Regulation of Protein Kinase C Theta by Phosphorylation

项目摘要 拟议研究的总体愿景是全面了解磷酸化如何 调节免疫信号传导的关键调节因子Ser/Thr蛋白激酶C（PKC）的活性和功能 Theta（θ）。这种激酶选择性地在造血细胞中表达，在造血细胞中它转导导致T细胞增殖的信号。 细胞和血小板活化。1，2其失调与多种病理生理条件有关 包括血癌、3、4炎性疾病、5血栓形成、6和止血。7尽管如此， 蛋白激酶C β的功能尚不清楚，需要进一步研究。PKC β的磷酸化 在调节其成熟、催化活性和亚细胞定位方面起着重要作用，8所有这些都是 对于T细胞和血小板中的PKC β功能至关重要。该提案旨在了解磷酸化在 已知的保守引发位点（激活环，转向基序，疏水基序），9一个生物信息学鉴定的新的 潜在的引发位点（Ser 662）和未表征的激活诱导位点（Ser 685）调节成熟， 活性和/或PKC β的定位。无偏见的磷酸蛋白质组学方法已经揭示， 然而，在T细胞10和血小板11中，Ser 685的磷酸化响应于刺激而显著增加， 其职能尚未确定，部分原因是缺乏可用的研究工具。这个网站和Ser 662 位于一个关键的调控片段，C-尾，并在进化上是保守的。核心假设 推动这一提议的是，S662的磷酸化参与了PKC β的成熟，而PKC β的激活- 诱导的S685磷酸化促进激活的PKC β的再自抑制，以促进信号终止。 为此，我将研究这些残基上的非磷酸化或磷酸化模拟突变如何影响 蛋白激酶C的生物化学特性、细胞活性、亚细胞定位和下游信号传导（Aim 1）。 此外，我将研究Jurkat细胞和血小板中PKC β的磷酸化蛋白质组， 激动剂诱导位点Ser 685的磷酸化影响下游信号传导。我亦会致力找出 使用各种磷酸化蛋白质组学方法研究调节PKC β Ser 685磷酸化的激酶（目的2）。 这些关键的研究将阐明PKC β磷酸化在关键残基的功能影响，以及这是如何影响蛋白质表达的。 影响下游信号。此外，该建议将阐明底物和信号网络 由PKC β调节。揭示PKC β的调节和功能，T细胞和血小板的关键调节因子， 对于理解正常和疾病状态下的T细胞和血小板信号传导至关重要。

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.