흰쥐 말초 림프구의 분자량 22kDa 인산화 단백질의 특성

Abstract

Lymphocytes were found to possess all of the enzymatic machinery needed to phosphorylate and dephosphorylate proteins. At least four groups of protein kinases participate in T cell activation and interact with each other in a complex and as yet unknown manner. Increased phosphorylation in lymphocytes following stimulation with mitogens or interleukin-2, and the pivotal role of protein kinase C(PKC) in the initial biochemical reaction have been reported. But the exact role of PKC in T cell activation and the substrate of PKC are not well known. This study was attempted to clarify the characteristics of 22 KDa phosphoprotein obtained from rat peripheral blood lymphocytes(rPBL) stimulated with phorbol 12-myristate 13-acetate(PMA), using various kinase inhibitors as well as kinase activators. The lymphocytes were incubated with 32P orthophosphate before PMA stimulation. The migration pattern of the phosphorylated proteins of PMA-treated rPBL in the two dimensional electrophortic fields were analyzed after autoradiography. And the phosphorylation sites of 22 kDa protein were analyzed by high performance liquid chromatography(HPLC) and scintilation counting. The results are as follows: 1) Increased phosphorylation of 22 kDa protein was observed with PMA. 2) Forskolin, an activator of adenylyl cyclase, causes no significant change of phosphorylation of 22 kDa. 3) A 23187, a Ca" ionophore, has no noticed effect on the phosphorylation of 22 kDa protein. 4) Staurosporine, a potent PKC inhibitor, showed inhibitory action on PMA-stimulated phosphorylation of 22 kDa. 5) Calphostin C, specific PKC inhibitor inhibited the PMA-stimulated phosphorylation of 22 kDa. 6) Among trypic peptide fractions of 22 kDa by HPLC, one 32P phosphopeptide peak was observed at about 20% acetonitrile. From the above results, it could be suggested that the 22 kDa phosphoprotein of rat peripheral blood lymphocytes would be a substrate of PKC, and has one phosphorylation on serine residue.