REGULATION OF PROTEIN KINASE C IN TRANSFORMED CELLS

转化细胞中蛋白激酶 C 的调节

• 批准号: 3807264
• 负责人: SUSAN JAKEN
• 金额: --
• 依托单位: ADIRONDACK BIOMEDICAL RESEARCH INSTITUTE
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/3807264
• 关键词: adrenocorticotropic hormone; biological signal transduction; calcium; cortisol; enzyme induction /repression; enzyme inhibitors; enzyme substrate; hormone regulation /control mechanism; immunocytochemistry; lithium; monoclonal antibody; neoplastic cell; peptide hormone biosynthesis; phorbols; phosphorylation; pituitary gland; protein kinase C; receptor sensitivity; tissue /cell culture; tumor promoters

Exposure of cultured pituitary corticotrophs (AtT20-D16v, D16 cells) to phorbol esters causes a rapid increase in ACTH secretion which is followed by a refractory period (t 1/2 = 0.85 h). Desensitization appears to be due to uncoupling of PDBu receptor occupancy from activation of PKC and phosphorylation of substrates important for secretion. We will compare the biochemical properties of PDBu receptors and PKC from cytosols and membranes of control and desensitized cultures with respect to Ca2+ and phospholipid requirements, activation by PDBu, substrate specificity, phosphorylation state, subcellular localization by immunocytochemical techniques, and PKC isozyme content to determine if changes in PKC or isozyme composition may accompany desensitization. Our studies are greatly facilitated due to our success in producing PKC type-specific monoclonal and polyclonal antibodies. We will also determine if desensitization is associated with changes in levels of 2 inhibitory substances we have identified in these cells, loss of PKC substrates, or increased phosphatase activity for PKC substrates. Other agents, including Li+, Ca 2+, and glucocorticoids also cause desensitization to PDBu-directed ACTH secretion. The effects of these agents on the parameters listed above will be assessed to determine if they work through common or independent pathways. Prolonged treatment of D16 cells with PDBu causes loss of cellular PDBu receptors. This down-modulation also contributes to attenuation of PDBu- directed responses, but is clearly distinct from desensitization. We will determine whether down-modulation is associated with increased proteolysis of PKC, and whether this is PKC type-specific. We will characterize the biochemical and immunochemical properties of the PKC and PDBu binding activities remaining in down-modulated cultures to determine if they represent a specific portion of the total PDBu receptor population. The significance of these studies is due to the importance of PKC in regulation of cell growth and differentiated functions, as well as its role in tumorigenesis. Biochemical pathways associated with attenuation of PDBu-directed responses are likely to also play a role in limiting responses to hormones and growth factors whose mechanism of action involves PKC. Further understanding of biochemical pathways associated with limiting phorbol ester-directed responses may lead to pharmacological approaches for interfering with PKC activity.

将培养的垂体促肾上腺皮质激素（AtT20-D16v、D16 细胞）暴露于 佛波酯导致 ACTH 分泌迅速增加，随后 不应期 (t 1/2 = 0.85 h)。 脱敏似乎是由于 PDBu 受体占据与 PKC 激活的解偶联 对分泌重要的底物的磷酸化。 我们将比较 PDBu 受体和 PKC 的生化特性 对照和脱敏培养物的膜相对于 Ca2+ 和 磷脂需求、PDBu 激活、底物特异性、 磷酸化状态，免疫细胞化学亚细胞定位 技术和 PKC 同工酶含量来确定 PKC 或 同工酶组合物可伴随脱敏。 我们的学习成绩很大 由于我们成功生产了 PKC 类型特异性单克隆抗体，这为我们带来了便利 和多克隆抗体。 我们还将确定是否需要脱敏 与 2 种抑制物质水平的变化有关 在这些细胞中发现，PKC 底物丢失或磷酸酶增加 PKC 底物的活性。 其他试剂，包括 Li+、Ca 2+ 和 糖皮质激素还会导致 PDBu 指导的 ACTH 分泌脱敏。 将评估这些药物对上述参数的影响 以确定它们是通过共同途径还是独立途径发挥作用。 用 PDBu 长时间处理 D16 细胞会导致细胞 PDBu 损失 受体。 这种下调也有助于衰减 PDBu- 定向反应，但与脱敏明显不同。 我们将 确定下调是否与蛋白水解增加相关 PKC，以及这是否是 PKC 类型特定的。 我们将表征 PKC 和 PDBu 结合的生化和免疫化学特性 下调培养物中剩余的活动以确定它们是否 代表总 PDBu 受体群体的特定部分。 这些研究的意义在于 PKC 在 细胞生长和分化功能的调节及其作用 在肿瘤发生中。 与减弱相关的生化途径 PDBu 定向反应也可能在限制 对激素和生长因子的反应，其作用机制涉及 PKC。 进一步了解与相关的生化途径 限制佛波酯定向反应可能会导致药理学 干扰 PKC 活性的方法。