Studies of cell structure and signaling are converging with the realization that the shape of a cell, its cytoskeletal architecture, and its attachment to extracellular matrix alter cell function. These parameters, in turn, govern crucial decisions in normal and neoplastic cells, e.g., differentiation, proliferation, apoptosis, and invasion and metastasis. To influence cellular decisions, cell structure must be sensed or interpreted by molecules linked to signaling pathways; these include the cytoskeletal-associated tyrosine kinases. Juliano and the PI and Guan and Hynes first described one such interface, integrin- dependent tyrosine phosphorylation, and, with Parsons, demonstrated the involvement of a then novel tyrosine kinase, the focal adhesion kinase p125FAK. While investigating a novel Calcium-Dependent Tyrosine Kinase, CADTK, the PI's group purified, then sequenced this second member of the cytoskeletal-associated tyrosine kinase family. This kinase was also isolated by four other groups using molecular techniques and was named Pyk2, CAKbeta, RAFTK, and FAK2. CADTK and p125FAK have the same domain structure and are 45 percent identical. However, they are often expressed in the same cells, exhibiting differences in localization, activation, and signaling. For example, CADTK distribution can extend beyond focal contacts to actin stress fibers. In adherent cells, CADTK is inactive until stimulated by G-protein coupled receptors or growth factors; p125FAK is constitutively tyrosine phosphorylated. With respect to signaling, CADTK appears to regulate c-Jun N terminal kinase and tyrosine phosphorylates both JIP-1 (a cytoplasmic JNK inhibitor) and Vav (a guanine nucleotide exchange factor that activates Rho family members); p125FAK does not. Unopposed, sustained CADTK signaling leads to cell death, whereas p125FAK appears to protect neoplastic cells from apoptosis. Conversely, more transient CADTK activation by hormones or growth factors is combined with other signals to stimulate proliferation. Our objective is to define functional differences and interactions between these two cytoskeletal-associated kinases, particularly in epithelial cells in which both are expressed. How is their expression changed by and what do they contribute to the transformed phenotype? How can we manipulate CADTK-dependent apoptosis? Specific Aims include: (1) studying CADTK subcellular localization and identifying interacting proteins that are substrates or participate in CADTK localization and signaling; (2) examining the mechanism of CADTK activation; (3) dissecting the CADTK to JNK signaling pathway, determining whether a cell-type specific CADTK yields MAPK pathway exists, and studying the role of CADTK-dependent Vav phosphorylation; and (4) defining the role of CADTK and co-stimulatory signals in proliferation, transformation, and apoptosis.
