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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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Expression of PSGAP in various cells. (A) Characterization of anti–PSGAP antibody. A GST fusion protein containing the mouse PSGAP SH3 domain (amino acids 731–786) was used to generate rabbit polyclonal antibodies as described in Materials and Methods. Lysates (10 μg protein) of 293 cells expressing Flag-tagged PSGAP (PSGAP-m and PSGAP-s) and Graf, and other indicated cell lysates (20 μg protein) were resolved on SDS-PAGE and subjected to immunoblotting with anti–PSGAP antibodies, antibodies preabsorbed with the GST-PSGAP antigen, or an anti–Flag antibody. (B) Western blot analysis of PSGAP expression. Lysates (20 μg protein) of various cells were resolved on SDS-PAGE and subjected to immunoblotting with anti–PSGAP antibodies (6 μg protein of HEK293 cells expressing PSGAP was loaded). (Solid arrows) Transfected PSGAP and endogenously expressed PSGAP. Molecular weight markers are indicated (left).
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Figure 3: Expression of PSGAP in various cells. (A) Characterization of anti–PSGAP antibody. A GST fusion protein containing the mouse PSGAP SH3 domain (amino acids 731–786) was used to generate rabbit polyclonal antibodies as described in Materials and Methods. Lysates (10 μg protein) of 293 cells expressing Flag-tagged PSGAP (PSGAP-m and PSGAP-s) and Graf, and other indicated cell lysates (20 μg protein) were resolved on SDS-PAGE and subjected to immunoblotting with anti–PSGAP antibodies, antibodies preabsorbed with the GST-PSGAP antigen, or an anti–Flag antibody. (B) Western blot analysis of PSGAP expression. Lysates (20 μg protein) of various cells were resolved on SDS-PAGE and subjected to immunoblotting with anti–PSGAP antibodies (6 μg protein of HEK293 cells expressing PSGAP was loaded). (Solid arrows) Transfected PSGAP and endogenously expressed PSGAP. Molecular weight markers are indicated (left).

Mentions: To characterize PSGAP protein distribution, specific antibodies were raised in rabbits using a recombinant PSGAP protein (amino acids 731–786) as an antigen. Western blot analysis detected three protein bands of ∼140, 95, and 85 kD (Fig. 3 A). These bands were recognized by the antibodies, but not preimmune sera (data not shown). Preabsorption of antibodies with the antigen inhibited their ability to detect all three bands (Fig. 3 A). Furthermore, although Graf and PSGAP share a high homology of sequences, the PSGAP antibodies did not cross react with Graf, whose expression was evident by immunoblotting with anti–Flag antibodies (Fig. 3 A). These results suggest that the three bands represent specifically three variants of PSGAP, which may be generated by mRNA splicing as revealed by Northern blot analysis. The 95- and 85-kD proteins appeared to be encoded by the two identified splice variants, PSGAP-m (786 amino acids) and PSGAP-s (683 amino acids), respectively, since expression of both transcripts yielded proteins of similar molecular masses (Fig. 3 A). In addition, the three PSGAP isoforms exhibited a unique expression pattern. The 95-kD protein (PSGAP-m) was expressed in fibroblastic cell lines, including 10T1/2, 3Y1, and Swiss 3T3 fibroblasts, whereas the 140- and 85-kD (PSGAP-s) proteins appeared to be expressed ubiquitously with a relatively high level in tumor cell lines including prostate (PC3 and LnCap) and colon (DLD-1) cancer cells (Fig. 3 B).


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)

Expression of PSGAP in various cells. (A) Characterization of anti–PSGAP antibody. A GST fusion protein containing the mouse PSGAP SH3 domain (amino acids 731–786) was used to generate rabbit polyclonal antibodies as described in Materials and Methods. Lysates (10 μg protein) of 293 cells expressing Flag-tagged PSGAP (PSGAP-m and PSGAP-s) and Graf, and other indicated cell lysates (20 μg protein) were resolved on SDS-PAGE and subjected to immunoblotting with anti–PSGAP antibodies, antibodies preabsorbed with the GST-PSGAP antigen, or an anti–Flag antibody. (B) Western blot analysis of PSGAP expression. Lysates (20 μg protein) of various cells were resolved on SDS-PAGE and subjected to immunoblotting with anti–PSGAP antibodies (6 μg protein of HEK293 cells expressing PSGAP was loaded). (Solid arrows) Transfected PSGAP and endogenously expressed PSGAP. Molecular weight markers are indicated (left).
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Figure 3: Expression of PSGAP in various cells. (A) Characterization of anti–PSGAP antibody. A GST fusion protein containing the mouse PSGAP SH3 domain (amino acids 731–786) was used to generate rabbit polyclonal antibodies as described in Materials and Methods. Lysates (10 μg protein) of 293 cells expressing Flag-tagged PSGAP (PSGAP-m and PSGAP-s) and Graf, and other indicated cell lysates (20 μg protein) were resolved on SDS-PAGE and subjected to immunoblotting with anti–PSGAP antibodies, antibodies preabsorbed with the GST-PSGAP antigen, or an anti–Flag antibody. (B) Western blot analysis of PSGAP expression. Lysates (20 μg protein) of various cells were resolved on SDS-PAGE and subjected to immunoblotting with anti–PSGAP antibodies (6 μg protein of HEK293 cells expressing PSGAP was loaded). (Solid arrows) Transfected PSGAP and endogenously expressed PSGAP. Molecular weight markers are indicated (left).
Mentions: To characterize PSGAP protein distribution, specific antibodies were raised in rabbits using a recombinant PSGAP protein (amino acids 731–786) as an antigen. Western blot analysis detected three protein bands of ∼140, 95, and 85 kD (Fig. 3 A). These bands were recognized by the antibodies, but not preimmune sera (data not shown). Preabsorption of antibodies with the antigen inhibited their ability to detect all three bands (Fig. 3 A). Furthermore, although Graf and PSGAP share a high homology of sequences, the PSGAP antibodies did not cross react with Graf, whose expression was evident by immunoblotting with anti–Flag antibodies (Fig. 3 A). These results suggest that the three bands represent specifically three variants of PSGAP, which may be generated by mRNA splicing as revealed by Northern blot analysis. The 95- and 85-kD proteins appeared to be encoded by the two identified splice variants, PSGAP-m (786 amino acids) and PSGAP-s (683 amino acids), respectively, since expression of both transcripts yielded proteins of similar molecular masses (Fig. 3 A). In addition, the three PSGAP isoforms exhibited a unique expression pattern. The 95-kD protein (PSGAP-m) was expressed in fibroblastic cell lines, including 10T1/2, 3Y1, and Swiss 3T3 fibroblasts, whereas the 140- and 85-kD (PSGAP-s) proteins appeared to be expressed ubiquitously with a relatively high level in tumor cell lines including prostate (PC3 and LnCap) and colon (DLD-1) cancer cells (Fig. 3 B).

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