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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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SH3 domain of PSGAP is essential for the interaction with PYK2. (A) Mapping of the binding region in PSGAP by yeast two-hybrid assays. Yeast cells were cotransformed with a vector encoding the Gal4 binding domain fused to different PSGAP constructs and Gal4AD fused to PYK2. Transformed yeast cells were seeded in Leu−, Trp−, and His− plates and assayed for β-Gal activity. Transformed yeast cells were also grown in Leu− and Trp− medium for liquid β-Gal assays. (B) Analysis of PSGAP binding to PYK2 by in vitro GST pull-down assays. HEK293 cells were transfected with Myc-tagged PYK2. Lysates of transfected cells were incubated with various GST-PSGAP fusion proteins (SH3, amino acids 731–786; PH-GAP, amino acids 265–590; and PH, amino acids 265–454) immobilized on beads. Lysate input and bound proteins were resolved on SDS-PAGE and subjected to immunoblotting with an anti–Myc antibody (top). An equal amount of GST fusion proteins was used as revealed by Coomassie staining (bottom).
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Figure 4: SH3 domain of PSGAP is essential for the interaction with PYK2. (A) Mapping of the binding region in PSGAP by yeast two-hybrid assays. Yeast cells were cotransformed with a vector encoding the Gal4 binding domain fused to different PSGAP constructs and Gal4AD fused to PYK2. Transformed yeast cells were seeded in Leu−, Trp−, and His− plates and assayed for β-Gal activity. Transformed yeast cells were also grown in Leu− and Trp− medium for liquid β-Gal assays. (B) Analysis of PSGAP binding to PYK2 by in vitro GST pull-down assays. HEK293 cells were transfected with Myc-tagged PYK2. Lysates of transfected cells were incubated with various GST-PSGAP fusion proteins (SH3, amino acids 731–786; PH-GAP, amino acids 265–590; and PH, amino acids 265–454) immobilized on beads. Lysate input and bound proteins were resolved on SDS-PAGE and subjected to immunoblotting with an anti–Myc antibody (top). An equal amount of GST fusion proteins was used as revealed by Coomassie staining (bottom).

Mentions: To understand the mechanism of the PSGAP–PYK2 interaction, we mapped the domain in PSGAP required for interaction in yeast two-hybrid assays. As shown in Fig. 4 A, deletion of the SH3 domain (PSGAPΔSH3) inhibited almost completely PSGAP binding to PYK2. Moreover, PSGAP SH3 domain (PSGAP-SH3) alone was able to bind to PYK2. These data indicated that the SH3 domain of PSGAP was required and sufficient for the binding to PYK2 in yeast two-hybrid system.


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)

SH3 domain of PSGAP is essential for the interaction with PYK2. (A) Mapping of the binding region in PSGAP by yeast two-hybrid assays. Yeast cells were cotransformed with a vector encoding the Gal4 binding domain fused to different PSGAP constructs and Gal4AD fused to PYK2. Transformed yeast cells were seeded in Leu−, Trp−, and His− plates and assayed for β-Gal activity. Transformed yeast cells were also grown in Leu− and Trp− medium for liquid β-Gal assays. (B) Analysis of PSGAP binding to PYK2 by in vitro GST pull-down assays. HEK293 cells were transfected with Myc-tagged PYK2. Lysates of transfected cells were incubated with various GST-PSGAP fusion proteins (SH3, amino acids 731–786; PH-GAP, amino acids 265–590; and PH, amino acids 265–454) immobilized on beads. Lysate input and bound proteins were resolved on SDS-PAGE and subjected to immunoblotting with an anti–Myc antibody (top). An equal amount of GST fusion proteins was used as revealed by Coomassie staining (bottom).
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Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2198805&req=5

Figure 4: SH3 domain of PSGAP is essential for the interaction with PYK2. (A) Mapping of the binding region in PSGAP by yeast two-hybrid assays. Yeast cells were cotransformed with a vector encoding the Gal4 binding domain fused to different PSGAP constructs and Gal4AD fused to PYK2. Transformed yeast cells were seeded in Leu−, Trp−, and His− plates and assayed for β-Gal activity. Transformed yeast cells were also grown in Leu− and Trp− medium for liquid β-Gal assays. (B) Analysis of PSGAP binding to PYK2 by in vitro GST pull-down assays. HEK293 cells were transfected with Myc-tagged PYK2. Lysates of transfected cells were incubated with various GST-PSGAP fusion proteins (SH3, amino acids 731–786; PH-GAP, amino acids 265–590; and PH, amino acids 265–454) immobilized on beads. Lysate input and bound proteins were resolved on SDS-PAGE and subjected to immunoblotting with an anti–Myc antibody (top). An equal amount of GST fusion proteins was used as revealed by Coomassie staining (bottom).
Mentions: To understand the mechanism of the PSGAP–PYK2 interaction, we mapped the domain in PSGAP required for interaction in yeast two-hybrid assays. As shown in Fig. 4 A, deletion of the SH3 domain (PSGAPΔSH3) inhibited almost completely PSGAP binding to PYK2. Moreover, PSGAP SH3 domain (PSGAP-SH3) alone was able to bind to PYK2. These data indicated that the SH3 domain of PSGAP was required and sufficient for the binding to PYK2 in yeast two-hybrid system.

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