Cdk1‐dependent phosphorylation of leukemia‐associated RhoGEF during mitosis (802.30)

Abstract

Rho GTPases are crucial players in the regulation of actin cytoskeleton‐dependent processes such as mitosis. Our laboratory has discovered a novel role and localization of the leukemia‐associated Rho guanine‐nucleotide exchange factor (LARG), a regulator of G protein signaling (RGS)‐RhoGEF, during mitosis. Rho and LARG have been implicated in diseases such as cancer and heart disease, thus highlighting the importance for further research. The purpose of this study is to determine the molecular mechanism and role of mitotic‐dependent phosphorylation of LARG. Using mitotic‐shift assays and kinase inhibitors, we report that LARG undergoes a mitotic‐dependent and Cyclin‐dependent kinase 1 (Cdk1) inhibitor‐sensitive phosphorylation. Cell synchronization followed by Western blot also identified the temporal feasibility of Cdk1 as a potential kinase. Furthermore, using in vitro kinase assays, we show that LARG can be directly phosphorylated by Cdk1. Using both N‐ and C‐terminal deletion and multi‐site phosphorylation mutants, we demonstrate that the mitotic‐dependent shift of LARG relies on phosphorylation occurring in both termini. Using custom phosphospecific antibodies, we confirm that two sites that are phosphorylated during mitosis and also in a Cdk1‐dependent manner. We conclude that Cdk1 phosphorylates LARG during mitosis, and phosphorylation of LARG may play a role in LARG activity and function.Grant Funding Source: Supported by NIH GM062884