RAFTK/Pyk2 activation is mediated by trans-acting autophosphorylation in a Src-independent manner.
The related adhesion focal tyrosine kinase (RAFTK), also known as Pyk2, undergoes autophosphorylation upon its stimulation. This leads to cascades of intracellular signaling that result in the regulation of various cellular activities. However, the molecular mechanism of RAFTK autophosphorylation is not yet known. Using various RAFTK constructs fused with two different tags, we found that the autophosphorylation of RAFTK was mediated by a trans-acting mechanism, not a cis-acting mechanism. In addition, overexpression of kinase-mutated RAFTK inhibited wild type RAFTK autophosphorylation in a dose-dependent manner by a trans-acting interaction. Trans-acting autophosphorylation was also observed between endogenous and exogenous RAFTK upon potassium depolarization of neuroendocrine PC12 cells. Using immunoprecipitation and affinity chromatography, we detected RAFTK self-association that was not affected by deletion of a single region or domain of RAFTK. Furthermore, RAFTK autophosphorylation occurred only at site Tyr402 in a Src kinase activity-independent manner. However, Src significantly enhanced RAFTK-mediated paxillin phosphorylation, suggesting a key role for Src in RAFTK activation and phosphorylation of downstream substrates. Our results indicate that the activation of RAFTK occurs in several steps. First, upon stimulus, RAFTK trans-autophosphorylates Tyr402. Second, phosphorylated Tyr402 recruits and activates Src kinase that in turn phosphorylates RAFTK and enhances its kinase activity. Lastly, the enhanced RAFTK activity induces the activation of downstream signaling molecules. Taken together, these studies provide insights into the molecular mechanism of RAFTK autophosphorylation and the specific role of Src in the regulation of RAFTK activation.
Park
Shin-Young
SY
Division of Experimental Medicine, Beth Israel Deaconess Medical Center and Harvard Medical School, Boston, Massachusetts 02115, USA.
Avraham
Hava Karsenty
HK
Avraham
Shalom
S
D000818:Animals; D002846:Chromatography, Affinity; D003598:Cytoskeletal Proteins / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D004789:Enzyme Activation; D051418:Focal Adhesion Kinase 2; D006801:Humans; D007163:Immunosorbent Techniques; D051379:Mice; D016297:Mutagenesis, Site-Directed; D016716:PC12 Cells; D051419:Paxillin; D010750:Phosphoproteins / Q000378:metabolism; D010766:Phosphorylation; D011505:Protein-Tyrosine Kinases / Q000235:genetics / Q000378:metabolism*; D016392:Proto-Oncogene Proteins pp60(c-src) / Q000378:metabolism*; D051381:Rats; D015398:Signal Transduction; D015533:Transcriptional Activation; D014162:Transfection; D014443:Tyrosine / Q000378:metabolism
D003598:Cytoskeletal Proteins; C496061:PXN protein, human; D051419:Paxillin; D010750:Phosphoproteins; C496062:Pxn protein, mouse; C496063:Pxn protein, rat; D014443:Tyrosine; D011505:Protein-Tyrosine Kinases; D051418:Focal Adhesion Kinase 2; D016392:Proto-Oncogene Proteins pp60(c-src); C496053:Ptk2b protein, mouse; C496054:Ptk2b protein, rat