Isolation and characterization of protein kinase C from Y-1 adrenal cell cytoskeleton.
Isolation and characterization of protein kinase C from Y-1 adrenal cell cytoskeleton.
复制标题
DOI：
10.1083/jcb.108.2.553
复制
发表时间：
1989-02
期刊：
影响因子：
--
通讯作者：
Hall PF
中科院分区：
文献类型：
--
作者：
Papadopoulos V;Hall PF
Y-1小鼠肾上腺肿瘤细胞的细胞骨架含有钙和磷脂依赖性蛋白激酶（蛋白激酶C），其结合足够紧密，可抵抗0.5% Triton的提取，但不能抵抗1.0% Triton的提取。该酶已从细胞骨架和胞质溶胶中纯化至接近均一。它显示了这种类型的激酶的典型特征，即需要Ca 2+和磷脂，由肿瘤促进剂刺激，但不受非肿瘤促进佛波酯的刺激，以及由三氟拉嗪抑制。该酶对细胞骨架中发现的四种底物具有特异性，即80、33、20和18 kD。前三种底物被酶磷酸化;第四种底物被脱磷酸化，因此间接受激酶影响。80-kD蛋白是激酶本身，其在体外和细胞骨架中自磷酸化。20-kD蛋白是肌球蛋白轻链。33-和18-kD蛋白是未鉴定的。当Y-1细胞用毛地黄皂苷透化并与[γ-32 P]ATP和佛波醇-12-肉豆蔻酸酯-13-乙酸酯孵育时，相同的底物被磷酸化。当将部分纯化的蛋白激酶C加入到先前提取的细胞骨架中以除去内源性蛋白激酶C时，改变了相同底物的磷酸化程度。加入Ca 2+、磷脂酰丝氨酸和佛波醇-12-肉豆蔻酸酯-13-乙酸酯到细胞骨架中，以及加入这三种试剂加上蛋白激酶C到提取的细胞骨架中，导致这些结构经历快速和广泛的圆化。通过添加佛波酯在完整细胞中诱导类似的变化。它的结论是蛋白激酶C是能够改变肾上腺细胞的形状的行动，涉及自磷酸化和磷酸化的肌球蛋白轻链。这种反应可能反过来与ACTH和环AMP的类固醇生成反应有关。
The cytoskeletons of Y-1 mouse adrenal tumor cells contain a calcium and phospholipid-dependent protein kinase (protein kinase C) that is bound sufficiently tight to resist extraction by 0.5% Triton but not by 1.0% Triton. The enzyme has been purified to near homogeneity from cytoskeleton and cytosol. It shows features typical of this type of kinase, namely a requirement for Ca2+ and phospholipid, stimulation by tumor promoters but not by nontumor-promoting phorbol esters, and inhibition by trifluoperazine. The enzyme shows specificity for four substrates found in the cytoskeleton, namely 80, 33, 20, and 18 kD. The first three substrates are phosphorylated by the enzyme; the fourth is dephosphorylated and is therefore affected by the kinase indirectly. The 80-kD protein is the kinase enzyme itself which is autophosphorylated in vitro and in the cytoskeleton. The 20-kD protein is myosin light chain. The 33- and 18-kD proteins are unidentified. The same substrates were phosphorylated when Y-1 cells were permeabilized with digitonin and incubated with [gamma-32P]ATP and phorbol-12- myristate-13-acetate. Partly purified protein kinase C changes the extent of phosphorylation of the same substrates when added to cytoskeletons previously extracted to remove endogenous protein kinase C. Addition of Ca2+, phosphatidylserine, and phorbol-12-myristate-13- acetate to cytoskeletons, and addition of these three agents plus protein kinase C to extracted cytoskeletons, causes these structures to undergo a rapid and extensive rounding. A similar change is induced in intact cells by addition of phorbol ester. It is concluded that protein kinase C is capable of changing the shape of adrenal cells by an action that involves autophosphorylation and phosphorylation of myosin light chain. This response may in turn be related to the steroidogenic responses to ACTH and cyclic AMP.