Mitotic-dependent phosphorylation of leukemia-associated RhoGEF (LARG) by Cdk1.
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.
Department of Biochemistry and Molecular Biology, Thomas Jefferson University, Philadelphia, PA, USA.
Michelle C
MC
Helms
Department of Biochemistry and Molecular Biology, Thomas Jefferson University, Philadelphia, PA, USA.
Elda
E
Grabocka
Department of Biochemistry and Molecular Biology, Thomas Jefferson University, Philadelphia, PA, USA.
Matthew K
MK
Martz
Department of Biochemistry and Molecular Biology, Thomas Jefferson University, Philadelphia, PA, USA.
Christopher C
CC
Fischer
Department of Signal Dynamics, University of Tokyo, Meguro, Tokyo, Japan.
Nobuchika
N
Suzuki
Department of Biochemistry and Molecular Biology, Thomas Jefferson University, Philadelphia, PA, USA. Electronic address: philip.wedegaertner@jefferson.edu.
Philip B
PB
Wedegaertner
D008841:Actin Cytoskeleton / Q000378:metabolism; D016203:CDC2 Protein Kinase / Q000378:metabolism*; D006367:HeLa Cells; D006801:Humans; D007938:Leukemia / Q000378:metabolism*; D008938:Mitosis / Q000502:physiology*; D010766:Phosphorylation; D064067:Rho Guanine Nucleotide Exchange Factors / Q000235:genetics / Q000378:metabolism*; D020741:rho GTP-Binding Proteins / Q000378:metabolism; D020742:rhoA GTP-Binding Protein / Q000378:metabolism
C577222:ARHGEF12 protein, human; D064067:Rho Guanine Nucleotide Exchange Factors; D016203:CDC2 Protein Kinase; D020741:rho GTP-Binding Proteins; D020742:rhoA GTP-Binding Protein
Cdk1; Cytokinesis; Mitosis; Phosphorylation; RhoA; RhoGEF; Subcellular localization