293T cells with 5 μM forskolin, an activator of adenylyl cyclase, induced dephosphorylation of CAP1 at S307/S309. (B) Treatment of HEK293T cells with isoproterenol, a physiological agonist and an external stimulus of cAMP signaling, caused dephosphorylation on CAP1 at S307/S309. (C) H89, a specific inhibitor of PKA, considerably blocked dephosphorylation of CAP1 induced by forskolin in HEK293T cells. (D) Stable knockdown of the catalytic subunit alpha of PKA in HeLa cells using two independent shRNA constructs increased CAP1 phosphorylation at S307/S309 and also partially blocked CAP1 dephosphorylation induced by forskolin. #1 and #2, two independent shRNA constructs; C1 and C2, two stable clones derived from construct #1. (E) Overexpression of Epac1 in both HEK293T cells and HeLa cells led to enhanced dephosphorylation of CAP1 at S307/S309. (F) GST-cofilin pulldown assays show that forskolin treatment, which induced CAP1 dephosphorylation, enhanced CAP1 binding with cofilin. The graph shows the quantitated results and statistical analysis using Student’s t test. The error bars represent standard deviations. **, P < 0.01.

Article Snippet: Recombinant 6×His-tagged CAP1 (full length or truncated) purified from E. coli cells and conjugated to nickel beads (Qiagen, Hilden, Germany) was incubated with active CDK5 or CDK2 purchased from SignalChem (Richmond, Canada) in kinase assay buffer (5 mM MOPS [morpholinepropanesulfonic acid; pH 7.2], 2.5 mM β-glycerol-phosphate, 5 mM MgCl 2 , 1 mM EGTA, and 250 μm ATP) for 30 min at 30°C.

Techniques: Activation Assay, De-Phosphorylation Assay, shRNA, Construct, Clone Assay, Derivative Assay, Over Expression, Binding Assay

Journal: Molecular and Cellular Biology

Article Title: Dynamic Phosphorylation and Dephosphorylation of Cyclase-Associated Protein 1 by Antagonistic Signaling through Cyclin-Dependent Kinase 5 and cAMP Are Critical for the Protein Functions in Actin Filament Disassembly and Cell Adhesion

doi: 10.1128/MCB.00282-19

Figure Lengend Snippet: Inhibition of the activity of protein phosphatases in cells did not block the dephosphorylation of CAP1 induced by activated cAMP signaling. (A) Pretreatment of HEK293T cells with fostriecin (FST), an inhibitor of the protein phosphatases PP2A and PP4, did not block the dephosphorylation of CAP1 at S307/S309 induced by forskolin. (B) Pretreatment of HEK293T cells with protein phosphatase PP1 inhibitor tautomycetin (TMC) did not block the dephosphorylation of CAP1 at S307/S309 induced by forskolin either. (C) Inhibiting PP2A and PP1 using okadaic acid (OA) in HEK293T cells did not block the dephosphorylation of CAP1 induced by forskolin. The cells were pretreated with OA or the vehicle DMSO for 1 h, followed by forskolin treatment for the indicated time durations, and cell lysates were prepared for analysis in Western blotting. (D) Inhibition of protein phosphatase PP2B with the specific inhibitor FK506 did not inhibit but instead enhanced dephosphorylation of CAP1 at S307/S309 in both HeLa and HEK293T cells. (E) Inhibition of PP2B with another specific inhibitor, cyclosporine (CsA), similarly enhanced the dephosphorylation of CAP1 at S307/S309 in both HeLa and HEK293T cells.

Article Snippet: Recombinant 6×His-tagged CAP1 (full length or truncated) purified from E. coli cells and conjugated to nickel beads (Qiagen, Hilden, Germany) was incubated with active CDK5 or CDK2 purchased from SignalChem (Richmond, Canada) in kinase assay buffer (5 mM MOPS [morpholinepropanesulfonic acid; pH 7.2], 2.5 mM β-glycerol-phosphate, 5 mM MgCl 2 , 1 mM EGTA, and 250 μm ATP) for 30 min at 30°C.

Techniques: Inhibition, Activity Assay, Blocking Assay, De-Phosphorylation Assay, Western Blot

Journal: Molecular and Cellular Biology

Article Title: Dynamic Phosphorylation and Dephosphorylation of Cyclase-Associated Protein 1 by Antagonistic Signaling through Cyclin-Dependent Kinase 5 and cAMP Are Critical for the Protein Functions in Actin Filament Disassembly and Cell Adhesion

doi: 10.1128/MCB.00282-19

Figure Lengend Snippet: Prevention of access of CAP1 to CDK5/p35 is a mechanism underlying the dephosphorylation of CAP1 induced by cAMP signaling. (A) Treatment of HEK293T cells with forskolin did not affect the expression levels or activity of GSK3 isoforms, as assessed by inhibitory phosphorylation of GSK3 in Western blotting. (B and C) Coimmunoprecipitation of CAP1 with HA-CDK5 (B) and p35 (C) shows that treatment of HEK293T cells with forskolin prevented association of CAP1 with the CDK5/p35 complex. In contrast, the PKC activator PMA, which also induces dephosphorylation of CAP1 at S307/S309, as we reported previously, did not have this effect. (D) Activated cAMP signaling also inhibited the association between GFP-CAP1 and CDK5/p35, as detected by coimmunoprecipitation with the HA tag or p35. Longer treatment with forskolin led to further reduced association between CAP1 and CDK5/p35. HEK293T cells were transfected with GFP-CAP1, HA-CDK5, and p35. After 24 h, the cells were treated with forskolin for the indicated time durations before cell lysates were prepared and used in coimmunoprecipitation with either the anti-HA or anti-p35 antibody. The precipitates were analyzed in Western blotting to detect coprecipitated GFP-CAP1 using an anti-GFP antibody. (E and F) Activation of CDK5 through the coexpression of p35 attenuated or delayed dephosphorylation of CAP1 induced by both forskolin (E) and PMA (F).

Article Snippet: Recombinant 6×His-tagged CAP1 (full length or truncated) purified from E. coli cells and conjugated to nickel beads (Qiagen, Hilden, Germany) was incubated with active CDK5 or CDK2 purchased from SignalChem (Richmond, Canada) in kinase assay buffer (5 mM MOPS [morpholinepropanesulfonic acid; pH 7.2], 2.5 mM β-glycerol-phosphate, 5 mM MgCl 2 , 1 mM EGTA, and 250 μm ATP) for 30 min at 30°C.

Techniques: De-Phosphorylation Assay, Expressing, Activity Assay, Western Blot, Transfection, Activation Assay

Journal: Molecular and Cellular Biology

Article Title: Dynamic Phosphorylation and Dephosphorylation of Cyclase-Associated Protein 1 by Antagonistic Signaling through Cyclin-Dependent Kinase 5 and cAMP Are Critical for the Protein Functions in Actin Filament Disassembly and Cell Adhesion

doi: 10.1128/MCB.00282-19

Figure Lengend Snippet: A schematic model of how CAP1-phosphoregulatory cell signals CDK5 and cAMP antagonistically regulate the phosphorylation state of CAP1 at S307/S309 to control the functions of the protein in actin filament disassembly and regulation of cell adhesion. CDK5 phosphorylates the S307/S309 regulatory site, while cAMP induces dephosphorylation of CAP1, with the cAMP signals being mediated by both of the cAMP effectors PKA and Epac. This transient phosphorylation, or the cycling between the phosphorylated and dephosphorylated forms, is essential to the regulation of CAP1 functions in promoting actin filament disassembly and regulating cell adhesion. Preventing this physiological regulation of CAP1 at the S307/S309 site, as in the case of the phosphorylation mutants that resist transition between phosphorylated and dephosphorylated forms, disrupts CAP1 functions in these cellular processes. Our data support the idea that activated cAMP signaling prevents the access of CAP1 to its kinase, CDK5, which at least partially underlies the dephosphorylation of the cellular pool of CAP1 induced by cAMP. No evidence was found supporting a role for activated serine/threonine protein phosphatase(s) in dephosphorylating CAP1 downstream from the cAMP signaling cascade; however, some degree of involvement of a protein phosphatase is not entirely ruled out.

Article Snippet: Recombinant 6×His-tagged CAP1 (full length or truncated) purified from E. coli cells and conjugated to nickel beads (Qiagen, Hilden, Germany) was incubated with active CDK5 or CDK2 purchased from SignalChem (Richmond, Canada) in kinase assay buffer (5 mM MOPS [morpholinepropanesulfonic acid; pH 7.2], 2.5 mM β-glycerol-phosphate, 5 mM MgCl 2 , 1 mM EGTA, and 250 μm ATP) for 30 min at 30°C.

Techniques: De-Phosphorylation Assay