Pietromonaco S F, Simons P C, Altman A, Elias L
Department of Internal Medicine, University of New Mexico School of Medicine, Albuquerque, New Mexico 87131, USA.
J Biol Chem. 1998 Mar 27;273(13):7594-603. doi: 10.1074/jbc.273.13.7594.
Moesin, a member of the ezrin-radixin-moesin (ERM) family of membrane/cytoskeletal linkage proteins, is known to be threonine-phosphorylated at Thr558 in activated platelets within its conserved putative actin-binding domain. The pathway leading to this phosphorylation step and its control have not been previously elucidated. We have detected and characterized reactions leading to moesin phosphorylation in human leukocyte extracts. In vitro phosphorylation of endogenous moesin, which was identified by peptide microsequencing, was dependent on phosphatidylglycerol (PG) or to a lesser extent, phosphatidylinositol (PI), but not phosphatidylserine (PS) and diacylglycerol (DAG). Analysis of charge shifts, phosphoamino acid analysis, and stoichiometry was consistent with a single phosphorylation site. By using mass spectroscopy and direct microsequencing of CNBr fragments of phospho-moesin, the phosphorylation site was identified as KYKT*LRQIR (where * indicates the phosphorylation site) (Thr558), which is conserved in the ERM family. Recombinant moesin demonstrated similar in vitro phospholipid-dependent phosphorylation compared with the endogenous protein. The phosphorylation site sequence of moesin displays a high degree of conservation with the pseudosubstrate sequences of the protein kinase C (PKC) family. We identified the kinase activity as PKC-theta on the basis of immunodepletion of the moesin kinase activity and copurification of PKC-theta with the enzymic activity. We further demonstrate that PKC-theta displays a preference for PG vesicles over PI or PS/DAG, with minimal activation by DAG, as well as specificity for moesin compared with myelin basic protein, histone H1, or other cellular proteins. Expression of a human His6-tagged PKC-theta in Jurkat cells and purification by Ni2+ chelate chromatography yield an active enzyme that phosphorylates moesin. PG vesicle binding experiments with expressed PKC-theta and moesin demonstrate that both bind to vesicles independently of one another. Thus, PKC-theta is identified as a major kinase within cells with specificity for moesin and with activation under non-classical PKC conditions. It appears likely that this activity corresponds to a specific intracellular pathway controlling the function of moesin as well as other ERM proteins.
埃兹蛋白（Ezrin）-根蛋白（Radixin）-膜突蛋白（Moesin）（ERM）家族是一类膜/细胞骨架连接蛋白，其中的膜突蛋白已知在其保守的假定肌动蛋白结合域内，于活化血小板的苏氨酸558位点发生磷酸化。此前尚未阐明导致这一磷酸化步骤的途径及其调控机制。我们在人白细胞提取物中检测并鉴定了导致膜突蛋白磷酸化的反应。通过肽微测序鉴定的内源性膜突蛋白的体外磷酸化依赖于磷脂酰甘油（PG），在较小程度上也依赖于磷脂酰肌醇（PI），但不依赖于磷脂酰丝氨酸（PS）和二酰基甘油（DAG）。电荷迁移分析、磷酸氨基酸分析和化学计量分析结果与单一磷酸化位点相符。通过对磷酸化膜突蛋白的溴化氰片段进行质谱分析和直接微测序，确定磷酸化位点为KYKTLRQIR（其中表示磷酸化位点）（苏氨酸558），该位点在ERM家族中保守。与内源性蛋白相比，重组膜突蛋白在体外表现出类似的磷脂依赖性磷酸化。膜突蛋白的磷酸化位点序列与蛋白激酶C（PKC）家族的假底物序列高度保守。基于膜突蛋白激酶活性的免疫去除以及PKC-θ与酶活性的共纯化，我们确定该激酶活性为PKC-θ。我们进一步证明，与PI或PS/DAG相比，PKC-θ对PG囊泡具有偏好性，对DAG的激活作用最小，并且与髓鞘碱性蛋白、组蛋白H1或其他细胞蛋白相比，对膜突蛋白具有特异性。在Jurkat细胞中表达人His6标记的PKC-θ并通过Ni2+螯合层析纯化，得到一种可使膜突蛋白磷酸化的活性酶。用表达的PKC-θ和膜突蛋白进行PG囊泡结合实验表明，两者彼此独立地与囊泡结合。因此，PKC-θ被确定为细胞内一种主要的激酶，对膜突蛋白具有特异性，且在非经典PKC条件下被激活。这种活性很可能对应于一条控制膜突蛋白以及其他ERM蛋白功能的特定细胞内途径。
