application) The long-term goal of this research has been to elucidate the function of specific protein kinase C (PKC) isozymes in the control of vascular smooth muscle (VSM) cell contractility and growth. An understanding of these signaling pathways could provide a basis for explaining or treating the pathology underlying pervasive vascular diseases including hypertension, atherosclerosis, and restenosis. The mitogen-activated protein kinase (MAPK) signaling pathway is essential for controlling cell growth and gene transcription and has also been implicated in the control of VSM cell motility and contractility. Studies indicate that a pool of VSM cell PKC-delta, a Ca2+-independent isozyme of PKC, and Raf-1 (an upstream activator of the MAPK) co-immunoprecipitate, suggesting a physical interaction.
One specific aim the proposed research is to test the hypothesis that the pool of interacting PKC-delta and Raf-1 is coupled functionally (i.e. PKC-delta activation leads to stimulation of Raf serine/threonine kinase activity) and determine to what degree this contributes to MAPK activation in response to tyrosine kinase receptor-coupled growth factors and G-protein receptor coupled ligands. Tyrosine phosphorylated focal adhesion kinase (FAK) and paxillin, also co-immunoprecipitate with PKC-delta from VSM cell lysates. This has led to the hypothesis that PKC-delta and Raf physically interact with focal adhesion complexes, which are structures required for cell adhesion, migration, and force transmission.
The second aim of the research is to determine: A) whether integrin-dependent MAPK signaling stimulated by cell adhesion or stretch is mediated specifically by the pool of interacting PKC-delta/Raf; B) whether the interaction of PKC-delta and Raf with focal adhesion complex proteins is modulated by integrin-dependent VSM cell adhesion and/or stretch of adherent cells; and C) whether PKC-delta regulates integrin-dependent VSM cell adhesion or migration.
The final aim i s to determine if Src- or a related tyrosine-kinase phosphorylates PKC-delta thereby regulating PKC-delta /Raf/focal adhesion complex interactions and the integrin-dependent signaling pathway. This research will lead to a better understanding of the mechanisms by which PKC regulates focal adhesion assembly/disassembly and provide insight into the integrin-dependent signaling pathways which control VSM cell migration and patterns of gene expression.
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