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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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Cytoskeletal reorganization in fibroblasts expressing PSGAP. (A) 10T1/2 cells were transfected with the indicated constructs and stained with anti–PSGAP (green) and anti–paxillin (red) antibodies. Bar, 50 μm. (B) Schematic representation of PSGAP and PSGAP mutants. The numbers represent the amino acid residues deleted from PSGAP-m. The PH domain, rhoGAP domain, and SH3 domain are indicated. Cytoskeletal reorganization index (mean ± SEM) was determined by counting the morphologically altered PSGAP-expressing cells divided by the total number of PSGAP-expressing cells, and listed at right.
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Figure 11: Cytoskeletal reorganization in fibroblasts expressing PSGAP. (A) 10T1/2 cells were transfected with the indicated constructs and stained with anti–PSGAP (green) and anti–paxillin (red) antibodies. Bar, 50 μm. (B) Schematic representation of PSGAP and PSGAP mutants. The numbers represent the amino acid residues deleted from PSGAP-m. The PH domain, rhoGAP domain, and SH3 domain are indicated. Cytoskeletal reorganization index (mean ± SEM) was determined by counting the morphologically altered PSGAP-expressing cells divided by the total number of PSGAP-expressing cells, and listed at right.

Mentions: To study potential roles of PSGAP in regulating cytoskeletal organization, we examined the cytoskeletal organization and cellular morphology in 10T1/2 fibroblasts expressing PSGAP or its mutants. Expression of wild-type PSGAP disrupted focal adhesions and cell morphology in approximately half of transfected cells (Fig. 11). The number (73–75%) of cells with disorganized cytoskeleton or abnormal morphology increased in cells expressing PSGAPΔN or PSGAPΔPH, indicating that the NH2 terminus and PH domains were not required for these events. To examine whether rhoGAP activity was required for PSGAP-mediated disorganization of cytoskeleton, we generated a rhoGAP-inactive mutant (PSGAPΔN-GD) containing an arginine 424-to-glutamine mutation in the GAP domain (Fig. 11 B). Arginine 424 in PSGAP is conserved with arginine 85 in p50rhoGAP, a critical residue for the direct contacts with CDC42 (Barrett et al. 1997; Rittinger et al. 1997). Mutation of this residue rendered many rhoGAP proteins enzymatically inactive (Barrett et al. 1997; Rittinger et al. 1997; Taylor et al. 1999). Indeed, PSGAPΔN-GD was unable to stimulate CDC42 or RhoA GTPase activity in vitro (data not shown). Remarkably, a majority of cells expressing PSGAPΔN-GD exhibited normal focal adhesions and cellular morphology (Fig. 11), indicating that the rhoGAP activity was required for PSGAP-mediated cytoskeletal reorganization.


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)

Cytoskeletal reorganization in fibroblasts expressing PSGAP. (A) 10T1/2 cells were transfected with the indicated constructs and stained with anti–PSGAP (green) and anti–paxillin (red) antibodies. Bar, 50 μm. (B) Schematic representation of PSGAP and PSGAP mutants. The numbers represent the amino acid residues deleted from PSGAP-m. The PH domain, rhoGAP domain, and SH3 domain are indicated. Cytoskeletal reorganization index (mean ± SEM) was determined by counting the morphologically altered PSGAP-expressing cells divided by the total number of PSGAP-expressing cells, and listed at right.
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Related In: Results  -  Collection

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Figure 11: Cytoskeletal reorganization in fibroblasts expressing PSGAP. (A) 10T1/2 cells were transfected with the indicated constructs and stained with anti–PSGAP (green) and anti–paxillin (red) antibodies. Bar, 50 μm. (B) Schematic representation of PSGAP and PSGAP mutants. The numbers represent the amino acid residues deleted from PSGAP-m. The PH domain, rhoGAP domain, and SH3 domain are indicated. Cytoskeletal reorganization index (mean ± SEM) was determined by counting the morphologically altered PSGAP-expressing cells divided by the total number of PSGAP-expressing cells, and listed at right.
Mentions: To study potential roles of PSGAP in regulating cytoskeletal organization, we examined the cytoskeletal organization and cellular morphology in 10T1/2 fibroblasts expressing PSGAP or its mutants. Expression of wild-type PSGAP disrupted focal adhesions and cell morphology in approximately half of transfected cells (Fig. 11). The number (73–75%) of cells with disorganized cytoskeleton or abnormal morphology increased in cells expressing PSGAPΔN or PSGAPΔPH, indicating that the NH2 terminus and PH domains were not required for these events. To examine whether rhoGAP activity was required for PSGAP-mediated disorganization of cytoskeleton, we generated a rhoGAP-inactive mutant (PSGAPΔN-GD) containing an arginine 424-to-glutamine mutation in the GAP domain (Fig. 11 B). Arginine 424 in PSGAP is conserved with arginine 85 in p50rhoGAP, a critical residue for the direct contacts with CDC42 (Barrett et al. 1997; Rittinger et al. 1997). Mutation of this residue rendered many rhoGAP proteins enzymatically inactive (Barrett et al. 1997; Rittinger et al. 1997; Taylor et al. 1999). Indeed, PSGAPΔN-GD was unable to stimulate CDC42 or RhoA GTPase activity in vitro (data not shown). Remarkably, a majority of cells expressing PSGAPΔN-GD exhibited normal focal adhesions and cellular morphology (Fig. 11), indicating that the rhoGAP activity was required for PSGAP-mediated cytoskeletal reorganization.

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