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.
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
Steven S
SS
Wu
Rodrigo O
RO
Jácamo
Sheung K
SK
Vong
Enrique
E
Rozengurt
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