Mogi A, Hatai M, Soga H, Takenoshita S, Nagamachi Y, Fujimoto J, Yamamoto T, Yokota J, Yaoi Y
Biology Division, National Cancer Center Research Institute, Tokyo, Japan.
FEBS Lett. 1995 Oct 9;373(2):135-40. doi: 10.1016/0014-5793(95)01014-6.
Effects of various types of protein kinase inhibitor on the adhesion and spreading of BALB/c mouse 3T3 cells and on the phosphorylation and stability of focal adhesion kinase (FAK) in the cells were studied. Inhibitors of protein tyrosine kinases, methyl 2,5-dihydroxycinnamate and herbimycin A, inhibited tyrosine-phosphorylation of FAK and the adhesion of 3T3 cells to fibronectin. Among inhibitors of serine/threonine kinases tested, calphostin C, a specific inhibitor of protein kinase C, inhibited cell spreading rather than cell adhesion, and it induced the decrease of intracellular FAK within 30 min. Inhibitors of tyrosine kinase, A kinase, G kinase, and myosin light chain kinase did not induce such a rapid and specific decrease of FAK. When calphostin C (20 microM) was added to sub-confluent monolayer cultures, serine-phosphorylation of FAK was inhibited by 67% within 2 h, and decrease in the amount of FAK and rounding up of the cells began after 4 h. Label-chase experiments indicated that about 60% of 35S-labeled FAK degraded within 1-2 h after addition of calphostin C to monolayer cultures. These results indicated that serine-phosphorylation of FAK induced by protein kinase C was important in the regulation of metabolic stability of FAK.
研究了各种类型的蛋白激酶抑制剂对BALB/c小鼠3T3细胞黏附与铺展以及对细胞中黏着斑激酶（FAK）磷酸化和稳定性的影响。蛋白酪氨酸激酶抑制剂2,5-二羟基肉桂酸甲酯和除莠霉素A抑制了FAK的酪氨酸磷酸化以及3T3细胞与纤连蛋白的黏附。在所测试的丝氨酸/苏氨酸激酶抑制剂中，蛋白激酶C的特异性抑制剂钙泊三醇C抑制细胞铺展而非细胞黏附，并且在30分钟内诱导细胞内FAK减少。酪氨酸激酶、A激酶、G激酶和肌球蛋白轻链激酶的抑制剂未诱导FAK如此快速且特异性的减少。当将钙泊三醇C（20微摩尔）添加到亚汇合单层培养物中时，2小时内FAK的丝氨酸磷酸化被抑制67%，4小时后FAK量减少且细胞变圆。标记追踪实验表明，在向单层培养物中添加钙泊三醇C后1 - 2小时内，约60%的35S标记的FAK降解。这些结果表明，蛋白激酶C诱导的FAK丝氨酸磷酸化在FAK代谢稳定性的调节中起重要作用。
