PKC zeta as a Target for Ceramide

描述（由申请人提供）： 神经酰胺是一种脂质衍生的第二信使，在体外和体内抑制血管平滑肌（VSM）细胞有丝分裂。多个实验室，包括我们自己的，已经确定蛋白激酶C ζ（PKCzeta）作为神经酰胺的目标。我们已经发表的数据表明，神经酰胺激活的PKCzeta是VSM细胞生长停滞所必需的。已确定两种信号通路有助于神经酰胺的抗促有丝分裂作用。神经酰胺直接激活PKCzeta，至1。）诱导SAPK信号复合物形成;和2.）抑制AKT 1信号级联。阐明神经酰胺激活PKCzeta的生物物理和生物化学机制是这项新提议的重点。现在假设神经酰胺刺激离散脂质微结构域或筏内的PKCzeta;活性。我们将在以下具体目标中检验这一假设。在特定目标1中，我们将确定富含神经酰胺的脂筏作为PKCzeta被磷酸化并被上游激酶最佳激活的位点。在具体目标2中，我们将表征和验证PKCzeta上的神经酰胺结合结构域或裂缝。这些研究将阐明炎症性细胞因子受体诱导的神经酰胺形成限制炎症性血管疾病模型中细胞增殖的生物物理和生化机制。这些研究的长期创新是将神经酰胺类似物直接递送到血管成形术和支架置入术后观察到的VSM生长失调部位的临床潜力。此外，这一提议有望定义适合作为进一步药物发现的治疗剂或筛选工具的拟脂质体。

DESCRIPTION (provided by applicant): Ceramide is a lipid-derived second messenger that suppresses vascular smooth muscle (VSM) cell mitogenesis in vitro and in vivo. Multiple laboratories, including our own, have identified protein kinase C zeta (PKCzeta) as a target of ceramide. We have published data demonstrating that ceramide-activated PKCzeta is necessary for VSM cell growth arrest. Two signaling pathways have been identified that contribute to the anti-mitogenic actions of ceramide. Ceramide directly activates PKCzeta, to 1.) induce SAPK signal complex formation; and 2.) inhibit AKT1 signaling cascades. Elucidation of the biophysical and biochemical mechanisms by which ceramide activates PKCzeta is the focus of this new proposal. It is now hypothesized that ceramide stimulates PKCzeta; activity within discrete lipid microdomains or rafts. We will test this hypothesis in the following Specific Aims. In Specific Aim 1, we will identify ceramide-enriched lipid rafts as the site at which PKCzeta, is phosphorylated and optimally activated by upstream kinases. In Specific Aim 2, we will characterize and validate ceramide-binding domains or clefts on PKCzeta. These studies will elucidate the biophysical and biochemical mechanisms by which inflammatory cytokine receptor-induced ceramide formation limits cellular proliferation in models of inflammatory vascular diseases. The long-term innovation of these studies is the clinical potential of direct delivery of ceramide analogues to the site of dysregulated VSM growth observed after angioplasty and stent placement. Additionally, this proposal holds the promise of defining lipidomimetics suitable as therapeutics or screening tools for further drug discovery.