Keranen L M, Dutil E M, Newton A C
Department of Pharmacology, University of California at San Diego, La Jolla 92093-0640, USA.
Curr Biol. 1995 Dec 1;5(12):1394-1403. doi: 10.1016/s0960-9822(95)00277-6.
Protein kinase Cs are a family of enzymes that transduce the plethora of signals promoting lipid hydrolysis. Here, we show that protein kinase C must first be processed by three distinct phosphorylations before it is competent to respond to second messengers.
We have identified the positions and functions of the in vivo phosphorylation sites of protein kinase C by mass spectrometry and peptide sequencing of native and phosphatase-treated kinase from the detergent-soluble fraction of cells. Specifically, the threonine at position 500 (T500) on the activation loop, and T641 and S660 on the carboxyl terminus of protein kinase C beta II are phosphorylated in vivo. T500 and S660 are selectively dephosphorylated in vitro by protein phosphatase 2A to yield an enzyme that is still capable of lipid-dependent activation, whereas all three residues are dephosphorylated by protein phosphatase 1 to yield an inactive enzyme. Biochemical analysis reveals that protein kinase C autophosphorylates on S660, that autophosphorylation on S660 follows T641 autophosphorylation, that autophosphorylation on S660 is accompanied by the release of protein kinase C into the cytosol, and that T500 is not an autophosphorylation site.
Structural and biochemical analyses of native and phosphatase-treated protein kinase C indicate that protein kinase C is processed by three phosphorylations. Firstly, trans-phosphorylation on the activation loop (T500) renders it catalytically competent to autophosphorylate. Secondly, a subsequent autophosphorylation on the carboxyl terminus (T641) maintains catalytic competence. Thirdly, a second autophosphorylation on the carboxyl terminus (S660) regulates the enzyme's subcellular localization. The conservation of each of these residues (or an acidic residue) in conventional, novel and atypical protein kinase Cs underscores the essential role for each in regulating the protein kinase C family.
蛋白激酶C是一类能转导大量促进脂质水解信号的酶。在此，我们表明蛋白激酶C在能够响应第二信使之前，必须先经过三种不同的磷酸化修饰。
我们通过质谱分析以及对细胞去污剂可溶部分中天然和经磷酸酶处理的激酶进行肽测序，确定了蛋白激酶C体内磷酸化位点的位置和功能。具体而言，蛋白激酶CβII激活环上第500位的苏氨酸（T500）以及羧基末端的T641和S660在体内被磷酸化。T500和S660在体外被蛋白磷酸酶2A选择性去磷酸化，产生的酶仍具有脂质依赖性激活能力，而所有三个残基被蛋白磷酸酶1去磷酸化后产生无活性的酶。生化分析表明，蛋白激酶C在S660上进行自身磷酸化，S660上的自身磷酸化发生在T641自身磷酸化之后，S660上的自身磷酸化伴随着蛋白激酶C释放到细胞质中，且T500不是自身磷酸化位点。
对天然和经磷酸酶处理的蛋白激酶C的结构和生化分析表明，蛋白激酶C经过三种磷酸化修饰。首先，激活环上的反式磷酸化（T500）使其具有催化自身磷酸化的能力。其次，随后羧基末端的自身磷酸化（T641）维持催化活性。第三，羧基末端的第二次自身磷酸化（S660）调节酶的亚细胞定位。在传统型、新型和非典型蛋白激酶C中，这些残基（或酸性残基）中的每一个的保守性强调了它们在调节蛋白激酶C家族中的重要作用。
