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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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Regulation of PSGAP by PYK2, but not FAK. (A) Tyrosine phosphorylation of PSGAP by PYK2, but not FAK. HEK293 cells were transfected with indicated constructs. Cell lysates were immunoprecipitated with Flag antibodies (for PSGAP) and immunoblotted with antibodies against phosphotyrosine (Ptyr) and Flag. Open arrows indicate PSGAP. (B) Inhibition of PSGAP's effect on CDC42 by PYK2, but not FAK. HEK293 cells were transfected with indicated constructs. Cell lysates were incubated with GST-PBD immobilized on beads. Bound active CDC42 was resolved on SDS-PAGE and detected by immunoblotting. Equal amounts of CDC42 were expressed as indicated. The expression of PYK2, FAK, and PSGAP are also shown. (Solid arrows) CDC42; (open arrows) PYK2 or PSGAP.
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Figure 8: Regulation of PSGAP by PYK2, but not FAK. (A) Tyrosine phosphorylation of PSGAP by PYK2, but not FAK. HEK293 cells were transfected with indicated constructs. Cell lysates were immunoprecipitated with Flag antibodies (for PSGAP) and immunoblotted with antibodies against phosphotyrosine (Ptyr) and Flag. Open arrows indicate PSGAP. (B) Inhibition of PSGAP's effect on CDC42 by PYK2, but not FAK. HEK293 cells were transfected with indicated constructs. Cell lysates were incubated with GST-PBD immobilized on beads. Bound active CDC42 was resolved on SDS-PAGE and detected by immunoblotting. Equal amounts of CDC42 were expressed as indicated. The expression of PYK2, FAK, and PSGAP are also shown. (Solid arrows) CDC42; (open arrows) PYK2 or PSGAP.

Mentions: While both PYK2 and FAK interacted with PSGAP, effects of PYK2 and/or FAK on PSGAP were unknown. To address this question, we first examined whether PSGAP could be tyrosine phosphorylated by PYK2 or FAK. PSGAP was cotransfected in HEK293 cells with wild-type, mutant PYK2, or FAK. Tyrosine phosphorylation was revealed by immunoprecipitation and immunoblotting using antibodies against phosphotyrosine. As shown in Fig. 8 A, PSGAP was tyrosine phosphorylated in cells coexpressing PYK2. Tyrosine phosphorylation of PSGAP was diminished in cells coexpressing PYK2-KD (a catalytically inactive PYK2) or PYK2-Y402F (an autophosphorylation mutant), suggesting the involvement of PYK2 kinase activity and autophosphorylation in this event. In contrast, PSGAP was not tyrosine phosphorylated by FAK (Fig. 8 A). These results suggest that the regulation of PSGAP by PYK2 may be different from that by FAK. To further address the differential effects of PYK2 and FAK on PSGAP, we examined whether PYK2 and FAK affected PSGAP's regulation of GTPase activity of CDC42. GST-PBD binding to CDC42 in cell lysates coexpressing PSGAP and PYK2 or FAK was carried out. While PSGAP exhibited significant reduction of the precipitated GTP-bound CDC42 (Fig. 8 B), coexpressing PYK2 increased the GTP-bound CDC42, suggesting that PSGAP's negative effect on CDC42-GTP loading was abolished when PYK2 was coexpressed (Fig. 8 B). PYK2-mediated inhibition of PSGAP depended on PYK2 binding to PSGAP, since mutations in PYK2 (PYK2-P859A) or PSGAP (PSGAPΔSH3) that block the interaction of PYK2 with PSGAP failed to exhibit such inhibitory effect (Fig. 8 B). Interestingly, although FAK was able to interact with PSGAP, it seemed unable to regulate PSGAP (Fig. 8 B). These results further indicated that PYK2 and FAK regulated PSGAP differently.


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)

Regulation of PSGAP by PYK2, but not FAK. (A) Tyrosine phosphorylation of PSGAP by PYK2, but not FAK. HEK293 cells were transfected with indicated constructs. Cell lysates were immunoprecipitated with Flag antibodies (for PSGAP) and immunoblotted with antibodies against phosphotyrosine (Ptyr) and Flag. Open arrows indicate PSGAP. (B) Inhibition of PSGAP's effect on CDC42 by PYK2, but not FAK. HEK293 cells were transfected with indicated constructs. Cell lysates were incubated with GST-PBD immobilized on beads. Bound active CDC42 was resolved on SDS-PAGE and detected by immunoblotting. Equal amounts of CDC42 were expressed as indicated. The expression of PYK2, FAK, and PSGAP are also shown. (Solid arrows) CDC42; (open arrows) PYK2 or PSGAP.
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Figure 8: Regulation of PSGAP by PYK2, but not FAK. (A) Tyrosine phosphorylation of PSGAP by PYK2, but not FAK. HEK293 cells were transfected with indicated constructs. Cell lysates were immunoprecipitated with Flag antibodies (for PSGAP) and immunoblotted with antibodies against phosphotyrosine (Ptyr) and Flag. Open arrows indicate PSGAP. (B) Inhibition of PSGAP's effect on CDC42 by PYK2, but not FAK. HEK293 cells were transfected with indicated constructs. Cell lysates were incubated with GST-PBD immobilized on beads. Bound active CDC42 was resolved on SDS-PAGE and detected by immunoblotting. Equal amounts of CDC42 were expressed as indicated. The expression of PYK2, FAK, and PSGAP are also shown. (Solid arrows) CDC42; (open arrows) PYK2 or PSGAP.
Mentions: While both PYK2 and FAK interacted with PSGAP, effects of PYK2 and/or FAK on PSGAP were unknown. To address this question, we first examined whether PSGAP could be tyrosine phosphorylated by PYK2 or FAK. PSGAP was cotransfected in HEK293 cells with wild-type, mutant PYK2, or FAK. Tyrosine phosphorylation was revealed by immunoprecipitation and immunoblotting using antibodies against phosphotyrosine. As shown in Fig. 8 A, PSGAP was tyrosine phosphorylated in cells coexpressing PYK2. Tyrosine phosphorylation of PSGAP was diminished in cells coexpressing PYK2-KD (a catalytically inactive PYK2) or PYK2-Y402F (an autophosphorylation mutant), suggesting the involvement of PYK2 kinase activity and autophosphorylation in this event. In contrast, PSGAP was not tyrosine phosphorylated by FAK (Fig. 8 A). These results suggest that the regulation of PSGAP by PYK2 may be different from that by FAK. To further address the differential effects of PYK2 and FAK on PSGAP, we examined whether PYK2 and FAK affected PSGAP's regulation of GTPase activity of CDC42. GST-PBD binding to CDC42 in cell lysates coexpressing PSGAP and PYK2 or FAK was carried out. While PSGAP exhibited significant reduction of the precipitated GTP-bound CDC42 (Fig. 8 B), coexpressing PYK2 increased the GTP-bound CDC42, suggesting that PSGAP's negative effect on CDC42-GTP loading was abolished when PYK2 was coexpressed (Fig. 8 B). PYK2-mediated inhibition of PSGAP depended on PYK2 binding to PSGAP, since mutations in PYK2 (PYK2-P859A) or PSGAP (PSGAPΔSH3) that block the interaction of PYK2 with PSGAP failed to exhibit such inhibitory effect (Fig. 8 B). Interestingly, although FAK was able to interact with PSGAP, it seemed unable to regulate PSGAP (Fig. 8 B). These results further indicated that PYK2 and FAK regulated PSGAP differently.

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.

Show MeSH
Related in: MedlinePlus