Phosphorylation of TIMAP by Glycogen Synthase Kinase-3β Activates Its Associated Protein Phosphatase 1

TIMAP (TGF-β1 inhibited, membrane-associated protein) is a prenylated, endothelial cell-predominant protein phosphatase 1 (PP1c) regulatory subunit that localizes to the plasma membrane of filopodia. Here, we determined whether phosphorylation regulates TIMAP-associated PP1c function. Phosphorylation of TIMAP was observed in cells metabolically labeled with [32P]orthophosphate and was reduced by inhibitors of protein kinase A (PKA) and glycogen synthase kinase-3 (GSK-3). In cell-free assays, immunopurified TIMAP was phosphorylated by PKA and, after PKA priming, by GSK-3β. Site-specific Ser to Ala substitution identified amino acid residues Ser333/Ser337 as the likely PKA/GSK-3β phosphorylation site. Substitution of Ala for Val and Phe in the KVSF motif of TIMAP (TIMAPV64A/F66A) abolished PP1c binding and TIMAP-associated PP1c activity. TIMAPV64A/F66A was hyper-phosphorylated in cells, indicating that TIMAP-associated PP1c auto-dephosphorylates TIMAP. Constitutively active GSK-3β stimulated phosphorylation of TIMAPV64A/F66A, but not wild-type TIMAP, suggesting that the PKA/GSK-3β site may be subject to dephosphorylation by TIMAP-associated PP1c. Substitution of Asp or Glu for Ser at amino acid residues 333 and 337 to mimic phosphorylation reduced the PP1c association with TIMAP. Conversely, GSK-3 inhibitors augmented PP1c association with TIMAP-PP1c in cells. The 333/337 phosphomimic mutations also increased TIMAP-associated PP1c activity in vitro and against the non-integrin laminin receptor 1 in cells. Finally, TIMAP mutants with reduced PP1c activity strongly stimulated endothelial cell filopodia formation, an effect mimicked by the GSK-3 inhibitor LiCl. We conclude that TIMAP is a target for PKA-primed GSK-3β-mediated phosphorylation. This phosphorylation controls TIMAP association and activity of PP1c, in turn regulating extension of filopodia in endothelial cells. TIMAP (TGF-β1 inhibited, membrane-associated protein) is a prenylated, endothelial cell-predominant protein phosphatase 1 (PP1c) regulatory subunit that localizes to the plasma membrane of filopodia. Here, we determined whether phosphorylation regulates TIMAP-associated PP1c function. Phosphorylation of TIMAP was observed in cells metabolically labeled with [32P]orthophosphate and was reduced by inhibitors of protein kinase A (PKA) and glycogen synthase kinase-3 (GSK-3). In cell-free assays, immunopurified TIMAP was phosphorylated by PKA and, after PKA priming, by GSK-3β. Site-specific Ser to Ala substitution identified amino acid residues Ser333/Ser337 as the likely PKA/GSK-3β phosphorylation site. Substitution of Ala for Val and Phe in the KVSF motif of TIMAP (TIMAPV64A/F66A) abolished PP1c binding and TIMAP-associated PP1c activity. TIMAPV64A/F66A was hyper-phosphorylated in cells, indicating that TIMAP-associated PP1c auto-dephosphorylates TIMAP. Constitutively active GSK-3β stimulated phosphorylation of TIMAPV64A/F66A, but not wild-type TIMAP, suggesting that the PKA/GSK-3β site may be subject to dephosphorylation by TIMAP-associated PP1c. Substitution of Asp or Glu for Ser at amino acid residues 333 and 337 to mimic phosphorylation reduced the PP1c association with TIMAP. Conversely, GSK-3 inhibitors augmented PP1c association with TIMAP-PP1c in cells. The 333/337 phosphomimic mutations also increased TIMAP-associated PP1c activity in vitro and against the non-integrin laminin receptor 1 in cells. Finally, TIMAP mutants with reduced PP1c activity strongly stimulated endothelial cell filopodia formation, an effect mimicked by the GSK-3 inhibitor LiCl. We conclude that TIMAP is a target for PKA-primed GSK-3β-mediated phosphorylation. This phosphorylation controls TIMAP association and activity of PP1c, in turn regulating extension of filopodia in endothelial cells. PP1c 2The abbreviations used are:PP1cprotein phosphatase 1 catalytic subunitGSK-3βglycogen synthase kinase 3βWTwild-typeTGF-β1transforming growth factor-β1PKAprotein kinase AMYPTmyosin phosphatase target/regulatory subunitaaamino acid(s)TIMAPTGF-β1-inhibited membrane-associated proteinLAMR1laminin receptor 1GENglomerular endothelialMDCKMadin-Darby canine kidney cellPBSphosphate-buffered salineGFPgreen fluorescent proteinEGFPenhanced GFPIPimmunoprecipitationMOPS4-morpholinepropanesulfonic acidMES4-morpholineethanesulfonic acid. 2The abbreviations used are:PP1cprotein phosphatase 1 catalytic subunitGSK-3βglycogen synthase kinase 3βWTwild-typeTGF-β1transforming growth factor-β1PKAprotein kinase AMYPTmyosin phosphatase target/regulatory subunitaaamino acid(s)TIMAPTGF-β1-inhibited membrane-associated proteinLAMR1laminin receptor 1GENglomerular endothelialMDCKMadin-Darby canine kidney cellPBSphosphate-buffered salineGFPgreen fluorescent proteinEGFPenhanced GFPIPimmunoprecipitationMOPS4-morpholinepropanesulfonic acidMES4-morpholineethanesulfonic acid. belongs to the family of serine/threonine phosphatases (1Cohen P.T. J. Cell Sci. 2002; 115: 241-256Crossref PubMed Google Scholar) that includes PP2A, PP4-PP7, and calcineurin (PP2B). Ser/Thr phosphatases counteract essentially every signal involving Ser/Thr kinases, and like the kinases their activity is tightly regulated. Because of extreme sequence conservation (>90%) among PP1c isoforms, substrate- and location-specific actions are not inherent in PP1c itself but dictated by more than 50 PP1c regulatory (targeting) subunits (1Cohen P.T. J. Cell Sci. 2002; 115: 241-256Crossref PubMed Google Scholar). Among these, the myosin phosphatase-targeting (MYPT) subunits regulate muscle and non-muscle cell contraction through dephosphorylation of regulatory myosin light chains (2Hartshorne D.J. Ito M. Erdödi F. J. Muscle Res. Cell Motil. 1998; 19: 325-341Crossref PubMed Scopus (344) Google Scholar). Most PP1c regulatory subunits share the PP1c binding motif RVXF, but their structures are otherwise diverse, serving in each case highly specific and localized functions (1Cohen P.T. J. Cell Sci. 2002; 115: 241-256Crossref PubMed Google Scholar). protein phosphatase 1 catalytic subunit glycogen synthase kinase 3β wild-type transforming growth factor-β1 protein kinase A myosin phosphatase target/regulatory subunit amino acid(s) TGF-β1-inhibited membrane-associated protein laminin receptor 1 glomerular endothelial Madin-Darby canine kidney cell phosphate-buffered saline green fluorescent protein enhanced GFP immunoprecipitation 4-morpholinepropanesulfonic acid 4-morpholineethanesulfonic acid. protein phosphatase 1 catalytic subunit glycogen synthase kinase 3β wild-type transforming growth factor-β1 protein kinase A myosin phosphatase target/regulatory subunit amino acid(s) TGF-β1-inhibited membrane-associated protein laminin receptor 1 glomerular endothelial Madin-Darby canine kidney cell phosphate-buffered saline green fluorescent protein enhanced GFP immunoprecipitation 4-morpholinepropanesulfonic acid 4-morpholineethanesulfonic acid. The function of PP1c targeting subunits is regulated by changes in expression, by allosteric effects of substrates, and by phosphorylation (1Cohen P.T. J. Cell Sci. 2002; 115: 241-256Crossref PubMed Google Scholar). For example, phosphorylation at or near the RVXF motif of NIPP-1 reduces PP1c binding, and phosphorylation elsewhere in the molecule can increase or decrease PP1c activity (3Boudrez A. Beullens M. Waelkens E. Stalmans W. Bollen M. J. Biol. Chem. 2002; 277: 31834-31841Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar). The activity of the myosin II phosphatase targeting subunit MYPT1 is similarly regulated by phosphorylation (2Hartshorne D.J. Ito M. Erdödi F. J. Muscle Res. Cell Motil. 1998; 19: 325-341Crossref PubMed Scopus (344) Google Scholar, 4Hirano K. Hirano M. Kanaide H. J. Smooth Muscle Res. 2004; 40: 219-236Crossref PubMed Scopus (67) Google Scholar). We identified TIMAP (TGF-β1-inhibited, membrane-associated protein) as an endothelium-restricted PP1c regulatory subunit subject to TGF-β1-mediated transcriptional repression (5Cao W. Mattagajasingh S.N. Xu H. Kim K. Fierlbeck W. Deng J. Lowenstein C.J. Ballermann B.J. Am. J. Physiol. 2002; 283: C327-CC37Crossref PubMed Scopus (40) Google Scholar). TIMAP belongs to the MYPT family of PP1c regulatory subunits based on domain conservation in the N terminus where a PP1c binding motif is immediately followed by several ankyrin repeats (5Cao W. Mattagajasingh S.N. Xu H. Kim K. Fierlbeck W. Deng J. Lowenstein C.J. Ballermann B.J. Am. J. Physiol. 2002; 283: C327-CC37Crossref PubMed Scopus (40) Google Scholar). Within the MYPT family of PP1c-targeting subunits, TIMAP most closely resembles the domain structure of MYPT3 (6Skinner J.A. Saltiel A.R. Biochem. J. 2001; 356: 257-267Crossref PubMed Scopus (36) Google Scholar, 7Ito M. Nakano T. Erdodi F. Hartshorne D.J. Mol. Cell Biochem. 2004; 259: 197-209Crossref PubMed Scopus (377) Google Scholar). Both TIMAP and MYPT3 lack the C-terminal domain that confers myosin-binding activity and phosphorylation-dependent regulation in MYPT1 and its homologs (7Ito M. Nakano T. Erdodi F. Hartshorne D.J. Mol. Cell Biochem. 2004; 259: 197-209Crossref PubMed Scopus (377) Google Scholar). Also, by contrast to other MYPT family members, TIMAP and MYPT3 are prenylated at their C terminus, allowing for interaction with the plasma membrane (5Cao W. Mattagajasingh S.N. Xu H. Kim K. Fierlbeck W. Deng J. Lowenstein C.J. Ballermann B.J. Am. J. Physiol. 2002; 283: C327-CC37Crossref PubMed Scopus (40) Google Scholar, 6Skinner J.A. Saltiel A.R. Biochem. J. 2001; 356: 257-267Crossref PubMed Scopus (36) Google Scholar, 8Kim K. Li L. Kozlowski K. Suh H.S. Cao W. Ballermann B.J. Biochem. Biophys. Res. Commun. 2005; 338: 1327-1334Crossref PubMed Scopus (30) Google Scholar). MYPT3 inhibits PP1c activity toward the regulatory myosin light chain 2 in vitro (6Skinner J.A. Saltiel A.R. Biochem. J. 2001; 356: 257-267Crossref PubMed Scopus (36) Google Scholar), and its associated PP1c activity against myosin light chain 2 in cells is stimulated by PKA-mediated phosphorylation at Ser353 (9Yong J. Tan I. Lim L. Leung T. J. Biol. Chem. 2006; 281: 31202-31211Abstract Full Text Full Text PDF PubMed Scopus (24) Google Scholar). Currently, it is not known whether TIMAP can act as a myosin light chain phosphatase-targeting subunit. However, we have previously shown that TIMAP associates with the plasma membrane of endothelial cell filopodia, where it interacts with, and serves to dephosphorylate the non-integrin laminin receptor 1 (LAMR1) (8Kim K. Li L. Kozlowski K. Suh H.S. Cao W. Ballermann B.J. Biochem. Biophys. Res. Commun. 2005; 338: 1327-1334Crossref PubMed Scopus (30) Google Scholar). LAMR1 is involved in regulating cell motility (10Vande Broek I. Vanderkerken K. De Greef C. Asosingh K. Straetmans N. Van Camp B. Van Riet I. Br. J. Cancer. 2001; 85: 1387-1395Crossref PubMed Scopus (54) Google Scholar, 11Ménard S. Castronovo V. Tagliabue E. Sobel M.E. J. Cell Biochem. 1997; 67: 155-165Crossref PubMed Scopus (130) Google Scholar) and angiogenesis (12Gebarowska D. Stitt A.W. Gardiner T.A. Harriott P. Greer B. Nelson J. Am. J. Pathol. 2002; 160: 307-313Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar, 13Grant K. B. Full Text PDF PubMed Scopus Google Scholar) through The LAMR1 also serves as the receptor that and and S. S. C. C. S. D. S. J. 2001; PubMed Scopus Google Scholar, P. Biochem. Biophys. 2004; PubMed Scopus Google Scholar). In we for regulation of by phosphorylation. We that TIMAP is phosphorylated by PKA and, after PKA priming, by GSK-3β. phosphorylation by GSK-3β regulates the association of PP1c with TIMAP and its activity in vitro and in cells. of TIMAP that its associated PP1c activity the of endothelial cell filopodia. The that TIMAP function is regulated by phosphorylation and that TIMAP may function in the of endothelial cell filopodia. and of and otherwise was by (8Kim K. Li L. Kozlowski K. Suh H.S. Cao W. Ballermann B.J. Biochem. Biophys. Res. Commun. 2005; 338: 1327-1334Crossref PubMed Scopus (30) Google Scholar). as previously (5Cao W. Mattagajasingh S.N. Xu H. Kim K. Fierlbeck W. Deng J. Lowenstein C.J. Ballermann B.J. Am. J. Physiol. 2002; 283: C327-CC37Crossref PubMed Scopus (40) Google Scholar). was was and Cell glomerular endothelial cells as previously B.J. Am. J. Physiol. PubMed Google Scholar) and in with 2 and filopodia formation, the cells on after with TIMAP mutants and and cells in with and In cells with or for to with in for The plasma membrane was with 1 The of filopodia was by cells that their at the plasma For each and and 50 cells in each of was by of TIMAP and of TIMAP was the A the was with and in and are for and sequence is to in cells. The was was by was by This was used to several TIMAP mutants the II TIMAP mutants the previously (5Cao W. Mattagajasingh S.N. Xu H. Kim K. Fierlbeck W. Deng J. Lowenstein C.J. Ballermann B.J. Am. J. Physiol. 2002; 283: C327-CC37Crossref PubMed Scopus (40) Google Scholar) as used for specific mutations are in was in a of the of each the of and of in For was at for followed by of for for 1 and for of the was used to cells. was by used for in a For cells and to The cells to the The cells with the and with and immunoprecipitation and inhibitors phosphorylated 1 and A in on and at for at The with (8Kim K. Li L. Kozlowski K. Suh H.S. Cao W. Ballermann B.J. Biochem. Biophys. Res. Commun. 2005; 338: 1327-1334Crossref PubMed Scopus (30) Google Scholar), or of for 1 at followed by at The at for at with with II and with The in and for The on or and with and was with cells with and in the for 1 The was with and for the of A was in In the dephosphorylation of cells labeled with as previously (8Kim K. Li L. Kozlowski K. Suh H.S. Cao W. Ballermann B.J. Biochem. Biophys. Res. Commun. 2005; 338: 1327-1334Crossref PubMed Scopus (30) Google Scholar). with and in and phosphatase The as that also and in for followed by in the to and to with to In by metabolically labeled cells that with A the of the TIMAP cell and TIMAP in the of phosphatase with phosphatase inhibitors and The in of PP1c 1 for at in or with of in the or of the or A. TIMAP was the with and to and In was or cells with TIMAP as that phosphatase inhibitors the and The TIMAP on was with the kinase The with of kinase II 50 of of GSK-3β of PKA catalytic subunit 50 or of protein kinase C catalytic 1 in the of and of specific at for in a of The with phosphatase TIMAP was in 50 of and for For GSK-3 in vitro phosphorylation the TIMAP with of PKA for at in PKA The at for to with GSK-3 and or as The for at with or or in GSK-3 and The with phosphatase and the with 50 of to a and to and PP1c in PP1c activity was the by PP1c, fluorescent with at TIMAP the cells with or TIMAP was to PP1c the other for The with 50 in of PP1c for at The was at every a and as a or of A to the was used to determined cells was TIMAP in TIMAP was cells, a with a of was observed in cells that lack TIMAP, was TIMAP that was and the TIMAP was by with a and TIMAP are phosphorylated in cells. PKA TIMAP in and in of the TIMAP sequence with N. S. S. J. Mol. Biol. PubMed Scopus Google Scholar) phosphorylation and phosphorylation in the residues to and to of TIMAP. In with that TIMAP may be a for kinase and protein kinase C not cells to with the PKA inhibitor the GSK-3 inhibitor the of of TIMAP was and reduced with TIMAP cells not to inhibitors that TIMAP phosphorylation was reduced by and with in the of or the protein kinase C inhibitor C the inhibitor and the kinase inhibitor effect on by TIMAP in cells. PKA and GSK-3 to be kinases for TIMAP phosphorylation. TIMAP was immunopurified and cells to whether it can as a in vitro for kinases by in phosphatase inhibitors the and the was TIMAP the phosphatase inhibitors TIMAP immunoprecipitation for the in vitro phosphorylation in phosphorylation of TIMAP was observed in vitro in the of indicating that kinase was with TIMAP. phosphorylation in vitro was observed in the of but not with kinase protein kinase or GSK-3β The in vitro phosphorylation of immunopurified TIMAP was reduced by the PKA inhibitor in with a specific PKA-mediated phosphorylation. TIMAP after a PKA most GSK-3 substrates, phosphorylation by GSK-3 a phosphorylation by other kinases J. Cell Sci. PubMed Scopus Google Scholar). The lack of TIMAP phosphorylation by and GSK-3β in vitro be to the of a phosphorylation. Because PKA strongly TIMAP in vitro it that PKA as a kinase for or GSK-3β. shown in TIMAP was with GSK-3β in in the of TIMAP was as as was by a with not PKA-primed TIMAP. Also, was observed TIMAP was with in GSK-3 kinases, indicating that the phosphorylation observed in the of GSK-3β was not to activity of the PKA GSK-3β but not can TIMAP in but after PKA of GSK-3β Phosphorylation GSK-3β phosphorylation site was identified by The GSK-3 sequence J. Cell Sci. PubMed Scopus Google Scholar) of TIMAP is where as the PKA and as the GSK-3β phosphorylation site A of TIMAP, residues site was and cells. This reduced TIMAP phosphorylation to or Ser to Ala mutations at residues 333 and 337 and 333 and 337 of the or cells that metabolically labeled with [32P]orthophosphate that mutations phosphorylation of TIMAP with mutations not in vitro PKA-mediated phosphorylation of TIMAP indicating that other the Ser to Ala mutations at residues 333 337 abolished in vitro GSK-3β-mediated phosphorylation of TIMAP strongly that TIMAP is phosphorylated by GSK-3β at the with likely serving as the PKA site. of the PP1c PP1c and was that of the KVSF PP1c binding motif PP1c association (3Boudrez A. Beullens M. Waelkens E. Stalmans W. Bollen M. J. Biol. Chem. 2002; 277: 31834-31841Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar). also that phosphorylation of TIMAP at regulate the PP1c was used to TIMAP and TIMAPV64A/F66A, in cells. in PP1c with and but PP1c was in and TIMAPV64A/F66A TIMAP-associated phosphatase in of was essentially abolished in the TIMAPV64A/F66A of are with that the and residues the PP1c binding motif of PP1c regulatory subunits are for the association with PP1c (1Cohen P.T. J. Cell Sci. 2002; 115: 241-256Crossref PubMed Google Scholar). The that the substitution of Ser with Ala at not the PP1c association that phosphorylation at site not PP1c or that was not phosphorylated at TIMAP was not by the Ser to Ala substitution at of TIMAP by of TIMAP in cells or TIMAP in cells was strongly augmented the cells to the phosphatase inhibitor A the of [32P]orthophosphate with TIMAP by whether TIMAP itself can as a PP1c TIMAP was cells that metabolically labeled with [32P]orthophosphate of the with or with in the or of the PP1c inhibitor II or A. We observed that TIMAP was by in vitro and that PP1c inhibitors reduced We determined whether TIMAP be by TIMAP-associated PP1c. or TIMAPV64A/F66A in cells followed by [32P]orthophosphate and the and TIMAPV64A/F66A mutants hyper-phosphorylated with that the association of PP1c with TIMAP phosphorylated TIMAP of the PP1c catalytic domain to in their We determined whether GSK-3β-mediated phosphorylation of TIMAP is subject to regulation by TIMAP-associated PP1c. cells with active or GSK-3β in the of TIMAPV64A/F66A, or a TIMAP in the PP1c binding motif and the GSK-3β site phosphorylation of or was TIMAPV64A/F66A was more phosphorylated in the of active GSK-3β than with GSK-3β In TIMAPV64A/F66A phosphorylation was and in the of with GSK-3β. the reduced association of PP1c with TIMAP GSK-3β-mediated TIMAP phosphorylation in cells. phosphorylation of the was in the of active with GSK-3β TIMAP-associated PP1c may dephosphorylate the Ser333/Ser337 PKA/GSK-3β site or it may the phosphorylation. Phosphorylation of TIMAP by GSK-3β Both PP1c and with whether PP1c association with TIMAP or TIMAP-associated PP1c activity are the Ser333/Ser337 site is Ser at residues 333 and 337 was by acid or acid to mimic phosphorylation. with and PP1c was with and TIMAP-associated PP1c activity was in the and mutants with and of that phosphorylation of TIMAP at residues Ser333/Ser337 reduces PP1c association with TIMAP. the phosphomimic the activity of TIMAP-associated PP1c. In cells, GSK-3 and enhanced PP1c with with of the TIMAP-PP1c association by GSK-3β-mediated phosphorylation of TIMAP. enhanced the PP1c association with TIMAP in cells In in cells for to with A PP1c with TIMAP with that cells not with A that the association of PP1c with TIMAP is reduced TIMAP is phosphorylated by GSK-3β. at Ser333/Ser337 of LAMR1 in whether the changes in the TIMAP-associated PP1c activity observed in vitro also its activity in cells, TIMAPV64A/F66A, and the phosphomimic in cells. The cells metabolically labeled with [32P]orthophosphate or The previously shown to TIMAP and to be a for TIMAP in cells (8Kim K. Li L. Kozlowski K. Suh H.S. Cao W. Ballermann B.J. Biochem. Biophys. Res. Commun. 2005; 338: 1327-1334Crossref PubMed Scopus (30) Google Scholar) was followed by and for TIMAP Phosphorylation of LAMR1 was reduced in cells with with enhanced associated PP1c activity. The the of TIMAP and LAMR1 in the LAMR1 with essentially for the TIMAP-associated PP1c the of Cell is known to to endothelial cell filopodia, where it with its LAMR1 (8Kim K. Li L. Kozlowski K. Suh H.S. Cao W. Ballermann B.J. Biochem. Biophys. Res. Commun. 2005; 338: 1327-1334Crossref PubMed Scopus (30) Google Scholar). We determined whether of TIMAP in the PP1c binding the LAMR1 interaction domain (8Kim K. Li L. Kozlowski K. Suh H.S. Cao W. Ballermann B.J. Biochem. Biophys. Res. Commun. 2005; 338: 1327-1334Crossref PubMed Scopus (30) Google or the GSK-3β phosphorylation in of endothelial cell filopodia. or in cells In cells filopodia than observed in cells with cells The of filopodia in the other mutants was not In cells, the of filopodia was also on the TIMAP cell in cells, in cells, and in cells In cells or and filopodia was to that observed in cells shown are for filopodia and in cells. In cells a that with, and to dephosphorylate LAMR1 (8Kim K. Li L. Kozlowski K. Suh H.S. Cao W. Ballermann B.J. Biochem. Biophys. Res. Commun. 2005; 338: 1327-1334Crossref PubMed Scopus (30) Google Scholar), the of filopodia cells was cells Finally, in cells with the GSK-3 inhibitor a increase in filopodia was observed 2 and after of and likely that the PP1c-targeting subunit TIMAP regulates filopodia function in endothelial cells and that phosphorylation of TIMAP by GSK-3β controls This regulation of the endothelium-restricted PP1c-targeting subunit TIMAP. its family MYPT3 (9Yong J. Tan I. Lim L. Leung T. J. Biol. Chem. 2006; 281: 31202-31211Abstract Full Text Full Text PDF PubMed Scopus (24) Google Scholar), TIMAP can be phosphorylated by In a PKA-primed GSK-3β phosphorylation site not in MYPT3 is the phosphorylation of TIMAP. Phosphorylation at site reduces the association TIMAP and PP1c and PP1c activity. TIMAP-associated PP1c in turn serves to TIMAP. on and phosphomimic TIMAP TIMAP phosphorylation controls PP1c activity against the the known target of TIMAP (8Kim K. Li L. Kozlowski K. Suh H.S. Cao W. Ballermann B.J. Biochem. Biophys. Res. Commun. 2005; 338: 1327-1334Crossref PubMed Scopus (30) Google Scholar). In mutants of TIMAP with reduced PP1c activity enhanced the of filopodia in endothelial cells. We conclude that TIMAP is a PP1c targeting subunit involved in the regulation of endothelial cell filopodia A of that the function of MYPT family is regulated by phosphorylation (2Hartshorne D.J. Ito M. Erdödi F. J. Muscle Res. Cell Motil. 1998; 19: 325-341Crossref PubMed Scopus (344) Google Scholar). Phosphorylation of can in of the A. E. P. Hartshorne D.J. Erdödi F. PubMed Scopus Google Scholar), targeting function and C. Hartshorne D.J. Ito M. Res. 2002; PubMed Scopus Google Scholar, C. N. S. P. 2002; PubMed Scopus Google Scholar), as as changes in associated PP1c activity. was known that a of phosphorylation is highly TIMAP and MYPT3 and that PKA can MYPT3 (9Yong J. Tan I. Lim L. Leung T. J. Biol. Chem. 2006; 281: 31202-31211Abstract Full Text Full Text PDF PubMed Scopus (24) Google Scholar). Within (9Yong J. Tan I. Lim L. Leung T. J. Biol. Chem. 2006; 281: 31202-31211Abstract Full Text Full Text PDF PubMed Scopus (24) Google Scholar) identified residues and Ser353 to TIMAP and as the PKA phosphorylation of In with for we that TIMAP in endothelial cells as as TIMAP is that phosphorylation can be by that of the phosphorylation reduces TIMAP and that PKA can TIMAP in vitro 1 and phosphorylation a phosphorylation residues of the GSK-3 site J. Cell Sci. PubMed Scopus Google Scholar). We identified a GSK-3 site not observed in of the PKA site. with TIMAP was phosphorylated in vitro by but not by and phosphorylation was abolished by substitution of Ala for Ser at residues 333 phosphorylation in cells of TIMAP mutants in by Ala and of TIMAP phosphorylation by that can be phosphorylated in cells. Because TIMAP PP1c, it was of to whether TIMAP is subject to by its associated PP1c. L. P. A. F. S. P. J. Cell Sci. PubMed Google Scholar), of the KVSF motif (TIMAPV64A/F66A) reduced PP1c binding and abolished TIMAP-associated PP1c activity and In with dephosphorylation of TIMAP by its associated PP1c, and in cells as with TIMAP-associated PP1c is against the Ser333/Ser337 PKA/GSK-3β is strongly by the that but not is phosphorylated by active GSK-3β. the GSK-3β-mediated phosphorylation of TIMAP is to be it that the association of PP1c with TIMAP or the of PP1c activity be by phosphorylation of TIMAP at TIMAP mutants in residues at the 333/337 to mimic phosphorylation PP1c cells than wild-type TIMAP or mutants in Ala was at the 333/337 the that phosphorylation by PKA at be for the regulation of PP1c association with TIMAP, that substitution of residues a However, we also observed that of GSK-3 in cells increased PP1c with and We conclude that TIMAP phosphorylation by GSK-3β reduces its association with PP1c. The in vitro activity of TIMAP-associated PP1c was also in the phosphomimic mutants with or the activity in cells toward LAMR1 was also for the phosphomimic mutants with or and We to that phosphorylation of TIMAP at residues Ser333/Ser337 not the TIMAP-PP1c association but also PP1c activity. be to the by phosphorylation at the Ser333/Ser337 site can the TIMAP-PP1c association and increase PP1c activity. is that the Ser333/Ser337 site as an domain that the active site of PP1c in the The are also with the that TIMAP inhibits its associated PP1c as is the case for (6Skinner J.A. Saltiel A.R. Biochem. J. 2001; 356: 257-267Crossref PubMed Scopus (36) Google Scholar), and that phosphorylation at the Ser333/Ser337 site of PP1c TIMAP it more The that but not was phosphorylated by active GSK-3β with the of enhanced PP1c activity associated with the 333/337 phosphomimic mutants of TIMAP is with the that phosphorylation of TIMAP at the site is subject to effect of TIMAP-associated PP1c also be In endothelial cells, TIMAP associates with the plasma membrane of filopodia (5Cao W. Mattagajasingh S.N. Xu H. Kim K. Fierlbeck W. Deng J. Lowenstein C.J. Ballermann B.J. Am. J. Physiol. 2002; 283: C327-CC37Crossref PubMed Scopus (40) Google Scholar), where it with LAMR1 (8Kim K. Li L. Kozlowski K. Suh H.S. Cao W. Ballermann B.J. Biochem. Biophys. Res. Commun. 2005; 338: 1327-1334Crossref PubMed Scopus (30) Google Scholar). We previously that TIMAP and LAMR1 (8Kim K. Li L. Kozlowski K. Suh H.S. Cao W. Ballermann B.J. Biochem. Biophys. Res. Commun. 2005; 338: 1327-1334Crossref PubMed Scopus (30) Google Scholar) and that PP1c associates with LAMR1 in the but not in the of TIMAP. we that in cells LAMR1 is phosphorylated in the of the more active PKA/GSK-3β phosphomimic TIMAP than in the of can dephosphorylate LAMR1 its Ser333/Ser337 residues are phosphorylated or whether the effect is to whether LAMR1 is a for phosphorylated in the that LAMR1 is a likely target of in endothelial cells. In endothelial cells with a in TIMAP activity is filopodia, not observed in cells with The of filopodia in cells with a with reduced PP1c or with the with was also of GSK-3 with enhanced filopodia in cells. in endothelial cells with the or phosphomimic filopodia not more than in cells. We that TIMAP-associated PP1c activity is regulated by GSK-3β and that activity the phosphorylation of LAMR1 and the extension of filopodia LAMR1 is the that regulates filopodia its of is as In that the PP1c regulatory subunit TIMAP is phosphorylated in and in vitro by that its associated PP1c regulates TIMAP and that the phosphorylation regulates PP1c association and activity. The that TIMAP, known to to endothelial cell filopodia, regulates the that TIMAP may target its activity to endothelial cell involved in and of The GSK-3β by and W. of at

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