MilliporeSigma
  • Cdk1 phosphorylation negatively regulates the activity of Net1 towards RhoA during mitosis.

Cdk1 phosphorylation negatively regulates the activity of Net1 towards RhoA during mitosis.

Cellular signalling (2021-01-20)
Arzu Ulu, Wonkyung Oh, Yan Zuo, Jeffrey A Frost
ABSTRACT

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.

MATERIALS
Product Number
Brand
Product Description

Sigma-Aldrich
Anti-NET1 antibody produced in rabbit, Prestige Antibodies® Powered by Atlas Antibodies, affinity isolated antibody, buffered aqueous glycerol solution
Sigma-Aldrich
Roscovitine, A potent, reversible, and selective inhibitor of Cdks that exhibits about 10-fold greater efficacy towards p34-cdk1 and p33-cdk2 and 20-fold greater efficacy towards p33-cdk5 relative to Olomoucine.
Sigma-Aldrich
Anti-α-Tubulin antibody, Mouse monoclonal, clone DM1A, purified from hybridoma cell culture