Gastrointestinal Peptide Signaling through PKC/PKD

描述(由申请人提供)：G蛋白偶联受体(GPCR)激动剂，包括胃肠道(GI)肽，促进其目标细胞的快速收缩和/或分泌反应。胃肠肽还可以诱导细胞增殖的发现，为这些信息分子确定了一个新的作用。最近的证据表明，GPCR激动剂的促有丝分裂作用与许多关键的生物学过程有关，包括发育、代偿性生长和肿瘤发生。这项建议的广泛和长期的目标仍然是阐明介导GPCR诱导的细胞增殖的信号转导途径。蛋白激酶C(PKC)在信号转导通路中起着关键作用，这些信号转导通路介导了GI多肽引起的多种细胞反应，包括细胞增殖和GPCR信号的调节。然而，发生在特定PKC亚型下游的事件仍然难以捉摸，我们自己的结果表明存在导致有丝分裂发生的PKC非依赖途径。目前的挑战是确定参与细胞增殖调控的GPCRs的PKC依赖和PKC非依赖的下游靶点，这些GPCRs向细胞内部传递信号。最近，我们实验室克隆的蛋白激酶D(PKD)已经成为导致GPCR诱导细胞增殖的信号转导通路中的一个主要元件。我们之前的研究表明，在包括肠上皮细胞在内的多种生物系统中，通过依赖于PKC的PKD激活环在Ser744和Ser748上的磷酸化，PKD迅速激活。在目前的提案中，我们开发了一个模型，该模型设想了PKD的顺序激活，包括早期依赖PKC和晚期非依赖PKC的PKD激活阶段，通过转磷酸化和自磷酸化机制。另外，对培养和转基因小鼠的细胞进行的研究表明，无论是在体外还是在体内，PKD在肠道上皮细胞的增殖中都发挥着重要作用。因此，我们的中心假设是GPCR激动剂激活了PKD介导的磷酸化级联反应，该级联反应在GPCR诱导的细胞增殖的信号转导中发挥关键作用。另一个假设是，PKD信号也在调节肠上皮细胞的迁移中发挥作用。这项建议将研究以下具体目标：1)研究肠上皮细胞中PKD激活的早期和晚期；2)研究PKD在肠上皮细胞增殖和迁移中的作用；3)通过PKD的转基因表达，确定PKD在活体肠道细胞增殖中的作用。 与公共卫生相关：胃肠道(GI)肽是一种化学信使，在调节细胞增殖方面发挥关键作用，也与包括慢性炎症过程和癌症在内的重要疾病的发病机制有关。在目前的应用中，我们建议继续我们的研究，阐明蛋白激酶D(PKD)的调节和功能，PKD是一个在靶细胞中传递GI肽信号的关键分子。这些研究不仅对于了解胃肠道的基本调控机制，而且对于确定PKD作为治疗以细胞过度增殖或缺陷为特征的疾病的新靶点具有重要意义。

DESCRIPTION (provided by applicant): G protein-coupled receptors (GPCR) agonists, including gastrointestinal (GI) peptides, promote rapid contractile and/or secretory responses in their target cells. The discovery that GI peptides can also induce cell proliferation identified a novel role for these informational molecules. Recent evidence indicates that the mitogenic effects of GPCR agonists are relevant for a number of critical biological processes, including development, compensatory growth and tumorigenesis. The broad, long-term objective of this proposal remains to elucidate the signal transduction pathways that mediate GPCR-induced cell proliferation. 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. However, the events occurring downstream of specific isoforms of PKC remain elusive and our own results demonstrated the existence of PKC-independent pathways leading to mitogenesis. The present challenge is to identify PKC-dependent and PKC-independent downstream targets for GPCRs that transmit signals to the cell interior that participate in the regulation of cell proliferation. Recently, protein kinase D (PKD), cloned in our laboratory, has emerged as a major element in the signal transduction pathways leading to GPCR-induced cell proliferation. Our previous studies demonstrated rapid PKD activation in a broad range of biological systems, including intestinal epithelial cells, via PKC- dependent PKD activation loop phosphorylation on Ser744 and Ser748. In the current proposal, we develop a model that envisages sequential PKD activation involving an early PKC-dependent and a late PKC- independent phase of PKD activation via transphosphorylation and autophosphorylation mechanisms. Additional studies with cells in culture and transgenic mice that express elevated levels of PKD protein in the distal small intestinal and colonic epithelium indicate that PKD plays a major role in mediating proliferation of intestinal epithelial cells, both in vitro and in vivo. Consequently, our central hypothesis is that GPCR agonists activate a PKD-mediated phosphorylation cascade that plays a critical role in the signal transduction of GPCR-induced cellular proliferation. An additional hypothesis is that PKD signaling also operates in the regulation of migration of intestinal epithelial cells. This proposal will examine the following Specific Aims: 1) Characterize early and late phases of PKD activation in response to GPCR activation in intestinal epithelial cells; 2) Characterize the role of PKD in the proliferation and migration of intestinal epithelial cells; 3) Determine the role of PKD in intestinal cell proliferation in vivo using transgenic expression of PKD. Relevance to Public Health: Gastrointestinal (GI) peptides are chemical messengers that play critical roles in the regulation of cell proliferation and are also implicated in the pathogenesis of important diseases, including chronic inflammatory processes and cancer. In the current application, we propose to continue our studies elucidating the regulation and function of protein kinase D (PKD), a molecule that is emerging as a key player in transmitting GI peptide signals in their target cells. These studies have important implications not only for understanding fundamental regulatory mechanisms in the GI tract but also for identifying PKD as a novel target for therapeutic intervention in diseases characterized by either excessive or defective cell proliferation.