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 signaling functions beyond mitosis, it is important to understand the regulation of the
protein through modifications such as phosphorylation. 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 phosphonull mutants that contain non-phosphorylatable
alanine mutations at potential 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.