Phosphorylation of the protein kinase C-theta activation loop and hydrophobic motif regulates its kinase activity, but only activation loop phosphorylation is critical to in vivo nuclear-factor-κB induction
Phosphorylation of the protein kinase C-theta activation loop and hydrophobic motif regulates its kinase activity, but only activation loop phosphorylation is critical to in vivo nuclear-factor-κB induction
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DOI：
10.1042/bj3610255
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发表时间：
2002-01-15
影响因子：
4.1
通讯作者：
Shaw, S
中科院分区：
文献类型：
--
作者：
Liu, Y;Graham, C;Shaw, S
蛋白激酶C（PKC）-θ是PKC亚型的“新”亚家族的成员，在T淋巴细胞中的信号转导中具有独特的重要性。由于对新型PKC的调节磷酸化的理解是不完整的，并且与“经典”PKC亚型的发现不一致，我们研究了PKC-θ上的三个潜在磷酸化位点：激活环（Thr（538））、转角基序（Ser（676））和疏水基序（Ser（695））。来自磷酸特异性抗血清和MS的综合证据证明了所有三个位点的磷酸化。与其最接近的paralysts，PKC-δ，缺乏负电荷的活化环PKC-θ的结果在一个深刻的催化缺陷（> 100倍减少在T538 A突变）;高序列之间的相似性PKC-θ和-δ有助于制定结构假说，以解释这一重大差异。与其他PKC亚型提出的机制相反，其他两个位点的磷酸化不能重建催化活性。活化环磷酸化在体内是关键的，因为T538 A突变体完全丧失了其介导Jurkat T细胞中核因子κ B（NF-κ B）活化的T细胞受体刺激的能力。疏水基序磷酸化也显著影响PKC-θ催化活性（在S695 A突变体中降低5倍），但不损害Jurkat T细胞中的NF-κ B活化。其机制独立于对激活环磷酸化的次级影响，并且不能用热不稳定性来解释。Turn基序磷酸化对激酶活性的影响有限，但负调节PKC-θ功能的其他方面，因为S676 A突变体在Jurkat T细胞中诱导NF-κ B活化方面比野生型更有效。这些发现扩展了我们对磷酸化在新型PKC中的作用的理解，并表明PKC-θ是一种组成型活性激酶，其激活环的组成型磷酸化的结果。
Protein kinase C (PKC)-theta, a member of the 'novel' subfamily of PKC isoforms, is of singular importance in transducing signals in T-lymphocytes. Since understanding of regulatory phosphorylation of novel PKCs is fragmentary and inconsistent with findings for 'classical' PKC isoforms, we investigated three potential phosphorylation sites on PKC-theta; in the activation loop (Thr(538)), turn motif (Ser(676)) and hydrophobic motif (Ser(695)). Combined evidence from phospho-specific antisera and MS demonstrates phosphorylation at all three sites. Unlike its closest paralogue, PKC-delta, lack of negative charge in the activation loop of PKC-theta results in a profound catalytic defect (> 100-fold reduction in the T538A mutant); the high sequence similarity between PKC-theta and -delta assists in the formulation of structural hypotheses to account for this major difference. In contrast with mechanisms proposed for other PKC isoforms, phosphorylation at the other two sites does not reconstitute catalytic activity. Activation loop phosphorylation is critical in vivo, since the T538A mutant completely lost its capacity to mediate T-cell receptor-stimulation of nuclear factor kappaB (NF-kappaB) activation in Jurkat T-cells. Hydrophobic motif phosphorylation also substantially influences PKC-theta catalytic activity (5-fold reduction in the S695A mutant), but does not impair NF-kappaB activation in Jurkat T-cells. Its mechanism is independent of secondary effects on activation loop phosphorylation and cannot be explained by thermal instability. Turn motif phosphorylation has a limited effect on kinase activity, but negatively regulates other aspects of PKC-theta function, since the S676A mutant is more efficient than wild-type in inducing NF-kappaB activation in Jurkat T-cells. These findings expand our understanding of the roles of phosphorylation in novel PKCs, and indicate that PKC-theta is a constitutively competent kinase as a consequence of constitutive phosphorylation of its activation loop.