PKC/PKA Regulation in Prostate Cancer by SSeCKS/Gravin

描述（由申请人提供）：细胞生物学领域的一个主要谜题是具有看似相反功能的信号蛋白，如1蛋白激酶（PK） A和PKC，在有丝分裂刺激过程中如何在时间和空间上受到调节。脚手架蛋白被认为可以促进底物磷酸化的效率和特异性，并在时空上协调激酶和磷酸酶的作用。本研究的长期目标是了解SSeCKS（一种与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）如何帮助调节未转化成纤维细胞和上皮细胞中的信号串扰和G1-S进展，而在前列腺癌细胞中，SSeCKS的表达严重下调。我们的工作假设是SSeCKS（及其同源物GravinfAKAP25O）阻止PKCIPKA串扰：在静止细胞中，SSeCKS结合并抑制PKC活性，而在G1-S进程中，SSeCKS活化的磷酸化选择性地拮抗PKC结合；PKA的结合对磷酸化不敏感，因此被隔离在“非活性”位点。随着越来越多的数据表明SSeCKS在前列腺癌中具有转移抑制活性，我们设想解剖PKC/PKA/SSeCKS控制网络将进一步了解有丝分裂和肿瘤发生过程中的信号传导和细胞骨架控制。在Aim #1中，我们将使用定点诱变和体外结合试验来鉴定结合PKC和PKA并介导多聚的SSeCKS结构域。在Aim #2中，我们将研究SSeCKS结合如何影响特定底物上PKC和PKA的体外激酶活性。我们还将在未转化的NIH3T3和前列腺上皮细胞以及ssecks缺陷的人LNCaP前列腺癌细胞中，确定结合位点如何调节PKC和pka介导的G1-S信号传导和细胞骨架通路。我们将分析SSeCKS如何影响有丝分裂原和整合素介导的PKC和PKA的细胞定位。Aim #3将研究pkc诱导的磷酸化如何调节SSeCKS体内支架活性和参与G1-S调节。为此，我们将使用肽微测序和MALDI-TOF质谱相结合的方法，绘制促生长和致癌条件下SSeCKS上PKC的体内磷酸化位点。使用SSeCKS磷酸化特异性抗体和表达SSeCKS pkc磷酸化位点突变体的细胞系，将了解pkc诱导的磷酸化对SSeCKS支架/调节功能的作用。考虑到许多G1-S调控功能在肿瘤发生中被破坏，SSeCKS的研究结果应该拓宽我们对正常细胞和癌细胞细胞周期进程的时空控制的理解。

DESCRIPTION (provided by applicant): A major enigma in the field cell biology is how signaling proteins with seemingly antithetical functions, such as 1 protein kinase (PK) A and PKC, are regulated temporally and spatially during mitogenic 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, GravinfAKAP25O) prevents PKCIPKA 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 MALDI-TOF 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.