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Regulation of CDC42 GTPase by proline-rich tyrosine kinase 2 interacting with PSGAP, a novel pleckstrin homology and Src homology 3 domain containing rhoGAP protein.

Ren XR, Du QS, Huang YZ, Ao SZ, Mei L, Xiong WC - J. Cell Biol. (2001)

Bottom Line: Moreover, PSGAP is localized at cell periphery in fibroblasts in a pleckstrin homology domain-dependent manner.Over expression of PSGAP in fibroblasts results in reorganization of cytoskeletal structures and changes of cellular morphology, which requires rhoGTPase-activating activity.Taken together, our results suggest that PSGAP is a signaling protein essential for PYK2 regulation of cytoskeletal organization via Rho family GTPases.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathology and Cell Adhesion and Matrix Center, Pathology, and Physical Medicine and Rehabilitation, University of Alabama at Birmingham, Birmingham, Alabama 35294, USA.

ABSTRACT
Proline-rich tyrosine kinase 2 (PYK2), a tyrosine kinase structurally related to focal adhesion kinase (FAK), is implicated in regulating cytoskeletal organization. However, mechanisms by which PYK2 participates in and regulates cytoskeletal organization remain largely unknown. Here we report identification of PSGAP, a novel protein that interacts with PYK2 and FAK and contains multiple domains including a pleckstrin homology domain, a rhoGTPase-activating protein domain, and a Src homology 3 domain. PYK2 interacts with PSGAP Src homology 3 domain via the carboxyl-terminal proline-rich sequence. PSGAP is able to increase GTPase activity of CDC42 and RhoA in vitro and in vivo. Remarkably, PYK2, but not FAK, can activate CDC42 via inhibition of PSGAP-mediated GTP hydrolysis of CDC42. Moreover, PSGAP is localized at cell periphery in fibroblasts in a pleckstrin homology domain-dependent manner. Over expression of PSGAP in fibroblasts results in reorganization of cytoskeletal structures and changes of cellular morphology, which requires rhoGTPase-activating activity. Taken together, our results suggest that PSGAP is a signaling protein essential for PYK2 regulation of cytoskeletal organization via Rho family GTPases.

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Interaction of PSGAP with PYK2 and FAK in vivo. (A) Coimmunoprecipitation of PSGAP with PYK2 or FAK in mammalian-expressing cells. HEK293 cells cotransfected Myc-tagged PYK2 or FAK with Flag-tagged PSGAP or PSGAPΔSH3 were lysed. Immunoprecipitations (IP) with Flag antibody was revealed by immunoblotting (IB) with anti–Myc (top) or anti–Flag (middle) antibodies. Expression of PYK2 and FAK was demonstrated at bottom. (B) Coimmunoprecipitation of PSGAP with PYK2 in vivo. Cell lysates from HEK293 or 10T1/2 cells were immunoprecipitated with PSGAP antibodies and immunoblotted with PYK2 antibodies. (Solid arrow) PYK2; (open arrows) PSGAP.
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Figure 6: Interaction of PSGAP with PYK2 and FAK in vivo. (A) Coimmunoprecipitation of PSGAP with PYK2 or FAK in mammalian-expressing cells. HEK293 cells cotransfected Myc-tagged PYK2 or FAK with Flag-tagged PSGAP or PSGAPΔSH3 were lysed. Immunoprecipitations (IP) with Flag antibody was revealed by immunoblotting (IB) with anti–Myc (top) or anti–Flag (middle) antibodies. Expression of PYK2 and FAK was demonstrated at bottom. (B) Coimmunoprecipitation of PSGAP with PYK2 in vivo. Cell lysates from HEK293 or 10T1/2 cells were immunoprecipitated with PSGAP antibodies and immunoblotted with PYK2 antibodies. (Solid arrow) PYK2; (open arrows) PSGAP.

Mentions: To determine whether PYK2 and FAK interact with PSGAP in vivo, we examined their association in HEK293 cells transfected with Flag-tagged PSGAP with Myc-tagged PYK2 or FAK. Lysates of transfected cells were incubated with anti–Flag antibodies immobilized on beads. PSGAP-interacting proteins were purified by immunoprecipitation followed by immunoblotting with anti–Myc antibodies. PYK2 and FAK were detected in immunoprecipitates of PSGAP (Fig. 6 A), suggesting that PYK2 and FAK associated with PSGAP in mammalian cells. Again, the interaction of PYK2 with PSGAP required the SH3 domain of PSGAP because a mutant with deletion of the SH3 domain (PSGAPΔSH3) failed to interact with PYK2 (Fig. 6 A). This interaction was unaffected by the addition of 103 amino acid residues in the NH2 terminus of PSGAP-m isoform, since similar amounts of PSGAP-m and PSGAP-s were observed in PYK2 immunoprecipitates (data not shown). To further assess the ability of endogenous PYK2 to bind PSGAP, the in vivo coimmunoprecipitation between PSGAP and PYK2 was carried out in 10T1/2 fibroblasts and in HEK293 cells, where both PYK2 and PSGAP were expressed endogenously. As observed with transfected cells, 1–5% PYK2 was recovered in PSGAP immunocomplexes, but not in preimmune complexes (Fig. 6 B), indicating that PSGAP associates with PYK2 in vivo.


Regulation of CDC42 GTPase by proline-rich tyrosine kinase 2 interacting with PSGAP, a novel pleckstrin homology and Src homology 3 domain containing rhoGAP protein.

Ren XR, Du QS, Huang YZ, Ao SZ, Mei L, Xiong WC - J. Cell Biol. (2001)

Interaction of PSGAP with PYK2 and FAK in vivo. (A) Coimmunoprecipitation of PSGAP with PYK2 or FAK in mammalian-expressing cells. HEK293 cells cotransfected Myc-tagged PYK2 or FAK with Flag-tagged PSGAP or PSGAPΔSH3 were lysed. Immunoprecipitations (IP) with Flag antibody was revealed by immunoblotting (IB) with anti–Myc (top) or anti–Flag (middle) antibodies. Expression of PYK2 and FAK was demonstrated at bottom. (B) Coimmunoprecipitation of PSGAP with PYK2 in vivo. Cell lysates from HEK293 or 10T1/2 cells were immunoprecipitated with PSGAP antibodies and immunoblotted with PYK2 antibodies. (Solid arrow) PYK2; (open arrows) PSGAP.
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Figure 6: Interaction of PSGAP with PYK2 and FAK in vivo. (A) Coimmunoprecipitation of PSGAP with PYK2 or FAK in mammalian-expressing cells. HEK293 cells cotransfected Myc-tagged PYK2 or FAK with Flag-tagged PSGAP or PSGAPΔSH3 were lysed. Immunoprecipitations (IP) with Flag antibody was revealed by immunoblotting (IB) with anti–Myc (top) or anti–Flag (middle) antibodies. Expression of PYK2 and FAK was demonstrated at bottom. (B) Coimmunoprecipitation of PSGAP with PYK2 in vivo. Cell lysates from HEK293 or 10T1/2 cells were immunoprecipitated with PSGAP antibodies and immunoblotted with PYK2 antibodies. (Solid arrow) PYK2; (open arrows) PSGAP.
Mentions: To determine whether PYK2 and FAK interact with PSGAP in vivo, we examined their association in HEK293 cells transfected with Flag-tagged PSGAP with Myc-tagged PYK2 or FAK. Lysates of transfected cells were incubated with anti–Flag antibodies immobilized on beads. PSGAP-interacting proteins were purified by immunoprecipitation followed by immunoblotting with anti–Myc antibodies. PYK2 and FAK were detected in immunoprecipitates of PSGAP (Fig. 6 A), suggesting that PYK2 and FAK associated with PSGAP in mammalian cells. Again, the interaction of PYK2 with PSGAP required the SH3 domain of PSGAP because a mutant with deletion of the SH3 domain (PSGAPΔSH3) failed to interact with PYK2 (Fig. 6 A). This interaction was unaffected by the addition of 103 amino acid residues in the NH2 terminus of PSGAP-m isoform, since similar amounts of PSGAP-m and PSGAP-s were observed in PYK2 immunoprecipitates (data not shown). To further assess the ability of endogenous PYK2 to bind PSGAP, the in vivo coimmunoprecipitation between PSGAP and PYK2 was carried out in 10T1/2 fibroblasts and in HEK293 cells, where both PYK2 and PSGAP were expressed endogenously. As observed with transfected cells, 1–5% PYK2 was recovered in PSGAP immunocomplexes, but not in preimmune complexes (Fig. 6 B), indicating that PSGAP associates with PYK2 in vivo.

Bottom Line: Moreover, PSGAP is localized at cell periphery in fibroblasts in a pleckstrin homology domain-dependent manner.Over expression of PSGAP in fibroblasts results in reorganization of cytoskeletal structures and changes of cellular morphology, which requires rhoGTPase-activating activity.Taken together, our results suggest that PSGAP is a signaling protein essential for PYK2 regulation of cytoskeletal organization via Rho family GTPases.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathology and Cell Adhesion and Matrix Center, Pathology, and Physical Medicine and Rehabilitation, University of Alabama at Birmingham, Birmingham, Alabama 35294, USA.

ABSTRACT
Proline-rich tyrosine kinase 2 (PYK2), a tyrosine kinase structurally related to focal adhesion kinase (FAK), is implicated in regulating cytoskeletal organization. However, mechanisms by which PYK2 participates in and regulates cytoskeletal organization remain largely unknown. Here we report identification of PSGAP, a novel protein that interacts with PYK2 and FAK and contains multiple domains including a pleckstrin homology domain, a rhoGTPase-activating protein domain, and a Src homology 3 domain. PYK2 interacts with PSGAP Src homology 3 domain via the carboxyl-terminal proline-rich sequence. PSGAP is able to increase GTPase activity of CDC42 and RhoA in vitro and in vivo. Remarkably, PYK2, but not FAK, can activate CDC42 via inhibition of PSGAP-mediated GTP hydrolysis of CDC42. Moreover, PSGAP is localized at cell periphery in fibroblasts in a pleckstrin homology domain-dependent manner. Over expression of PSGAP in fibroblasts results in reorganization of cytoskeletal structures and changes of cellular morphology, which requires rhoGTPase-activating activity. Taken together, our results suggest that PSGAP is a signaling protein essential for PYK2 regulation of cytoskeletal organization via Rho family GTPases.

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Related in: MedlinePlus