Regulatory autophosphorylation sites on protein kinase C-delta at threonine-141 and threonine-295.
Regulatory autophosphorylation sites on protein kinase C-delta at threonine-141 and threonine-295.
复制标题
蛋白激酶 C-δ 苏氨酸 141 和苏氨酸 295 处的调节性自磷酸化位点。
DOI：
10.1021/bi802171c
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发表时间：
2009
期刊：
影响因子：
2.9
通讯作者：
Steinberg,SusanF
中科院分区：
文献类型：
--
作者：
Rybin,VitalyiO;Guo,Jianfen;Harleton,Erin;Feinmark,StevenJ;Steinberg,SusanF
蛋白激酶C-δ (PKCδ)是一种丝氨酸/苏氨酸激酶，调节广泛的细胞反应。本研究鉴定了新蛋白体外pkc δ自磷酸化位点位于假底物结构域附近的thr141位点，C1A-C1B结构域间的thr218位点，铰链区Ser295、Ser302和ser304位点，以及激活环中thr505附近的ser503位点。基于细胞的研究表明，thr141和thr295在体内也被磷酸化，并且thr141的磷酸化调节了COS7细胞中PKCδ下调的动力学。体外研究提示thr141和thr295自磷酸化是调节PKCδ活性的修饰。T141D取代显著增加基础脂质非依赖性PKCδ活性；PKCδ- t141d突变体在PMA体外处理中仅受到轻微的进一步刺激，这表明thr141磷酸化解除了限制PKCδ活性的自抑制约束。诱变研究也表明thr295位点的磷酸化有助于控制PKCδ底物特异性。我们之前证明，当PKCδ被脂质辅助因子变构激活时，它在Ser23/ ser24位点磷酸化心肌肌钙蛋白I (cTnI)，并且PKCδ的Thr505/ tyr311磷酸化形式（存在于Src试验中）获得了对cTnI- thr144的额外活性。本文报道的研究表明，T505A取代可减少pkc δ- thr295自磷酸化，T295A取代可导致src依赖性pkc δ- tyr311磷酸化和pkc δ依赖性ctni - thr144磷酸化缺陷。这些结果表明PKCδ- thr295自磷酸化是一种脂质依赖性修饰，将PKCδ- thr505磷酸化与PKCδ催化功能的src依赖性调节联系起来。总的来说，这些研究确定了PKCδ上新的调节自磷酸化，作为PKCδ活性的标记和调节因子。
Protein kinase C-δ (PKCδ) is a Ser/Thr kinase that regulates a wide range of cellular responses. This study identifies novelin vitroPKCδ autophosphorylation sites at Thr141adjacent to the pseudosubstrate domain, Thr218in the C1A-C1B interdomain, Ser295, Ser302, and Ser304in the hinge region, and Ser503adjacent to Thr505in the activation loop. Cell-based studies show that Thr141and Thr295also are phosphorylatedin vivoand that Thr141phosphorylation regulates the kinetics of PKCδ downregulation in COS7 cells.In vitrostudies implicate Thr141and Thr295autophosphorylation as modifications that regulate PKCδ activity. A T141D substitution markedly increases basal lipid-independent PKCδ activity; the PKCδ-T141D mutant is only slightly further stimulatedin vitroby PMA treatment, suggesting that Thr141phosphorylation relieves autoinhibitory constraints that limit PKCδ activity. Mutagenesis studies also indicate that a phosphorylation at Thr295contributes to the control of PKCδ substrate specificity. We previously demonstrated that PKCδ phosphorylates the myofilament protein cardiac troponin I (cTnI) at Ser23/Ser24when it is allosterically activated by lipid cofactors and that the Thr505/Tyr311-phosphorylated form of PKCδ (that is present in assays with Src) acquires as additional activity toward cTnI-Thr144. Studies reported herein show that a T505A substitution reduces PKCδ-Thr295autophosphorylation and that a T295A substitution leads to a defect in Src-dependent PKCδ-Tyr311phosphorylation and PKCδ-dependent cTnI-Thr144phosphorylation. These results implicate PKCδ-Thr295autophosphorylation as a lipid-dependent modification that links PKCδ-Thr505phosphorylation to Src-dependent regulation of PKCδ catalytic function. Collectively, these studies identify novel regulatory autophosphorylations on PKCδ that serve as markers and regulators of PKCδ activity.