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激动剂的有丝分裂效应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。该建议的广泛、长期目标仍然是阐明介导gpcr诱导细胞增殖的信号转导途径。蛋白激酶C （PKC）在信号转导途径中起着关键作用，介导由GI肽引发的许多细胞反应，包括细胞增殖和GPCR信号的调节。然而，发生在PKC特定同工异构体下游的事件仍然难以捉摸，我们自己的结果证明了PKC独立途径导致有丝分裂发生的存在。目前的挑战是确定GPCRs的pkc依赖性和pkc非依赖性下游靶标，这些靶标将信号传递到细胞内部，参与细胞增殖的调节。最近，在我们实验室克隆的蛋白激酶D （PKD）已成为导致gpcr诱导的细胞增殖的信号转导途径的主要元件。我们之前的研究表明，通过PKC依赖的PKD激活环磷酸化Ser744和Ser748， PKD在广泛的生物系统中快速激活，包括肠上皮细胞。在目前的建议中，我们开发了一个模型，该模型设想PKD的顺序激活涉及PKD激活的早期PKC依赖阶段和晚期PKC不依赖阶段，通过转磷酸化和自磷酸化机制。对培养细胞和转基因小鼠的进一步研究表明，PKD在小肠远端和结肠上皮中表达水平升高的PKD蛋白，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，该级联在GPCR诱导的细胞增殖的信号转导中起关键作用。另一种假设是PKD信号也参与肠上皮细胞迁移的调节。本提案将研究以下具体目标：1)表征肠上皮细胞中PKD激活响应GPCR激活的早期和晚期；2)表征PKD在肠上皮细胞增殖和迁移中的作用；3)利用转基因PKD在体内的表达，确定PKD在肠道细胞增殖中的作用。

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.