PKC/PKA in Prostate Cancer

描述：(申请人提供)细胞领域的一个重大谜团 生物学是如何传递具有看似相反功能的蛋白质的信号，如 作为蛋白激酶(PKA)和PKC，在时间和空间上都受到调控 丝裂原刺激。支架蛋白被认为有助于 底物磷酸化的效率和特异性，并协调 激酶和磷酸酶在时空中的作用。长期的 本研究的目的是了解SSeCKS，一种质膜-和 细胞骨架相关的PKC底物，结合PKC、PKA、钙调蛋白和 细胞周期蛋白，帮助调节信号串扰和G1-S的进展 与前列腺癌相比，未转化的成纤维细胞和上皮细胞 细胞中，SSeCKS表达严重下调。我们的工作 假设SSeCKS(及其同源基因Gravin/AKAP25O)阻止PKC/PKA 串扰：在静止细胞中，SSeCKS结合并抑制PKC活性 而在G1期S，丝裂原激活的SseCKs的磷酸化 选择性拮抗PKC结合；PKA，其结合是磷酸化 不敏感的，然后被隔离到“不活跃”的站点。随着不断增加的数据 SSeCKS在前列腺癌中具有转移抑制活性，我们设想 对PKC/PKA/SSeCKS控制网络的剖析将进一步了解 有丝分裂和肿瘤发生过程中的信号和细胞骨架调控。在AIM #1，我们将使用定点突变和体外结合试验来 识别绑定PKC和PKA的SSeCKS结构域以及 多聚合化。在目标2中，我们将研究SSeCKS绑定如何影响In 特定底物上PKC和PKA的体外激酶活性。我们还将 确定结合部位如何调节PKC和PKA介导的G1-S信号 和未转化的NIH3T3和前列腺体内的细胞骨架通路 上皮细胞和SSeCKS缺陷型人前列腺癌LNCaP细胞。我们 将分析SSeCKS如何影响丝裂原和整合素介导的细胞 PKC和PKA的本土化。Aim#3将研究PKC如何诱导 磷酸化调控SSeCKS在体内的支架活性和参与 在G1-S的调控下。为此，我们将绘制体内PKC的磷酸化图谱 在促生长和致癌条件下的SSeCKS上的位点 多肽微测序与MALDITOF质谱学的结合。角色 PKC诱导的SSeCKS支架/调控功能的磷酸化将 使用SSeCKS磷酸化特异性抗体和细胞系表达 SSeCKS PKC-磷酸化位点突变体。鉴于许多G1-S监管机构 这项拟议的研究的结果是，在肿瘤发生中功能受到破坏 关于SSeCKS应该拓宽我们对空间和时间控制的理解 控制正常细胞和癌细胞的细胞周期进程。

DESCRIPTION: (provided by applicant) A major enigma in the field of cell biology is how signaling proteins with seemingly antithetical functions, such as protein kinase (PKA) and PKC, are regulated temporally and spatially during mitoqenic stimulation. Scaffolding proteins are thought to facilitate the efficiency and specificity of substrate phosphorylation and also coordinate the actions of kinases and phosphatases in a spatiotemporal manner. The long-term goal of this research is to understand how SSeCKS, a plasma membrane-and cytoskeleton-associated PKC substrate that binds PKC, PKA, calmodulin and cyclins, helps regulate signaling crosstalk and G1-S progression in untransformed fibroblasts and epithelial cells, compared to prostate cancer cells, in which SSeCKS expression is downregulated severely. Our working hypothesis is that SSeCKS (and its orthologue, Gravin/AKAP25O) prevents PKC/PKA crosstalk: in quiescent cells, SSeCKS binds to and inhibits PKC activity whereas during G1 S progression, mitogen-activated phosphorylation of SSeCKS selectively antagonizes PKC binding; PKA, whose binding is phosphorylation insensitive, is then sequestered to "inactive" sites. With mounting data that SSeCKS has metastasis suppressor activity in prostate cancer, we envision that dissection of the PKC/PKA/SSeCKS control network will further understanding of signaling and cytoskeletal controls during mitogenesis and oncogenesis. In Aim #1, we will use site-directed mutagenesis and in vitro binding assays to identify the SSeCKS domains that bind PKC and PKA and that mediate multimerization. In Aim #2, we will examine how SSeCKS binding affects the in vitro kinase activity of PKC and PKA on specific substrates. We will also determine how the binding sites regulate PKC-and PKA-mediated G1-S signaling and cytoskeletal pathways in vivo in untransformed NIH3T3 and prostate epithelial cells, and in SSeCKS-deficient human LNCaP prostate cancer cells. We will analyze how SSeCKS affects the mitogen-and integrin-mediated cellular localization of PKC and PKA. Aim #3 will investigate how PKC-induced phosphorylation modulates SSeCKS in vivo scaffolding activity and involvement in G1-S regulation. To this end, we will map the in vivo PKC phosphorylation sites on SSeCKS under growth-promoting and oncogenic conditions using a combination of peptide microsequencing and MALDITOF mass spectrometry. The role of PKC-induced phosphorylation on SSeCKS' scaffolding/regulatory functions will be accessed using SSeCKS phospho-specific antibodies and cell lines expressing SSeCKS PKC-phosphorylation-site mutants. Given that many G1-S regulatory functions are undermined in oncogenesis, the results of the proposed research on SSeCKS should broaden our understanding of spatial and temporal controls governing cell cycle progression in normal and cancer cells.