Intracellular Na^+ Influx by Extracellular Stimuli and Signal Transduction

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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.