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，可促进其靶细胞的快速收缩和/或分泌反应。GI肽也可以诱导细胞增殖的发现确定了这些信息分子的新作用。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过程有关，包括发育、补偿性生长和肿瘤发生。该提议的广泛的长期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）在介导由GI肽引起的包括细胞增殖和GPCR信号调节的许多细胞应答的信号转导途径中占据关键作用。然而，下游发生的事件的具体亚型的PKC仍然难以捉摸，我们自己的结果表明存在的PKC独立的途径，导致有丝分裂。目前的挑战是确定PKC依赖性和PKC非依赖性的下游目标GPCR，传递信号到细胞内部，参与细胞增殖的调节。 最近，蛋白激酶D（PKD），在我们的实验室克隆，已成为一个主要因素的信号转导途径，导致GPCR诱导的细胞增殖。我们以前的研究表明，在广泛的生物系统，包括肠上皮细胞，通过PKC依赖性PKD激活环丝氨酸744和丝氨酸748磷酸化的快速PKD激活。在目前的建议中，我们开发了一个模型，设想顺序PKD激活涉及早期PKC依赖性和晚期PKC独立的阶段PKD激活通过转磷酸化和自磷酸化机制。用培养物中的细胞和在远端小肠和结肠上皮中表达升高水平的PKD蛋白的转基因小鼠进行的其他研究表明，PKD在体外和体内介导肠上皮细胞的增殖中起主要作用。因此，我们的中心假设是GPCR激动剂激活PKD介导的磷酸化级联反应，在GPCR诱导的细胞增殖的信号转导中起关键作用。另一个假设是PKD信号传导也在肠上皮细胞迁移的调节中起作用。本提案将研究以下具体目标：1）表征肠上皮细胞中响应于GPCR活化的PKD活化的早期和晚期; 2）表征PKD在肠上皮细胞增殖和迁移中的作用; 3）使用PKD的转基因表达确定PKD在体内肠细胞增殖中的作用。 与公共卫生的相关性：胃肠道（GI）肽是在细胞增殖调节中起关键作用的化学信使，并且还涉及重要疾病（包括慢性炎症过程和癌症）的发病机制。在本申请中，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是一种在靶细胞中传递GI肽信号的关键分子。这些研究不仅对理解胃肠道中的基本调节机制，而且对鉴定PKD作为以过度或有缺陷的细胞增殖为特征的疾病的治疗干预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.