Rapid activation of calmodulin-dependent protein kinase III in mitogen-stimulated human fibroblasts. Correlation with intracellular Ca2+ transients.
Rapid activation of calmodulin-dependent protein kinase III in mitogen-stimulated human fibroblasts. Correlation with intracellular Ca2+ transients.
复制标题
DOI：
10.1016/s0021-9258(18)48002-7
复制
发表时间：
1987-07
期刊：
影响因子：
--
通讯作者：
H. Palfrey;A. Nairn;L. Muldoon;M. Villereal
中科院分区：
文献类型：
--
作者：
H. Palfrey;A. Nairn;L. Muldoon;M. Villereal
生长停滞的人成纤维细胞对促有丝分裂刺激的反应是细胞质游离Ca 2+的快速、短暂增加。这一事件可能是至关重要的Na/H交换和随后的DNA合成的激活。以前的研究表明，钙调素（CaM）作为一个可能的调解人的影响，Ca 2+对这些过程。在这里，我们证明了一种特异性钙调素依赖性蛋白激酶（CaM-PK）系统在静止的成纤维细胞中被多种促分裂原刺激后迅速激活。两种人成纤维细胞类型的细胞质提取物中含有一个主要的Ca 2+刺激的磷蛋白的先生100，000和pI 6.8（先生100，000）。通过肽图谱和免疫学标准显示，该蛋白与先前在许多哺乳动物细胞和组织中鉴定的主要CaM-PK III底物相同（Palfrey，H. C. 04 The Dog of the Woman（1983）157，183-190; Nairn，A.C.，Bhagat，B.，和Palfrey，H.C.等人（1985）Proc. Acad. Sci. U.S.A.82，7939-7943）。刺激32 P标记的血清剥夺成纤维细胞与血清，个别的生长因子（缓激肽，加压素，表皮生长因子），或Ca 2+离子载体导致在一个快速的2- 10倍增加的磷酸化先生100，000通过免疫沉淀使用多克隆抗体。与血清或个人的生长因子，效果在0.5-1分钟达到峰值，然后下降到基线内5分钟。时间过程的研究表明，先生100,000的磷酸化状态密切相关，但稍微落后于Ca 2+瞬态（用Fura-2测量）。因此，在Ca 2+水平开始下降后不久，Mr 100，000的去磷酸化作用必然随之而来。血清，缓激肽，和加压素对细胞内Ca 2+和Mr 100，000的磷酸化的上升的影响是独立的外部Ca 2+，而表皮生长因子和A23187的影响需要外部Ca 2+。完整细胞中Mr 100，000的磷酸化发生在苏氨酸残基上，主要部分发生在体外标记的蛋白质中发现的相同的主要磷酸肽中。这些结果表明，人成纤维细胞的促有丝分裂活化导致Ca 2+与CaM的结合以及随后的CaM依赖性过程的活化。
Growth-arrested human fibroblasts respond to mitogenic stimulation with a rapid, transient increase in cytoplasmic free Ca2+. This event may be crucial to the activation of Na/H exchange and subsequent DNA synthesis. Previous studies have implicated calmodulin (CaM) as a possible mediator of the effects of Ca2+ on these processes. here, we demonstrate that a specific CaM-dependent protein kinase (CaM-PK) system is rapidly activated in quiescent fibroblasts stimulated by a variety of mitogens. Cytoplasmic extracts of two human fibroblast cell types contained a major Ca2+-stimulated phosphoprotein of Mr 100,000 and pI 6.8 (Mr 100,000). This protein was shown by peptide mapping and immunological criteria to be identical to the prominent CaM-PK III substrate previously identified in a number of mammalian cells and tissues (Palfrey, H. C. (1983) FEBS Lett. 157, 183-190; Nairn, A.C., Bhagat, B., and Palfrey, H.C. (1985) Proc. Natl. Acad. Sci. U.S.A. 82, 7939-7943). Stimulation of 32P-labeled serum-deprived fibroblasts with serum, individual growth factors (bradykinin, vasopressin, and epidermal growth factor), or Ca2+ ionophores resulted in a rapid 2- to 10-fold increase in the phosphorylation of Mr 100,000 as determined by immunoprecipitation using polyclonal antibodies. With serum or individual growth factors, the effect peaked at 0.5-1 min then declined back to base line within 5 min. Time course studies showed that the phosphorylation state of Mr 100,000 closely paralleled but lagged slightly behind the Ca2+ transient (measured with fura-2). Thus, dephosphorylation of Mr 100,000 must follow shortly after Ca2+ levels begin to decline. The effects of serum, bradykinin, and vasopressin on both the rise in intracellular Ca2+ and the phosphorylation of Mr 100,000 were independent of external Ca2+, whereas the effects of epidermal growth factor and A23187 required external Ca2+. Phosphorylation of Mr 100,000 in intact cells took place on threonine residues, a major portion occurring in the same major phosphopeptide found in the protein labeled in vitro. These results show that mitogenic activation of human fibroblasts leads to the binding of Ca2+ to CaM and the subsequent activation of CaM-dependent processes.