Identification of in vivo phosphorylation sites required for protein kinase D activation
Identification of in vivo phosphorylation sites required for protein kinase D activation
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DOI：
10.1074/jbc.273.42.27662
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发表时间：
1998-10-16
影响因子：
4.8
通讯作者：
Rozengurt, E
中科院分区：
文献类型：
--
作者：
Iglesias, T;Waldron, RT;Rozengurt, E
蛋白激酶D （PKD）在完整细胞中被磷酸酯、细胞渗透二酰基甘油、苔藓虫抑制素、神经肽和生长因子刺激而磷酸化激活，但PKD的关键激活残基尚未确定。通过自磷酸化和外源性合成肽-2肽底物磷酸化实验，研究人员发现，用丙氨酸（PKD- s744a /S748A）取代Ser（744）和Ser（748）完全阻断了phorbal -12,13-dibutyrate （PDB）处理完整细胞诱导的PKD激活。相反，用谷氨酸替代两个丝氨酸残基（PKD- s744e /S748E）显著增加了基础活性（与野生型PKD相比增加了7.5倍），PKD- s744e /S748E突变体在体内仅受到PDB处理的轻微进一步刺激，这表明这两个位点的磷酸化诱导了最大的PKD激活。从p -32标记的转染的COS-7细胞免疫沉淀的PKD突变体中获得的二维胰蛋白酶肽分析显示，在激酶缺乏的三突变体PKD- d733a /S744E/S748E中，pdb刺激的野生型PKD或激酶死亡的PKD- d733a磷酸肽图中存在的两个主要斑点完全消失。我们的研究结果表明，PKD是由Ser（744）和Ser（748）的磷酸化激活的，因此提供了第一个非ph激酶通过激活环中丝氨酸/苏氨酸残基的磷酸化而上调的例子。
Protein kinase D (PKD) is activated by phosphorylation in intact cells stimulated by phorbol esters, cell permeant diacylglycerols, bryostatin, neuropeptides, and growth factors, but the critical activating residues in PKD have not been identified. Here, we show that substitution of Ser(744) and Ser(748) with alanine (PKD-S744A/S748A) completely blocked PKD activation induced by phorbol-12,13-dibutyrate (PDB) treatment of intact cells as assessed by autophosphorylation and exogenous syntide-2 peptide substrate phosphorylation assays. Conversely, replacement of both serine residues with glutamic acid (PKD-S744E/S748E) markedly increased basal activity (7.5-fold increase compared with wild type PKD), PKD-S744E/S748E mutant was only slightly further stimulated by PDB treatment in vivo, suggesting that phosphorylation of these two sites induces maximal PKD activation. Two-dimensional tryptic phosphopeptide analysis obtained from PKD mutants immunoprecipitated from P-32-labeled transfected COS-7 cells showed that two major spots present in the PDB-stimulated wild type PKD or the kinase-dead PKD-D733A phosphopeptide maps completely disappeared in the kinase-deficient triple mutant PKD-D733A/S744E/S748E. Our results indicate that PKD is activated by phosphorylation of residues Ser(744) and Ser(748) and thus provide the first example of a non-PH kinase that is up-regulated by phosphorylation of serine/threonine residues within the activation loop.