Intracellular Na^+ Influx by Extracellular Stimuli and Signal Transduction

为了了解Na^+内流和细胞质碱化在各种生长因子和促肿瘤的佛波醇酯诱导下的作用，研究了离子强度和pH值对膜结合蛋白激酶活化的影响。在这些实验中，Ca^2+和磷脂依赖性蛋白激酶（蛋白激酶C）被胰蛋白酶样蛋白酶以离子强度（140-210 mM NACL）和pH（pH 7 -8）依赖性方式激活。该反应需要Ca^2+和磷脂，分子量为80，000的蛋白激酶C转化为分子量为45，000的该酶的活性片段（蛋白激酶M）。这种蛋白激酶C的蛋白水解激活也表现在再生大鼠肝部分切除后5小时。在再生肝中检测到的这种活化的激酶的性质与蛋白激酶M的性质非常相似，并且这种酶的活性用rebosomal蛋白和组蛋白检测。在多种生长因子和佛波醇酯刺激的细胞系统中，S6蛋白40 s rebosomal亚基的磷酸化是公认的。为了阐明蛋白激酶M的功能，用合成的蛋白激酶M的肽类似物测定了该酶对S6蛋白的磷酸化位点。通过对S6蛋白磷酸化肽段的分析，确定S6蛋白的主要磷酸化位点为Ser-236、Ser-240和Ser-242。这些位点对应于体内系统证实磷酸化的多个丝氨酸残基中的一些。这些证据有力地表明，蛋白激酶C在Na^+/H^+交换体刺激后水解产生的蛋白激酶M可能在细胞增殖的早期阶段起重要作用。

In an attempt to understand the role of Na^+-influx and cytoplasmic alkalinization induced by various growth factors and tumor-promoting phorbol esters, the effect of ionic strength and pH was examined on activation of membrane-bound protein kinases. During these experiments, Ca^<2+> and phospholipid-dependent protein kinase (protein kinase C) was shown to be activated by trypsin-like protease in an ionic strength (140-210 mM NACL) and pH (pH7-8)-dependent manner. This reaction required Ca^<2+> and phospholipid and protein kinase C with molecular mass of 80,000 was converted to the active fragment of this enzyme (protein kinase M) with molecular mass of 45,000. This kind of proteolytic activation of protein kinase C was also demonstrated in regenerating rat liver 5 h after partial hepatectomy. The properties of this activated kinase detected in regenerating liver was closelysimilar with those of protein kinase M and this enzyme activity was detected with rebosomal proteins and histones. It is well-established that the phosphorylation of S6 protein in 40s rebosomal subunit is observed in various cellular systems stimulated by many growth factors and phorbol esters. In order to elucidate the function of protein kinase M, the phosphorylated sites of S6 protein by this enzyme was determaned using synthetic peptide analogue of thie protein. By the analysis of the phoshporylated peptide, Ser-236, Ser-240 and Ser-242 were identified as major phosphorylated sites in S6 protein. These sites corresponded to some of the multiple serine residues whose phosphroylation were confirmed by in vivo system. These accumulated evidences strongly suggest that protein kinase M proteolytically produced from protein kinase C after stimulation of Na^+/H^+ exchanger may have an important role at the early stage of cell proliferation.