Einspahr K J, Abraham R T, Dick C J, Leibson P J
Department of Immunology, Mayo Clinic, Rochester, MN 55905.
J Immunol. 1990 Sep 1;145(5):1490-7.
Protein tyrosine kinases play fundamental roles in the transduction of signals that regulate cell growth, differentiation, and functional responses to a diversity of external stimuli. It is therefore likely that understanding protein tyrosine kinase activity in NK cells will be crucial in further defining the intracellular regulation of their unique and specialized functions. We investigated the role of protein tyrosine phosphorylation in receptor-mediated signal transduction using stimuli known to play major roles in regulating NK cell activation. Immunoblot analyses with antiphosphotyrosine antibodies demonstrated that IL-2, a potent stimulus for NK cell proliferation and an agent that enhances NK cytotoxic function, induced the tyrosine phosphorylation of at least eight proteins in clonal CD16+/CD3-human NK cells. In contrast, IL-4, which modulates NK cell function without inducing proliferation, had no apparent effect on protein tyrosine phosphorylation. Because protein kinase C (PKC) activation plays a prominent, yet distinct role in NK cell-mediated cytolytic reactions, we next investigated whether PKC activation affects NK cell protein tyrosine phosphorylation. Surprisingly, PKC-activating agents, including the phorbol esters 12-O-tetradecanoylphorbol-13-acetate and 4 beta-phorbol 12, 13-didecanoate, as well as the synthetic diacylglycerol,1-oleoyl-2-acetylglycerol, also induced the tyrosine phosphorylation of a distinct set of proteins. The 4 beta-phorbol 12,13-didecanoate homolog, 4 alpha-phorbol 12,13-didecanoate, which does not activate PKC, also failed to induce protein tyrosine phosphorylation. Further, the PKC inhibitor, 1-O-hexadecyl-2-O-methylglycerol blocked tyrosine phosphorylation induced by 1-oleoyl-2-acetylglycerol. In subsequent studies, both CD8+ and CD8- NK clones were found to express the src-family tyrosine kinase, p56lck, which was detected by immunoblot analysis with anti-p56lck antiserum. In both types of clonal NK cell lines, IL-2 and 12-O-tetradecanoyl-phorbol appeared to stimulate the differential phosphorylation of p56lck as evidenced by the appearance of higher molecular mass isoforms on SDS-polyacrylamide gels. Thus, our results identify and characterize a potential role for tyrosine phosphorylation and for the lymphocyte-specific tyrosine kinase p56lck in the signaling events that regulate NK cell activation.
蛋白酪氨酸激酶在调节细胞生长、分化以及对多种外部刺激的功能反应的信号转导中发挥着重要作用。因此，了解自然杀伤细胞（NK细胞）中的蛋白酪氨酸激酶活性对于进一步明确其独特和特殊功能的细胞内调节可能至关重要。我们使用已知在调节NK细胞活化中起主要作用的刺激物，研究了蛋白酪氨酸磷酸化在受体介导的信号转导中的作用。用抗磷酸酪氨酸抗体进行的免疫印迹分析表明，白细胞介素-2（IL-2）是NK细胞增殖的有效刺激物，也是增强NK细胞毒性功能的因子，它能诱导克隆的CD16+/CD3-人NK细胞中至少8种蛋白的酪氨酸磷酸化。相比之下，调节NK细胞功能但不诱导增殖的IL-4对蛋白酪氨酸磷酸化没有明显影响。由于蛋白激酶C（PKC）激活在NK细胞介导的溶细胞反应中起显著但不同的作用，我们接下来研究PKC激活是否影响NK细胞蛋白酪氨酸磷酸化。令人惊讶的是，PKC激活剂，包括佛波酯12-O-十四酰佛波醇-13-乙酸酯和4β-佛波醇12,13-十二烷酸酯，以及合成二酰甘油1-油酰-2-乙酰甘油，也能诱导一组不同蛋白的酪氨酸磷酸化。不激活PKC的4β-佛波醇12,13-十二烷酸酯同系物4α-佛波醇12,13-十二烷酸酯也不能诱导蛋白酪氨酸磷酸化。此外，PKC抑制剂1-O-十六烷基-2-O-甲基甘油可阻断1-油酰-2-乙酰甘油诱导的酪氨酸磷酸化。在随后的研究中，发现CD8+和CD8-NK克隆均表达src家族酪氨酸激酶p56lck，用抗p56lck抗血清进行免疫印迹分析可检测到。在这两种类型的克隆NK细胞系中，IL-2和12-O-十四酰佛波醇似乎能刺激p56lck的差异磷酸化，SDS-聚丙烯酰胺凝胶上出现的高分子量异构体证明了这一点。因此，我们的结果确定并描述了酪氨酸磷酸化以及淋巴细胞特异性酪氨酸激酶p56lck在调节NK细胞活化的信号事件中的潜在作用。
