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Identification and characterization of targets and potential effectors of the non-receptor protein tyrosine kinase Pyk2

Andreev, Julian

New York University 2000

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  • Título:
    Identification and characterization of targets and potential effectors of the non-receptor protein tyrosine kinase Pyk2
  • Autor: Andreev, Julian
  • Assuntos: Biochemistry ; Biology ; Chemistry ; Health Sciences ; Molecular ; Molecular biology ; Pharmacology
  • Descrição: Pyk2 is a non-receptor protein tyrosine kinase which belongs to Fak family. Using cytoplasmic yeast two-hybrid system we have identified a new Pyk2 binding protein designated Pap. Pap is a multidomain protein that contains an Arf-GAP domain. We demonstrate that Pap forms a stable complex with Pyk2 and that activation of Pyk2 leads to tyrosine phosphorylation of Pap in living cells. In vitro recombinant Pap exhibits GTPase activating protein (GAP) activity towards the small GTPase Arf1. Addition of recombinant Pap protein to Golgi preparations prevented Arf1-dependent generation of post-Golgi vesicles in vitro. Moreover, overexpression of Pap in cultured cells reduced the constitutive secretion of a marker protein. We propose that Pap functions as a GAP for Arf and that Pyk2 may be involved in regulation of ARF-mediated processes through its interactions with Pap. The crystal structure of the PAPβ ARF-GAP domain and the C-terminal ankyrin repeats has been determined at 2.1 Å resolution. An invariant arginine of ARF-GAP domain and several nearby hydrophobic residues are solvent-exposed and are predicted to be the site of interaction with ARFs. Site-directed mutagenesis of these residues confirms their importance in ARF-GAP activity. We investigated the possibility of Pyk2 being a substrate of protein tumor suppressor PTEN in vivo. Upon overexpression in 293 cells, PTEN efficiently dephosphorylated Pyk2. In U-87MG glioblastoma cells we have detected a physical association between Pyk2 and PTEN “trapping mutant”. In stably transfected inducible PC12 cell lines PTEN efficiently dephosphorylated endogenous PYK2 activated by agonists of GPCRs. Taken together, these experiments suggest that Pyk2 may be a substrate of PTEN in vivo. Using fibroblasts isolated from Src, Pyk2 or EGFR deficient mice we explored the role played by these tyrosine kinases in GPCR-induced activation of EGFR and MAP kinase. We show that Pyk2 and Src are essential for GPCR-induced activation of EGFR. By contrast, EGFR activation is dispensable for GPCR-induced activation of MAP kinase. In addition, experiments are presented demonstrating that upon GPCR stimulation, activated Pyk2 forms a complex with Src, which in turn phosphorylates EGFR directly. These experiments reveal a mechanism for tyrosine phosphorylation of EGFR by GPCR stimulation and demonstrate that EGFR activation is not required for linking GPCRs with MAP kinase signaling cascade.
  • Editor: New York University
  • Data de criação/publicação: 2000
  • Formato: 119
  • Idioma: Inglês
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  • Realização: ABCD-USP USP   Logos de Redes Sociais: Freepik

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