Phosphorylation of connexin 32, a hepatocyte gap-junction protein, by cAMP-dependent protein kinase, protein kinase C and Ca2+/calmodulin-dependent protein kinase II.
Phosphorylation of connexin 32, a hepatocyte gap-junction protein, by cAMP-dependent protein kinase, protein kinase C and Ca2+/calmodulin-dependent protein kinase II.
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DOI：
10.1111/j.1432-1033.1990.tb19223.x
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发表时间：
1990-09
期刊：
影响因子：
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通讯作者：
Juan C. Sáez;A. Nairn;A. Czernik;D. Spray;Elliot L. Hertzberg;Paul Greengard;Michael V. L. Bennett
中科院分区：
文献类型：
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作者：
Juan C. Sáez;A. Nairn;A. Czernik;D. Spray;Elliot L. Hertzberg;Paul Greengard;Michael V. L. Bennett
在纯化的肝间隙连接和肝细胞中研究了主要的肝间隙连接蛋白连接蛋白32的磷酸化。在分离的缝隙连接中，连接蛋白32被cAMP依赖性蛋白激酶（cAMP-PK）、蛋白激酶C（PKC）和Ca 2 +/钙调蛋白依赖性蛋白激酶II（Ca 2 +/CaM-PK II）磷酸化。连接蛋白26不被这三种蛋白激酶磷酸化。连接蛋白32的磷酸肽图谱表明，cAMP-PK和PKC主要磷酸化称为肽1的肽中的丝氨酰残基。PKC还磷酸化其他肽中的丝氨酰残基。CA 2 +/CaM-PK II磷酸化丝氨酸，并在较小程度上，苏氨酸，在不同的网站由其他两种蛋白激酶磷酸化。合成肽PSRKGSGFGHRL-胺（残基228-239的基础上推导的氨基酸序列的大鼠连接蛋白32）被磷酸化的cAMP-PK和PKC，与动力学性质是类似的其他生理底物磷酸化这些酶。Ca ~（2+）/CaM-PK Ⅱ不使肽磷酸化。磷酸化合成肽的磷酸肽图谱和氨基酸序列分析表明，连接蛋白32的Ser 233存在于肽1中，并被cAMP PK或PKC磷酸化。在肝细胞标记的[32 P]正磷酸，治疗与毛喉素或20-脱氧-20-氧代-12，13-二丁酸（PDBt）导致增加32 P-掺入连接蛋白32。磷酸肽映射和磷酸氨基酸分析表明，在肽1中的丝氨酰残基是最显着的基础条件下磷酸化。用毛喉素或PDBt处理刺激肽1的磷酸化。PDBt处理也增加了其他几种肽中丝氨酰残基的磷酸化。PDBt对肝细胞cAMP PK活性无影响。先前已经表明佛波酯在几种细胞类型中减少染料偶联，然而在大鼠肝细胞中，用PDBt处理不会减少染料偶联。因此，PKC的激活可能对不同细胞类型的连接通透性产生不同的影响;这种变异性的一个来源可能是不同间隙连接蛋白磷酸化位点的差异。
Phosphorylation of connexin 32, the major liver gap-junction protein, was studied in purified liver gap junctions and in hepatocytes. In isolated gap junctions, connexin 32 was phosphorylated by cAMP-dependent protein kinase (cAMP-PK), by protein kinase C (PKC) and by Ca2+/calmodulin-dependent protein kinase II (Ca2+/CaM-PK II). Connexin 26 was not phosphorylated by these three protein kinases. Phosphopeptide mapping of connexin 32 demonstrated that cAMP-PK and PKC primarily phosphorylated a seryl residue in a peptide termed peptide 1. PKC also phosphorylated seryl residues in additional peptides. CA2+/CaM-PK II phosphorylated serine and to a lesser extent, threonine, at sites different from those phosphorylated by the other two protein kinases. A synthetic peptide PSRKGSGFGHRL-amine (residues 228-239 based on the deduced amino acid sequence of rat connexin 32) was phosphorylated by cAMP-PK and by PKC, with kinetic properties being similar to those for other physiological substrates phosphorylated by these enzymes. Ca2+/CaM-PK II did not phosphorylate the peptide. Phosphopeptide mapping and amino acid sequencing of the phosphorylated synthetic peptide indicated that Ser233 of connexin 32 was present in peptide 1 and was phosphorylated by cAMP-PK or by PKC. In hepatocytes labeled with [32P]orthophosphoric acid, treatment with forskolin or 20-deoxy-20-oxophorbol 12,13-dibutyrate (PDBt) resulted in increased 32P-incorporation into connexin 32. Phosphopeptide mapping and phosphoamino acid analysis showed that a seryl residue in peptide 1 was most prominently phosphorylated under basal conditions. Treatment with forskolin or PDBt stimulated the phosphorylation of peptide 1. PDBt treatment also increased the phosphorylation of seryl residues in several other peptides. PDBt did not affect the cAMP-PK activity in hepatocytes. It has previously been shown that phorbol ester reduces dye coupling in several cell types, however in rat hepatocytes, dye coupling was not reduced by treatment with PDBt. Thus, activation of PKC may have differential effects on junctional permeability in different cell types; one source of this variability may be differences in the sites of phosphorylation in different gap-junction proteins.