Amino acid sequence of in vivo phosphorylation sites in the main intrinsic protein (MIP) of lens membranes.
Amino acid sequence of in vivo phosphorylation sites in the main intrinsic protein (MIP) of lens membranes.
复制标题
晶状体膜主要内在蛋白（MIP）体内磷酸化位点的氨基酸序列。
DOI：
10.1111/j.1432-1033.1990.tb15650.x
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发表时间：
1990
期刊：
影响因子：
--
通讯作者：
Johnson,RG
中科院分区：
文献类型：
--
作者：
Lampe,PD;Johnson,RG
透镜纤维细胞的主要内在膜蛋白MIP先前已显示在透镜碎片的制备中被磷酸化。磷酸化发生在分子细胞质C末端附近的丝氨酸残基上。由于MIP被认为是透镜质膜中的通道蛋白，可能是细胞间通道蛋白，磷酸化可以调节这些通道的组装或门控。我们试图确定磷酸化的特定丝氨酸，以帮助确定参与调节MIP功能的激酶。为此，我们从天然膜中纯化了一个肽片段，该肽片段没有受到任何外源激酶或激酶激活剂的作用。在这些片段中检测到的任何磷酸化必须是由于细胞磷酸化，因此被称为体内磷酸化。纯化的膜也用cAMP依赖性蛋白激酶磷酸化，以确定磷酸化和非磷酸化MIP衍生肽在不同HPLC柱上的迁移率，并确定可能的cAMP依赖性蛋白激酶磷酸化位点。用赖氨酰内肽酶C消化含有50-60% MIP蛋白的透镜膜。肽从MIP的C末端区域释放，并且21-22 kDa的主要产物保持膜结合。在C8反相HPLC上分离赖氨酰内肽酶-C-释放肽表明，其中一个片段（对应于MIP中的残基239-259）被部分磷酸化。经QAE高效液相色谱法分离出磷酸化和非磷酸化两种形式的多肽，并通过乙烯利磷酸丝氨酸修饰和序列分析，在243和245位残基上发现了磷酸化位点。丝氨酸240上从未检测到磷酸化。在标准测定条件下，通过将膜与cAMP依赖性蛋白激酶孵育，可以增加丝氨酸243的磷酸化水平。在酸性氨基酸附近发现的磷酸化丝氨酸的其他激酶必须负责丝氨酸245处的体内磷酸化。
The main intrinsic membrane protein of the lens fiber cell, MIP, has been previously shown to be phosphorylated in preparations of lens fragments. Phosphorylation occurred on serine residues near the cytoplasmic C‐terminus of the molecule. Since MIP is thought to function as a channel protein in lens plasma membranes, possibly as a cell‐to‐cell channel protein, phosphorylation could regulate the assembly or gating of these channels. We sought to identify the specific serines which are phosphorylated in order to help identify the kinases involved in regulating MIP function. To this end we purified a peptide fragment from native membranes that had not been subjected to any exogenous kinases or kinase activators. Any phosphorylation detected in these fragments must be due to cellular phosphorylation and thus is termedin vivophosphorylation. Purified membranes were also phosphorylated with cAMP‐dependent protein kinase to determine the mobility of phosphorylated and unphosphorylated MIP‐derived peptides on different HPLC columns and to determine possible cAMP‐dependent protein kinase phosphorylation sites. Lens membranes, which contain 50–60% of the protein as MIP, were digested with lysylendopeptidase C. Peptides were released from the C‐terminal region of MIP and a major product of 21–22 kDa remained membrane‐associated. Separation of the lysylendopeptidase‐C‐released peptides on C8reversed‐phase HPLC demonstrated that one of these fragments, corresponding to residues 239–259 in MIP, was partially phosphorylated. The phosphorylated and nonphosphorylated forms of this peptide were separated on QAE HPLC.In vivophosphorylation sites were found at residues 243 and 245 through phosphoserine modification via ethanethiol and sequence analysis. Phosphorylation was never detected on serine 240. The phosphorylation level of serine 243 could be increased by incubation of membranes with cAMP‐dependent protein kinase under standard assay conditions. Other kinases that phosphorylate serines found near acidic amino acids must be responsible for thein vivophosphorylation demonstrated at serine 245.