PKC/PKA Regulation in Prostate Cancer by SSeCKS/Gravin

SSeCKS/Gravin 对前列腺癌的 PKC/PKA 调节

• 批准号: 7110118
• 负责人: IRWIN H. GELMAN
• 金额: $ 28.6万
• 依托单位: ROSWELL PARK CANCER INSTITUTE CORP
• 依托单位国家: 美国
• 财政年份: 2002
• 资助国家: 美国
• 起止时间: 2002-08-01 至 2008-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7110118
• 关键词: active sites; binding sites; biological signal transduction; cell cell interaction; cytoskeletal proteins; enzyme activity; epithelium; integrins; laboratory mouse; laboratory rabbit; mass spectrometry; matrix assisted laser desorption ionization; mitogens; neoplastic process; phosphorylation; polymerase chain reaction; prostate neoplasms; protein kinase A; protein kinase C; protein protein interaction; site directed mutagenesis; transcription factor; yeast two hybrid system

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.

描述(申请人提供)：细胞生物学领域的一个主要谜团是，在有丝分裂刺激过程中，似乎具有相反功能的信号蛋白，如1蛋白激酶(PK)A和蛋白激酶C(PKC)如何在时间和空间上受到调节。支架蛋白被认为促进底物磷酸化的效率和特异性，并以一种时空方式协调激酶和磷酸酶的作用。这项研究的长期目标是了解与前列腺癌细胞相比，SSeCKS是如何帮助调节未转化的成纤维细胞和上皮细胞中的信号串扰和G1-S进展的。SSeCKS是一种质膜和细胞骨架相关的PKC底物，它结合了PKC、PKA、钙调蛋白和周期蛋白。我们的工作假设是，SSeCKS(及其同源物GravinfAKAP25O)防止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，丝裂原激活的SSeCKS选择性地拮抗PKC结合；PKA，其结合是磷酸化不敏感的，然后隔离到“不活跃”的位置。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，我们可以预见，对PKC/PKA/SSeCKS调控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。在目标1中，我们将使用定点突变和体外结合试验来鉴定与PKC和PKA结合的SSeCKS结构域以及介导多聚体的SSeCKS结构域。在目标2中，我们将研究SSeCKS结合如何影响特定底物上的PKC和PKA的体外激酶活性。我们还将在未转化的NIH3T3和前列腺上皮细胞中，以及在SSeCKS缺陷的人前列腺癌LNCaP细胞中，确定这些结合位点如何调节PKC和PKA介导的G1-S信号和细胞骨架通路。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。目的#3研究蛋白激酶C诱导的磷酸化如何调节SSeCKs在体内的支架活性，并参与G1-S的调节。为此，我们将结合多肽显微测序和MALDI-TOF质谱学，在促生长和致癌条件下定位体内SSeCKS上的PKC磷酸化位点。PKC诱导的磷酸化在SSeCKS支架/调节功能中的作用将通过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来获得。鉴于许多G1-S调控功能在肿瘤发生中被破坏，对SSeCKs的拟议研究结果将拓宽我们对正常细胞和癌细胞细胞周期进程的时空调控的理解。