Regulation of Protein Kinase C Theta by Phosphorylation

项目总结 拟议研究的总体愿景是全面了解磷酸化是如何 调节免疫信号的关键调节因子--丝氨酸/苏氨酸蛋白激酶C(PKC)的活性和功能 西塔()。该激酶选择性地在造血细胞中表达，在那里它传递信号导致T细胞 细胞和血小板的激活。1，2其失调与多种病理生理条件有关 包括血癌，3，4个炎症性疾病，5个血栓，6个和止血。 而PKC的功能在很大程度上还不清楚，需要进一步研究。蛋白激酶C的磷酸化 在调节其成熟度、催化活性和亚细胞定位方面起着重要作用。 在T细胞和血小板中对蛋白激酶C的功能至关重要。这项提议旨在了解磷酸化是如何在 已知的保守启动位点(激活环、转角基序、疏水基序)，9一个生物信息鉴定的新的 潜在的启动位点(Ser662)和一个未知的激活诱导位点(Ser685)调节成熟， PKC的活性和/或本地化。无偏向的磷蛋白质组学方法揭示了 然而，刺激后，T细胞10和血小板11中Ser685的磷酸化显著增加 它的功能尚未确定，部分原因是缺乏可用的研究工具。此站点和Ser662 位于关键的调控片段C-尾上，并且在进化上是保守的。中心假说 推动这一提议的是，S662的磷酸化参与了PKC的成熟，并且激活- S685的诱导磷酸化促进了激活的蛋白激酶C的再次自我抑制，从而促进了信号的终止。 为此，我将研究这些残基上的非磷酸化或拟磷化突变是如何影响 PKC生化特性、细胞活性、亚细胞定位和下游信号传递(目标1)。 此外，我将研究尤尔卡特细胞和血小板中pkc的磷酸蛋白质组，并研究如何 激动剂诱导位点Ser685的磷酸化影响下游信号转导。我还将致力于确定 S)使用不同的磷酸蛋白质组学方法调节PKCSer685的磷酸化(目标2)。 这些关键研究将阐明关键残基上PKC磷酸化的功能影响及其如何 影响下行信令。此外，该提案将阐明底物和信令网络。 受PKC调控。揭示T细胞和血小板的关键调节因子PKC的调节和功能 对于了解T细胞和血小板在正常和疾病状态下的信号至关重要。

PROJECT SUMMARY The overall vision of the proposed research is to gain a comprehensive understanding of how phosphorylation regulates the activity and function of a key regulator of immune signaling, the Ser/Thr protein kinase C (PKC) Theta (). This kinase is selectively expressed in hematopoietic cells where it transduces signals resulting in T cell and platelet activation.1,2 Its dysregulation is associated with a variety of pathophysiological conditions including blood cancers,3,4 inflammatory diseases,5 thrombosis,6 and hemostasis.7 Despite this, the regulation and function of PKC remains largely unknown and necessitates further investigation. Phosphorylation of PKC plays an essential role in regulating its maturation, catalytic activity, and subcellular localization,8 all of which are crucial for PKC function in T cells and platelets. This proposal aims to understand how phosphorylation at known conserved priming sites (activation loop, turn motif, hydrophobic motif),9 a bioinformatically-identified new potential priming site (Ser662), and an uncharacterized activation-induced site (Ser685), regulate the maturation, activity, and/or localization of PKC. Unbiased phosphoproteomics approaches have revealed that phosphorylation of Ser685 significantly increases in T cells10 and platelets11 in response to stimulation, however its function has not yet been determined due, in part, to a lack of available research tools. This site, and Ser662 are positioned on a key regulatory segment, the C-tail, and are evolutionarily conserved. The central hypothesis driving this proposal is that phosphorylation of S662 is involved in the maturation of PKC and that activation- induced phosphorylation of S685 promotes the re-autoinhibition of activated PKC to facilitate signal termination. To this end, I will investigate how nonphosphorylatable or phosphomimetic mutations at these residues impact PKC biochemical properties, cellular activity, subcellular localization, and downstream signaling (Aim 1). Additionally, I will examine the phosphoproteome of PKC in Jurkat cells and platelets and examine how phosphorylation at the agonist-induced site, Ser685, affects downstream signaling. I will also aim to identify the kinase(s) regulating PKC Ser685 phosphorylation using various phosphoproteomics approaches (Aim 2). These key studies will elucidate the functional impact of PKC phosphorylation at critical residues and how this influences downstream signaling. Moreover, this proposal will elucidate substrates and signaling networks regulated by PKC. Uncovering the regulation and function of PKC, a key regulator of T cells and platelets, is crucial to understanding T cell and platelet signaling in both normal and disease states.