Thuille Nikolaus, Heit Isabelle, Fresser Friedrich, Krumböck Nina, Bauer Birgit, Leuthaeusser Sabine, Dammeier Sascha, Graham Caroline, Copeland Terry D, Shaw Steve, Baier Gottfried
Department for Medical Genetics, Molecular and Clinical Pharmacology, Innsbruck Medical University, Innsbruck, Austria.
EMBO J. 2005 Nov 16;24(22):3869-80. doi: 10.1038/sj.emboj.7600856. Epub 2005 Oct 27.
Phosphopeptide mapping identified a major autophosphorylation site, phospho (p)Thr-219, between the tandem C1 domains of the regulatory fragment in protein kinase C (PKC)theta. Confirmation of this identification was derived using (p)Thr-219 antisera that reacted with endogenous PKCtheta in primary CD3+ T cells after stimulation with phorbol ester, anti-CD3 or vanadate. The T219A mutation abrogated the capacity of PKCtheta to mediate NF-kappaB, NF-AT and interleukin-2 promoter transactivation, and reduced PKCtheta's ability in Jurkat T cells to phosphorylate endogenous cellular substrates. In particular, the T219A mutation impaired crosstalk of PKCtheta with Akt/PKBalpha in NF-kappaB activation. Yet, this novel (p)Thr-219 site did not affect catalytic activity or second-messenger lipid-binding activity in vitro. Instead, the T219A mutation prevented proper recruitment of PKCtheta in activated T cells. The PKCthetaT219A mutant defects were largely rescued by addition of a myristoylation signal to force its proper membrane localization. We conclude that autophosphorylation of PKCtheta at Thr-219 plays an important role in the correct targeting and cellular function of PKCtheta upon antigen receptor ligation.
磷酸肽图谱分析确定了蛋白激酶C（PKC）θ调节片段串联C1结构域之间的一个主要自磷酸化位点，即磷酸化（p）苏氨酸-219。使用（p）苏氨酸-219抗血清对该位点进行了确认，在用佛波酯、抗CD3或钒酸盐刺激后，该抗血清可与原代CD3 + T细胞中的内源性PKCθ发生反应。T219A突变消除了PKCθ介导核因子κB（NF-κB）、活化T细胞核因子（NF-AT）和白细胞介素-2启动子反式激活的能力，并降低了PKCθ在Jurkat T细胞中磷酸化内源性细胞底物的能力。特别是，T219A突变损害了PKCθ在NF-κB激活过程中与Akt/PKBα的相互作用。然而，这个新的（p）苏氨酸-219位点在体外并不影响催化活性或第二信使脂质结合活性。相反，T219A突变阻止了PKCθ在活化T细胞中的正确募集。通过添加豆蔻酰化信号以促使其正确的膜定位，PKCθT219A突变体的缺陷在很大程度上得到了挽救。我们得出结论，PKCθ在苏氨酸-219处的自磷酸化在抗原受体连接后PKCθ的正确靶向和细胞功能中起重要作用。
