The tyrosine kinase Pyk2 regulates Arf1 activity by phosphorylation and inhibition of the Arf-GTPase-activating protein ASAP1.
Proline-rich tyrosine kinase 2 (Pyk2), a non-receptor tyrosine kinase structurally related to focal adhesion kinase, has been implicated in the regulation of mitogen-activated protein kinase cascades and ion channels, the induction of apoptosis, and in the modulation of the cytoskeleton. In order to understand how Pyk2 signaling mediates these diverse cellular functions, we performed a yeast two-hybrid screening using the C-terminal part of Pyk2 that contains potential protein-protein interaction sites as bait. A prominent binder of Pyk2 identified by this method was the Arf-GTPase-activating protein ASAP1. Pyk2-ASAP1 interaction was confirmed in pull-down as well as in co-immunoprecipitation experiments, and contact sites were mapped to the proline-rich regions of Pyk2 and the SH3 domain of ASAP1. Pyk2 directly phosphorylates ASAP1 on tyrosine residues in vitro and increases ASAP1 tyrosine phosphorylation when co-expressed in HEK293T cells. Phosphorylation of tyrosine 308 and 782 affects the phosphoinositide binding profile of ASAP1, and fluorimetric Arf-GTPase assays with purified proteins revealed an inhibition of ASAP1 GTPase-activating protein activity by Pyk2-mediated tyrosine phosphorylation. We therefore provide evidence for a functional interaction between Pyk2 and ASAP1 and a regulation of ASAP1 and hence Arf1 activity by Pyk2-mediated tyrosine phosphorylation.
Kruljac-Letunic
Anamarija
A
Institute of Pharmacology, University of Heidelberg, Im Neuenheimer Feld 366, D-69120 Heidelberg, Germany.
Moelleken
Jörg
J
Kallin
Anders
A
Wieland
Felix
F
Blaukat
Andree
A
D020823:ADP-Ribosylation Factor 1 / Q000378:metabolism*; D000818:Animals; D002352:Carrier Proteins / Q000378:metabolism*; D002460:Cell Line; D018076:DNA, Complementary / Q000378:metabolism; D051418:Focal Adhesion Kinase 2; D005982:Glutathione Transferase / Q000378:metabolism; D006801:Humans; D020935:MAP Kinase Signaling System; D016297:Mutagenesis, Site-Directed; D016716:PC12 Cells; D010449:Peptide Mapping; D010766:Phosphorylation; D011233:Precipitin Tests; D011392:Proline / Q000378:metabolism; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D011505:Protein-Tyrosine Kinases / Q000378:metabolism / Q000502:physiology*; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D013997:Time Factors; D014162:Transfection; D020798:Two-Hybrid System Techniques; D014443:Tyrosine / Q000737:chemistry; D018909:src Homology Domains
D002352:Carrier Proteins; D018076:DNA, Complementary; D011993:Recombinant Fusion Proteins; D014443:Tyrosine; D011392:Proline; D005982:Glutathione Transferase; D011505:Protein-Tyrosine Kinases; D051418:Focal Adhesion Kinase 2; C496054:Ptk2b protein, rat; D020823:ADP-Ribosylation Factor 1