The Neuroepithelial transforming gene 1 (Net1) is a RhoA subfamily guanine nucleotide exchange factor that is overexpressed in a number of cancers and contributes to cancer cell motility and proliferation. Net1 also plays a Rho GTPase independent role in mitotic progression, where it promotes centrosomal activation of Aurora A and Pak2, and aids in chromosome alignment during prometaphase. To understand regulatory mechanisms controlling the mitotic function of Net1, we examined whether it was phosphorylated by the mitotic kinase Cdk1. We observed that Cdk1 phosphorylated Net1 on multiple sites in its N-terminal regulatory domain and C-terminus in vitro. By raising phospho-specific antibodies to two of these sites, we also demonstrated that both endogenous and transfected Net1 were phosphorylated by Cdk1 in cells. Substitution of the major Cdk1 phosphorylation sites with aliphatic or acidic residues inhibited the interaction of Net1 with RhoA, and treatment of metaphase cells with a Cdk1 inhibitor increased Net1 activity. Cdk1 inhibition also increased Net1 localization to the plasma membrane and stimulated cortical F-actin accumulation. Moreover, Net1 overexpression caused spindle polarity defects that were reduced in frequency by acidic substitution of the major Cdk1 phosphorylation sites. These data indicate that Cdk1 phosphorylates Net1 during mitosis and suggest that this negatively regulates its ability to signal to RhoA and alter actin cytoskeletal organization.

神经上皮转化基因 1 (Net1) 是一种 RhoA 亚科鸟嘌呤核苷酸交换因子，在许多癌症中过度表达并有助于癌细胞运动和增殖。Net1 在有丝分裂进程中也起着独立的 Rho GTPase 作用，它促进 Aurora A 和 Pak2 的中心体激活，并在前中期帮助染色体对齐。为了了解控制 Net1 有丝分裂功能的调节机制，我们检查了它是否被有丝分裂激酶 Cdk1 磷酸化。我们在体外观察到 Cdk1 在其 N 端调节域和 C 端的多个位点磷酸化 Net1. 通过提高其中两个位点的磷酸化特异性抗体，我们还证明了内源性和转染的 Net1 都被细胞中的 Cdk1 磷酸化。用脂肪族或酸性残基取代主要的 Cdk1 磷酸化位点抑制了 Net1 与 RhoA 的相互作用，并且用 Cdk1 抑制剂处理中期细胞增加了 Net1 的活性。Cdk1 抑制也增加了 Net1 对质膜的定位并刺激了皮质 F-肌动蛋白的积累。此外，Net1 过表达导致主轴极性缺陷，其频率因主要 Cdk1 磷酸化位点的酸性取代而降低。这些数据表明 Cdk1 在有丝分裂期间磷酸化 Net1，并表明这会负调控其向 RhoA 发出信号并改变肌动蛋白细胞骨架组织的能力。