A novel phosphorylation site at Ser130 adjacent to the pseudosubstrate domain contributes to the activation of protein kinase C-δ.
A novel phosphorylation site at Ser130 adjacent to the pseudosubstrate domain contributes to the activation of protein kinase C-δ.
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DOI：
10.1042/bj20150812
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发表时间：
2016-02-01
期刊：
影响因子：
--
通讯作者：
Steinberg SF
中科院分区：
文献类型：
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作者：
Gong J;Holewinski RJ;Van Eyk JE;Steinberg SF
蛋白激酶C-δ(PKC-δ)是一种信号转导蛋白，调节多种细胞反应。虽然大多数研究都集中在激活膜上的蛋白激酶Cδ的变构机制上，但蛋白激酶Cδ也是通过多位点磷酸化来控制的。(2015)莫尔。牢房。比奥尔。35,1727-1740]。本研究使用基于MS的方法来确定PKCδ在Thr50和Ser645处的磷酸化(在静息和PMA处理的心肌细胞中)，以及Thr37、Thr38、Ser130、Thr164、Thr211、Thr215、Ser218、Thr295、Ser299和Thr656(作为随着PMA的增加而增加的位点)。我们重点研究了Ser130和Thr141(伪底物结构域的N端)磷酸化的后果。我们发现S130D和T141E替换协同提高了PKCδ的S基础脂非依赖性活性，Ser130/Thr141二磷酸化影响了PKCδ的S底物特异性。我们最近报道，PKCδ优先与磷酸受体丝氨酸残基一起磷酸化底物，这是由于Ser357的结构性磷酸化，Ser357是一个限制PKCδ的S苏氨酸激酶活性的G-环位点。(2015)莫尔。牢房。比奥尔。35,1727-1740]。本研究表明，S130D和T141E替换通过降低丝氨酸357位G环的磷酸化，间接提高了PKCδ的S苏氨酸激酶活性。S130F替换[模拟在某些人群中发现的S130F单核苷酸多态(SNP)]还可以增加PKCδ的S的最大脂质依赖催化活性，并赋予苏氨酸激酶活性。最后，我们发现Ser130/Thr141的磷酸化解除了在细胞环境中限制PKCδ的S活性和底物专一性的自抑制限制。由于磷酸化位置与其他PKC的假底物结构域相似，我们的结果提示该区域的磷酸化可能构成了控制PKC异构体活性的一般机制。
Protein kinase C-δ (PKCδ) is a signalling kinase that regulates many cellular responses. Although most studies focus on allosteric mechanisms that activate PKCδ at membranes, PKCδ also is controlled via multi-site phosphorylation [Gong et al. (2015) Mol. Cell. Biol. 35, 1727–1740]. The present study uses MS-based methods to identify PKCδ phosphorylation at Thr50 and Ser645 (in resting and PMA-treated cardiomyocytes) as well as Thr37, Thr38, Ser130, Thr164, Thr211, Thr215, Ser218, Thr295, Ser299 and Thr656 (as sites that increase with PMA). We focused on the consequences of phosphorylation at Ser130 and Thr141 (sites just N-terminal to the pseudosubstrate domain).We show that S130D and T141E substitutions co-operate to increase PKCδ’s basal lipid-independent activity and that Ser130/Thr141 di-phosphorylation influences PKCδ’s substrate specificity. We recently reported that PKCδ preferentially phosphorylates substrates with a phosphoacceptor serine residue and that this is due to constitutive phosphorylation at Ser357, an ATP-positioning G-loop site that limits PKCδ’s threonine kinase activity [Gong et al. (2015) Mol. Cell. Biol. 35, 1727–1740]. The present study shows that S130D and T141E substitutions increase PKCδ’s threonine kinase activity indirectly by decreasing G loop phosphorylation at Ser357. A S130F substitution [that mimics a S130F single-nt polymorphism (SNP) identified in some human populations] also increases PKCδ’s maximal lipid-dependent catalytic activity and confers threonine kinase activity. Finally, we show that Ser130/Thr141 phosphorylations relieve auto-inhibitory constraints that limit PKCδ’s activity and substrate specificity in a cell-based context. Since phosphorylation sites map to similar positions relative to the pseudosubstrate domains of other PKCs, our results suggest that phosphorylation in this region of the enzyme may constitute a general mechanism to control PKC isoform activity.