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+ con centration. We have identified a new Pyk2 binding protein designated Pap. P ap 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-term inal SH3 domain. We demonstrate that Pap forms a stable complex with Pyk2 a nd that activation of Pyk2 leads to tyrosine phosphorylation of Pap in livi ng 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-act ivating protein (GAP) activity towards the small GTPases Arf1 and Arf5 and weak activity towards Arf6. Addition of recombinant Pap protein to Golgi pr eparations prevented Arf-dependent generation of post-Golgi vesicles in vit ro. Moreover, overexpression of Pap in cultured cells reduced the constitut ive secretion of a marker protein. We propose that Pap functions as a GAP f or Arf and that Pyk2 may be involved in regulation of vesicular transport t hrough its interaction with Pap.