Differential regulation of Pyk2 phosphorylation at Tyr-402 and Tyr-580 in intestinal epithelial cells: roles of calcium, Src, Rho kinase, and the cytoskeleton.
The calcium-dependent proline-rich tyrosine kinase Pyk2 is activated by tyrosine phosphorylation, associates with focal adhesion proteins, and has been linked to proliferative and migratory responses in a variety of mesenchymal and epithelial cell types. Full Pyk2 activation requires phosphorylation at functionally distinct sites, including autophosphorylation site Tyr-402 and catalytic domain site Tyr-580, though the mechanisms involved are unclear. The pathways mediating Pyk2 phosphorylation at Tyr-402 and Tyr-580 were therefore investigated. Both sites were rapidly and transiently phosphorylated following cell stimulation by Ang II or LPA. However, only Tyr-580 phosphorylation was rapidly enhanced by intracellular Ca(2+) release, or inhibited by Ca(2+) depletion. Conversely, Tyr-402 phosphorylation was highly sensitive to inhibition of actin stress fibers, or of Rho kinase (ROK), an upstream regulator of stress fiber assembly. Ang II also induced a delayed (30-60 min) secondary phosphorylation peak occurring at Tyr-402 alone. Unlike the homologous focal adhesion kinase (FAK), Pyk2 phosphorylation was sensitive neither to the Src inhibitor PP2, nor to truncation of its N-terminal region, which contains a putative autoinhibitory FERM domain. These results better define the mechanisms involved in Pyk2 activation, demonstrating that autophosphorylation is ROK- and stress fiber-dependent, while transphosphorylation within the kinase domain is Ca(2+)-dependent and Src-independent in intestinal epithelial cells. This contrasts with the tight sequential coupling of phosphorylation seen in FAK activation, and further underlines the differences between these closely related kinases.
Wu
Steven S
SS
Department of Pediatrics, David Geffen School of Medicine, University of California-Los Angeles, 900 Veteran Avenue, Los Angeles, CA 90095, USA. swu@mednet.ucla.edu
Jácamo
Rodrigo O
RO
Vong
Sheung K
SK
Rozengurt
Enrique
E
D000804:Angiotensin II / Q000494:pharmacology; D000818:Animals; D002118:Calcium / Q000502:physiology*; D002478:Cells, Cultured; D003599:Cytoskeleton / Q000378:metabolism*; D051418:Focal Adhesion Kinase 2 / Q000737:chemistry / Q000187:drug effects / Q000378:metabolism*; D006801:Humans; D007413:Intestinal Mucosa / Q000737:chemistry / Q000166:cytology / Q000378:metabolism*; D047908:Intracellular Signaling Peptides and Proteins / Q000502:physiology*; D010766:Phosphorylation; D017346:Protein Serine-Threonine Kinases / Q000502:physiology*; D015398:Signal Transduction; D014443:Tyrosine; D054460:rho-Associated Kinases; D019061:src-Family Kinases / Q000502:physiology*
D047908:Intracellular Signaling Peptides and Proteins; D000804:Angiotensin II; D014443:Tyrosine; D051418:Focal Adhesion Kinase 2; D019061:src-Family Kinases; D017346:Protein Serine-Threonine Kinases; D054460:rho-Associated Kinases; D002118:Calcium