Identification of a new Pyk2 target protein with Arf-GAP activity.

Protein tyrosine kinase Pyk2 is activated by a variety of G-protein-coupled receptors and by extracellular signals that elevate intracellular Ca2+ concentration. We have identified a new Pyk2 binding protein designated Pap. Pap is a multidomain protein composed of an N-terminal alpha-helical region with a coiled-coil motif, followed by a pleckstrin ...
homology domain, an Arf-GAP domain, an ankyrin homology region, a proline-rich region, and a C-terminal SH3 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. Immunofluorescence experiments demonstrate that Pap is localized in the Golgi apparatus and at the plasma membrane, where it is colocalized with Pyk2. In addition, in vitro recombinant Pap exhibits strong GTPase-activating protein (GAP) activity towards the small GTPases Arf1 and Arf5 and weak activity towards Arf6. Addition of recombinant Pap protein to Golgi preparations prevented Arf-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 vesicular transport through its interaction with Pap.
Mesh Terms:
ADP-Ribosylation Factor 1, ADP-Ribosylation Factors, Adaptor Proteins, Signal Transducing, Amino Acid Sequence, Animals, COS Cells, Carrier Proteins, Cell Line, Transformed, Focal Adhesion Kinase 2, GTP-Binding Proteins, GTPase-Activating Proteins, Golgi Apparatus, Hela Cells, Humans, Intracellular Fluid, Mice, Molecular Sequence Data, PC12 Cells, Phosphorylation, Protein-Tyrosine Kinases, Proteins, Proto-Oncogene Proteins pp60(c-src), Rabbits, Rats, Tyrosine, src Homology Domains
Mol. Cell. Biol.
Date: Mar. 01, 1999
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