Mitotic-dependent phosphorylation of leukemia-associated RhoGEF (LARG) by Cdk1.
Mitotic-dependent phosphorylation of leukemia-associated RhoGEF (LARG) by Cdk1.
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DOI：
10.1016/j.cellsig.2015.10.004
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发表时间：
2016-01
影响因子：
4.8
通讯作者：
Wedegaertner PB
中科院分区：
文献类型：
--
作者：
Helms MC;Grabocka E;Martz MK;Fischer CC;Suzuki N;Wedegaertner PB
Rho GTP酶是肌动蛋白细胞骨架依赖的调控过程中不可或缺的一部分，包括有丝分裂。Rho和白血病相关的Rho鸟嘌呤核苷酸交换因子(LARG)，也被称为ARHGEF12，参与有丝分裂以及癌症和心脏病等疾病。由于LARG在有丝分裂和有丝分裂以外的多种信号功能中都有作用，因此了解通过磷酸化等修饰对蛋白质的调节是很重要的。我们的研究提供了关于G蛋白信号转导调节因子-Rhogef LARG的有丝分裂磷调节的进一步信息。在这里，我们报告了LARG经历了有丝分裂依赖和细胞周期蛋白依赖的激酶1(CDK1)抑制物敏感的磷酸化。此外，LARG在有丝分裂开始时被磷酸化，当细胞退出有丝分裂时去磷酸化，伴随着CDK1的活性。此外，使用体外激酶实验，我们发现LARG可以被CDK1直接磷酸化。通过表达N-端和C-端缺失以及在CDK1 S/TP位点上含有非磷酸化丙氨酸突变的磷酸突变体，我们证明了LARG在这两个末端都发生了磷酸化。使用磷酸特异性抗体，我们证实了丝氨酸190和丝氨酸1176这两个位点在有丝分裂过程中以依赖于CDK1的方式被磷酸化。此外，这些磷酸特异性抗体在特定的有丝分裂位置，即有丝分裂组织中心和中体两侧显示磷酸化的LARG。最后，RhoA活性分析表明，在细胞中，磷酸化LARG比仿磷化LARG更活跃。因此，我们的数据确定LARG在有丝分裂过程中是一种磷酸调节的Rhogef。
Rho GTPases are integral to the regulation of actin cytoskeleton-dependent processes, including mitosis. Rho and leukemia-associated Rho guanine-nucleotide exchange factor (LARG), also known as ARHGEF12, are involved in mitosis as well as diseases such as cancer and heart disease. Since LARG has a role in mitosis and diverse signalling functions beyond mitosis, it is important to understand the regulation of the protein through modifications such as phosphorylation. Our research provides further information about the mitotic phosphoregulation of the regulator of G protein signaling (RGS)-RhoGEF LARG. Here we report that LARG undergoes a mitotic-dependent and cyclin-dependent kinase 1 (Cdk1) inhibitor-sensitive phosphorylation. Additionally, LARG is phosphorylated at the onset of mitosis and dephosphorylated as cells exit mitosis, concomitant with Cdk1 activity. Furthermore, using an in vitro kinase assay, we show that LARG can be directly phosphorylated by Cdk1. Through expression of N- and C-terminal deletion and phosphonull mutants that contain non-phosphorylatable alanine mutations at Cdk1 S/TP sites, we demonstrate that LARG phosphorylation occurs in both termini. Using phosphospecific antibodies, we confirm that two sites, serine 190 and serine 1176, are phosphorylated during mitosis in a Cdk1-dependent manner. In addition, these phosphospecific antibodies show phosphorylated LARG at specific mitotic locations, namely the mitotic organizing centers and flanking the midbody. Lastly, RhoA activity assays reveal that phosphonull LARG is more active in cells than phosphomimetic LARG. Our data thus identifies LARG as a phosphoregulated RhoGEF during mitosis.