Regulation of cyclin D1/Cdk4 complexes by calcium/calmodulin-dependent protein kinase I
Regulation of cyclin D1/Cdk4 complexes by calcium/calmodulin-dependent protein kinase I
复制标题
DOI：
10.1074/jbc.m312543200
复制
发表时间：
2004-04-09
影响因子：
4.8
通讯作者：
Means, AR
中科院分区：
文献类型：
--
作者：
Kahl, CR;Means, AR
多功能钙/钙调蛋白依赖性激酶(CaMK)的选择性抑制剂KN-93可抑制G(1)中的多种细胞类型。然而，G(1)停滞点的生化性质和KN-93在G(1)中的生理靶点仍然存在争议。在这里，我们展示了在WI-38人二倍体成纤维细胞中，KN-93可逆地将细胞停滞在G(1)晚期，然后可检测到细胞周期蛋白依赖性蛋白依赖蛋白4(CDK4)的激活。在KN-93细胞停滞点，我们发现细胞周期蛋白D1/CDK4复合体已经与p21/p27组装在一起，并聚集在细胞核中，并在Thr-172上被磷酸化，但相对无效。凝胶过滤分析对CDK4复合体的进一步检测表明，在G(1)末期，含有细胞周期蛋白D1的复合体向低分子量(M-r)组分迁移，并伴随着CDK4活性的两个峰的出现，分别位于150-200和70 kDa。KN-93可抑制CDK4的激活，这种细胞周期蛋白D1迁移的改变和细胞周期蛋白D1/CDK4的过表达克服了KN-93细胞的这种抑制作用。为了确定哪些多功能CaMK在G(1)中起作用，我们表达了不同形式的CaMKI和CaMKII。过表达的CaMKI，而不是CaMKII，阻止了CDK4的激活，模拟了KN-93的停滞点。因此，我们假设KN-93阻止了周期蛋白D/CDK4激活中一个非常晚的、未确定的步骤，该步骤涉及CaMKI，并伴随着复杂的组装、核进入和磷酸化。
The selective inhibitor of the multifunctional calcium/calmodulin-dependent kinases (CaMK), KN-93, arrests a variety of cell types in G(1). However, the biochemical nature of this G(1) arrest point and the physiological target of KN-93 in G(1) remain controversial. Here we show that in WI-38 human diploid fibroblasts KN-93 reversibly arrested cells in late G(1) prior to detectable cyclin-dependent kinase 4 (cdk4) activation. At the KN-93 arrest point, we found that cyclin D1/cdk4 complexes had assembled with p21/p27, accumulated in the nucleus, and become phosphorylated on Thr-172, yet were relatively inactive. Additional examination of cdk4 complexes by gel filtration analysis demonstrated that, in late G(1), cyclin D1-containing complexes migrated toward lower molecular weight (M-r) fractions and this altered migration was accompanied by the appearance of two peaks of cdk4 activity, at 150-200 and 70 kDa, respectively. KN-93 prevented both the activation of cdk4, and this shift in cyclin D1 migration and overexpression of cyclin D1/cdk4 overcame the KN-93 arrest. To determine which multifunctional CaMK acts in G(1), we expressed kinase-deficient forms of CaMKI and CaMKII. Overexpression of kinase-deficient CaMKI, but not CaMKII, prevented cdk4 activation, mimicking the KN-93 arrest point. Therefore, we hypothesize that KN-93 prevents a very late, uncharacterized step in cyclin D/cdk4 activation that involves CaMKI and follows complex assembly, nuclear entry, and phosphorylation.