Differential Regulation of Pyk2 and Focal Adhesion Kinase (FAK)

Abstract

Pyk2 is a recently described cytoplasmic tyrosine ki-nase that is related to focal adhesion kinase (FAK) and can be activated by a variety of stimuli that elevate intracellular calcium. In this report, we showed that Pyk2 and FAK tyrosine phosphorylation are regulated differentially by integrin-mediated cell adhesion and soluble factors both in rat aortic smooth muscle cells, which express endogenous Pyk2 and FAK, and in trans-fected Chinese hamster ovary cells. We also found that Pyk2 is diffusely present throughout the cytoplasm, while FAK is localized in focal contacts as expected, suggesting that the different localization may account for their differential regulation. By analyzing a chimeric protein contain N-terminal and kinase domains of Pyk2 and C-terminal domain of FAK, we provided evidence that the distinctive C-terminal domains of Pyk2 and FAK were responsible for their differential regulation by integrins and soluble stimuli as well as their subcel-lular localization. Finally, we correlated FAK, Pyk2, and the chimeric protein binding to talin, but not paxillin, with their regulation by integrins and focal contact lo-calization. These results demonstrate that the distinctive C-terminal domain of Pyk2 and FAK confer their differential regulation by different subcellular localiza-tion and association with the cytoskeletal protein talin. Proline-rich tyrosine kinase 2 (Pyk2 1 ; also known as CAK, RAFTK, and CADTK) is a recently described cytoplasmic tyro-sine kinase that is related to the focal adhesion kinase (FAK) (1-4). Recent studies have shown that Pyk2 is involved in calcium-induced regulation of ion channel and mitogen-activated protein kinase activation (1), stress-induced c-Jun N-terminal kinase activation (5), and Src-mediated activation of the mitogen-activated protein kinase signaling pathway in PC12 cells (6). Although these studies indicate that Pyk2 is involved in several signal transduction pathways, many questions concerning the regulation and function of Pyk2, especially the role of Pyk2 in cell adhesion, remain unanswered. Pyk2 and FAK share a similar structural organization with a tyrosine kinase domain flanked by non-catalytic domains at both the N and C termini. These two kinases are approximately 60% identical in the central catalytic domain and share approximately 40% identity in both the N-and C-terminal domains (1-3). Because of the high sequence homology and similar overall organization between Pyk2 and FAK, it is especially interesting to compare the regulation of Pyk2 with that of FAK, in particular their responses to integrin-mediated cell adhesion. Several recent reports have presented somewhat conflicting data regarding regulation of Pyk2 by integrin-mediated cell adhesion in different cell types. It has been reported that Pyk2 displays an integrin-dependent phosphorylation and is localized in focal contacts in B lymphocytes, megakaryocytes, and transfected COS cells (7, 8); however, in transfected 3Y1 cells, Pyk2 phosphorylation is not increased upon plating on fi-bronectin (FN) and Pyk2 has been shown to localize in inter-cellular junctions (2). In addition, Siciliano et al. (9) reported that, in rat hippocampal slices and cortical synaptosomes, Pyk2 and FAK are regulated differentially by pathways involving calcium and protein kinase C. To determine whether induction of Pyk2 phosphorylation by integrins is as effective as that by soluble factors and how it compares with FAK phosphorylation in cell adhesion in the same cells, we investigated regulation of Pyk2 and FAK in rat aortic smooth muscle (RASM) cells, which express both ki-nases. Using a CHO cell transfection system, we also analyzed regulation of Pyk2, FAK, or a chimeric protein containing the N-terminal and kinase domains of Pyk2 fused to the C-terminal domain of FAK. Our results demonstrated that Pyk2 and FAK were regulated differentially in both RASM cells and transfected CHO cells and that the distinctive C-terminal domains are responsible for the differential regulation by directing different subcellular localization and association with the cytoskeletal protein talin.