Ferrell J E, Martin G S
Department of Molecular and Cell Biology, University of California, Berkeley 94720.
Mol Cell Biol. 1990 Jun;10(6):3020-6. doi: 10.1128/mcb.10.6.3020-3026.1990.
We have surveyed fibroblast lysates for protein kinases that might be involved in mitogenesis. The assay we have used exploits the ability of blotted, sodium dodecyl sulfate-denatured proteins to regain enzymatic activity after guanidine treatment. About 20 electrophoretically distinct protein kinases could be detected by this method in lysates from NIH 3T3 cells. One of the kinases, a 42-kilodalton serine(threonine) kinase (PK42), was found to possess two- to fourfold-higher in vitro activity when isolated from serum-stimulated cells than when isolated from serum-starved cells. This kinase comigrated on sodium dodecyl sulfate-gels with a protein (p42) whose phosphotyrosine content increased in response to serum stimulation. The time courses of p42 tyrosine phosphorylation and PK42 activation were similar, reaching maximal levels within 10 min and returning to basal levels within 5 h. Both p42 tyrosine phosphorylation and PK42 activation were stimulated by low concentrations of phorbol esters, and the responses of p42 and PK42 to TPA were abolished by chronic 12-O-tetradecanoylphorbol-13-acetate (TPA) treatment. Chronic TPA treatment had less effect on serum-induced p42 tyrosine phosphorylation and PK42 activation. PK42 and p42 bound to DEAE-cellulose, and both eluted at a salt concentration of 250 mM. Thus, PK42 and p42 comigrate and cochromatograph, and the kinase activity of PK42 correlates with the tyrosine phosphorylation of p42. These findings suggest that PK42 and p42 are related or identical, that PK42 is activated by tyrosine phosphorylation, and that this tyrosine phosphorylation can be regulated by protein kinase C.
我们已对成纤维细胞裂解物进行了调查，以寻找可能参与有丝分裂原生成的蛋白激酶。我们所采用的检测方法利用了经十二烷基硫酸钠变性并印迹的蛋白质在胍处理后恢复酶活性的能力。通过这种方法，在NIH 3T3细胞的裂解物中可检测到约20种电泳性质不同的蛋白激酶。其中一种激酶，即42千道尔顿的丝氨酸（苏氨酸）激酶（PK42），从血清刺激的细胞中分离出来时，其体外活性比从血清饥饿的细胞中分离出来时高两到四倍。这种激酶在十二烷基硫酸钠凝胶上与一种蛋白质（p42）迁移情况相同，该蛋白质的磷酸酪氨酸含量会随着血清刺激而增加。p42酪氨酸磷酸化和PK42激活的时间进程相似，在10分钟内达到最高水平，并在5小时内恢复到基础水平。低浓度的佛波酯可刺激p42酪氨酸磷酸化和PK42激活，而慢性12 - O - 十四烷酰佛波醇 - 13 - 乙酸酯（TPA）处理可消除p42和PK42对TPA的反应。慢性TPA处理对血清诱导的p42酪氨酸磷酸化和PK42激活影响较小。PK42和p42与二乙氨基乙基纤维素结合，二者均在250 mM的盐浓度下洗脱。因此，PK42和p42迁移情况相同且色谱行为一致，PK42的激酶活性与p42的酪氨酸磷酸化相关。这些发现表明PK42和p42相关或相同，PK42通过酪氨酸磷酸化被激活，且这种酪氨酸磷酸化可由蛋白激酶C调节。
