Gastrointestinal (GI) peptides including mammalian bombesin-like peptides, gastrin and cholecystokinin (CCK) integrate secretory, contractile and proliferative functions in the GI tract. The broad, long term objective of this proposal is to elucidate the signal transduction pathways that mediate GI peptide-mediated cell proliferation. GI peptides elicit their characteristics effects on cellular processes by binding to specific G protein-coupled receptors (GPCR) on the surface of their target cells. A rapid increase in the synthesis of lipid- derived second messengers with subsequent activation of protein phosphorylation cascades is a crucial mechanism for transducing GI peptide signals across the plasma membrane. Protein kinase C (PKC) occupies a pivotal role in the signal transduction pathways that mediate numerous cellular responses elicited by GI peptides including cell proliferation and modulation of GPCR signaling. PKC compromises a multi- gene family that encodes at least eleven distinct isoforms which are differentially expressed in cells and tissues. It is thought that individual PKC isoforms differ with respect to their roles in growth control and other biologic effects by executing distinct cellular functions at different subcellular locations. However, the events occurring downstream of specific isoforms of PKC remain elusive. Recently, we cloned a novel serine/threonine protein kinase termed protein kinase D (PKD) with distinct structural and enzymological properties that has emerged as a putative downstream target of novel isoforms of PKC. Our central hypothesis envisages a novel PKC/PKD phosphorylation cascade that is activated by the GI peptides in their target cells. This proposal will pursue the following specific aims 1) Characterize the activation of the PKC/PKD pathways using the bombesin/GRP and CCK/B receptors as general models for gastrointestinal GPCRs; 2) Determine the role of individual PKC isoforms in PKD activation via bombesin/GRP and CCK/B receptors; 3) Determine specific activating phosphorylation sites in PKD using a combination of two dimensional phosphopeptide mapping and mutational analysis; and 4) Characterize signal transduction induced by GI peptides in cells expressing wild type, constitutively activated or dominant-negative forms of PKD.
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