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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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Cloning of PSGAP, a novel rhoGAP-containing protein that interacts with PYK2. (A) Deduced amino acid sequences of mouse PSGAP-m and PSGAP-s. Filled arrows indicate the two methionines as the translational starting codons for the splice variants, PSGAP-m (786 amino acids) and PSGAP-s (683 amino acids), respectively. PSGAP-m contains a different amino terminus (∼103 amino acids) that is underlined with dots. The PH domain (amino acids 267–370 in PSGAP-m) is underlined, rhoGAP domain (amino acids 398–571 in PSGAP-m) is underlined with dashed lines, the proline rich motif is indicated by stars, and the SH3 domain (amino acids 733–786 in PSGAP-m) is boxed. The original clone isolated by yeast two-hybrid screen contains amino acids 265–786. (B) Comparison of the partial human and mouse PSGAP protein sequences. Partial human PSGAP (hu-PSGAP) (279 amino acids) were obtained by searching human EST databases, which corresponded to the residues of 508–786 of mouse PSGAP-m (mo-PSGAP-m) with 84% identity. Stars indicate identical residues. (C) Sequence alignment of PH domains of mouse PSGAP-m, human Graf, and oligophrenin-1. (D) Sequence alignment of the RhoGAP domains of mo-PSGAP-m, hu-Graf, Oligophrenin-1, HAT-2. (E) Sequence alignment of the SH3 domains of mo-PSGAP-m, hu-Graf, and myosin 1C. Alignments were determined using the program pileup from the Genetics Computer Group. (C, D, and E) Identical residues are boxed. (F) Comparison of domain structures of mo-PSGAP-m, mo-PSGAP-s, hu-Graf, and oligophrenin-1. The PH, rhoGAP, and SH3 domains are indicated. The amino acid identities (%) within a specific domain are shown.
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Figure 1: Cloning of PSGAP, a novel rhoGAP-containing protein that interacts with PYK2. (A) Deduced amino acid sequences of mouse PSGAP-m and PSGAP-s. Filled arrows indicate the two methionines as the translational starting codons for the splice variants, PSGAP-m (786 amino acids) and PSGAP-s (683 amino acids), respectively. PSGAP-m contains a different amino terminus (∼103 amino acids) that is underlined with dots. The PH domain (amino acids 267–370 in PSGAP-m) is underlined, rhoGAP domain (amino acids 398–571 in PSGAP-m) is underlined with dashed lines, the proline rich motif is indicated by stars, and the SH3 domain (amino acids 733–786 in PSGAP-m) is boxed. The original clone isolated by yeast two-hybrid screen contains amino acids 265–786. (B) Comparison of the partial human and mouse PSGAP protein sequences. Partial human PSGAP (hu-PSGAP) (279 amino acids) were obtained by searching human EST databases, which corresponded to the residues of 508–786 of mouse PSGAP-m (mo-PSGAP-m) with 84% identity. Stars indicate identical residues. (C) Sequence alignment of PH domains of mouse PSGAP-m, human Graf, and oligophrenin-1. (D) Sequence alignment of the RhoGAP domains of mo-PSGAP-m, hu-Graf, Oligophrenin-1, HAT-2. (E) Sequence alignment of the SH3 domains of mo-PSGAP-m, hu-Graf, and myosin 1C. Alignments were determined using the program pileup from the Genetics Computer Group. (C, D, and E) Identical residues are boxed. (F) Comparison of domain structures of mo-PSGAP-m, mo-PSGAP-s, hu-Graf, and oligophrenin-1. The PH, rhoGAP, and SH3 domains are indicated. The amino acid identities (%) within a specific domain are shown.

Mentions: To identify proteins that bind to PYK2, we used the yeast two-hybrid system. Using the PYK2 COOH terminus (amino acids 781–1009) as bait, a positive clone was isolated from a mouse brain cDNA library. Sequence analysis revealed that this clone contained an insert of ∼2.5 kb with a predicted open reading frame of 526 amino acids contiguous with the GAL4 activation domain (Gal4AD), missing an apparent NH2 terminus. RACE-PCR was used to isolate 5′cDNA from a mouse brain cDNA. Two splice variants that overlapped with the original cDNA clone were isolated. Both share a common COOH-terminal coding sequence, but vary in the positions of their starting codons and in the sequences of their respective 5′-untranslated regions. Whereas one isoform encoded a protein of 786 amino acids, the other splice variant appeared use methionine 104 as the translational starting codon (indicated by an arrow), resulting in an open reading frame for a protein of 683 amino acids (Fig. 1 A). By searching the human EST databases, we were able to obtain a partial human cDNA clone that corresponded to the residues of 508–786 of the mouse clone with 84% identity (Fig. 1 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)

Cloning of PSGAP, a novel rhoGAP-containing protein that interacts with PYK2. (A) Deduced amino acid sequences of mouse PSGAP-m and PSGAP-s. Filled arrows indicate the two methionines as the translational starting codons for the splice variants, PSGAP-m (786 amino acids) and PSGAP-s (683 amino acids), respectively. PSGAP-m contains a different amino terminus (∼103 amino acids) that is underlined with dots. The PH domain (amino acids 267–370 in PSGAP-m) is underlined, rhoGAP domain (amino acids 398–571 in PSGAP-m) is underlined with dashed lines, the proline rich motif is indicated by stars, and the SH3 domain (amino acids 733–786 in PSGAP-m) is boxed. The original clone isolated by yeast two-hybrid screen contains amino acids 265–786. (B) Comparison of the partial human and mouse PSGAP protein sequences. Partial human PSGAP (hu-PSGAP) (279 amino acids) were obtained by searching human EST databases, which corresponded to the residues of 508–786 of mouse PSGAP-m (mo-PSGAP-m) with 84% identity. Stars indicate identical residues. (C) Sequence alignment of PH domains of mouse PSGAP-m, human Graf, and oligophrenin-1. (D) Sequence alignment of the RhoGAP domains of mo-PSGAP-m, hu-Graf, Oligophrenin-1, HAT-2. (E) Sequence alignment of the SH3 domains of mo-PSGAP-m, hu-Graf, and myosin 1C. Alignments were determined using the program pileup from the Genetics Computer Group. (C, D, and E) Identical residues are boxed. (F) Comparison of domain structures of mo-PSGAP-m, mo-PSGAP-s, hu-Graf, and oligophrenin-1. The PH, rhoGAP, and SH3 domains are indicated. The amino acid identities (%) within a specific domain are shown.
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Figure 1: Cloning of PSGAP, a novel rhoGAP-containing protein that interacts with PYK2. (A) Deduced amino acid sequences of mouse PSGAP-m and PSGAP-s. Filled arrows indicate the two methionines as the translational starting codons for the splice variants, PSGAP-m (786 amino acids) and PSGAP-s (683 amino acids), respectively. PSGAP-m contains a different amino terminus (∼103 amino acids) that is underlined with dots. The PH domain (amino acids 267–370 in PSGAP-m) is underlined, rhoGAP domain (amino acids 398–571 in PSGAP-m) is underlined with dashed lines, the proline rich motif is indicated by stars, and the SH3 domain (amino acids 733–786 in PSGAP-m) is boxed. The original clone isolated by yeast two-hybrid screen contains amino acids 265–786. (B) Comparison of the partial human and mouse PSGAP protein sequences. Partial human PSGAP (hu-PSGAP) (279 amino acids) were obtained by searching human EST databases, which corresponded to the residues of 508–786 of mouse PSGAP-m (mo-PSGAP-m) with 84% identity. Stars indicate identical residues. (C) Sequence alignment of PH domains of mouse PSGAP-m, human Graf, and oligophrenin-1. (D) Sequence alignment of the RhoGAP domains of mo-PSGAP-m, hu-Graf, Oligophrenin-1, HAT-2. (E) Sequence alignment of the SH3 domains of mo-PSGAP-m, hu-Graf, and myosin 1C. Alignments were determined using the program pileup from the Genetics Computer Group. (C, D, and E) Identical residues are boxed. (F) Comparison of domain structures of mo-PSGAP-m, mo-PSGAP-s, hu-Graf, and oligophrenin-1. The PH, rhoGAP, and SH3 domains are indicated. The amino acid identities (%) within a specific domain are shown.
Mentions: To identify proteins that bind to PYK2, we used the yeast two-hybrid system. Using the PYK2 COOH terminus (amino acids 781–1009) as bait, a positive clone was isolated from a mouse brain cDNA library. Sequence analysis revealed that this clone contained an insert of ∼2.5 kb with a predicted open reading frame of 526 amino acids contiguous with the GAL4 activation domain (Gal4AD), missing an apparent NH2 terminus. RACE-PCR was used to isolate 5′cDNA from a mouse brain cDNA. Two splice variants that overlapped with the original cDNA clone were isolated. Both share a common COOH-terminal coding sequence, but vary in the positions of their starting codons and in the sequences of their respective 5′-untranslated regions. Whereas one isoform encoded a protein of 786 amino acids, the other splice variant appeared use methionine 104 as the translational starting codon (indicated by an arrow), resulting in an open reading frame for a protein of 683 amino acids (Fig. 1 A). By searching the human EST databases, we were able to obtain a partial human cDNA clone that corresponded to the residues of 508–786 of the mouse clone with 84% identity (Fig. 1 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