Assay and functional properties of the tyrosine kinase Pyk2 in regulation of Arf1 through ASAP1 phosphorylation.
The Arf-GTPase-activating protein (GAP) ASAP1 has been identified in a yeast-two-hybrid screen as a prominent binder of the proline-rich tyrosine kinase 2 (Pyk2) via SH3 domain-mediated interaction. Following binding, Pyk2 directly phosphorylates ASAP1 on tyrosine residues 308 and 782 in vitro and in cells. To understand the functional impact of this interaction and subsequent phosphorylation, nonphosphorylated and Pyk2-phosphorylated ASAP1 have been generated. This material can be used for lipid-protein overlay assays and fluorimetric Arf-GTPase tests to show that the Pyk2-mediated tyrosine phosphorylation of ASAP1 modulates its GAP activity towards Arf1 in vitro. These studies provide the first evidence for a regulation of ASAP1 and hence Arf1 activity by tyrosine phosphorylation and suggest a functional link between tyrosine kinases and Arf GTPases. Furthermore, an assay to study cellular Arf activation and thus GAP as well as guanine nucleotide exchange factor (GEF) activities and their regulation is described.
Kruljac-Letunic
Anamarija
A
European Molecular Biology Laboratory, Cell Biology and Biophysics Unit, Heidelburg, Germany.
Blaukat
Andree
A
D020823:ADP-Ribosylation Factor 1 / Q000378:metabolism*; D048868:Adaptor Proteins, Signal Transducing / Q000302:isolation & purification / Q000378:metabolism*; D022181:COP-Coated Vesicles / Q000502:physiology; D002478:Cells, Cultured; D051418:Focal Adhesion Kinase 2 / Q000378:metabolism*; D020558:GTP Phosphohydrolases / Q000032:analysis; D006801:Humans; D008081:Liposomes / Q000378:metabolism; D019814:Myristic Acid / Q000378:metabolism; D010743:Phospholipids / Q000378:metabolism; D025941:Protein Interaction Mapping; D020798:Two-Hybrid System Techniques; D018909:src Homology Domains
C117891:ASAP1 protein, human; D048868:Adaptor Proteins, Signal Transducing; D008081:Liposomes; D010743:Phospholipids; D019814:Myristic Acid; D051418:Focal Adhesion Kinase 2; D020558:GTP Phosphohydrolases; D020823:ADP-Ribosylation Factor 1