C-terminal domain phosphorylation of ERK3 controlled by Cdk1 and Cdc14 regulates its stability in mitosis.
ERK3 (extracellular-signal-regulated kinase 3) is an atypical MAPK (mitogen-activated protein kinase) that is suggested to play a role in cell-cycle progression and cellular differentiation. However, it is not known whether the function of ERK3 is regulated during the cell cycle. In the present paper, we report that ERK3 is stoichiometrically hyperphosphorylated during entry into mitosis and is dephosphorylated at the M-->G1 transition. The phosphorylation of ERK3 is associated with the accumulation of the protein in mitosis. In vitro phosphorylation of a series of ERK3-deletion mutants by mitotic cell extracts revealed that phosphorylation is confined to the unique C-terminal extension of the protein. Using MS analysis, we identified four novel phosphorylation sites, Ser684, Ser688, Thr698 and Ser705, located at the extreme C-terminus of ERK3. All four sites are followed by a proline residue. We have shown that purified cyclin B-Cdk1 (cyclindependent kinase 1) phosphorylates these sites in vitro and demonstrate that Cdk1 acts as a major Thr698 kinase in vivo. Reciprocally, we found that the phosphatases Cdc14A and Cdc14B (Cdc is cell-division cycle) bind to ERK3 and reverse its C-terminal phosphorylation in mitosis. Importantly, alanine substitution of the four C-terminal phosphorylation sites markedly decreased the half-life of ERK3 in mitosis, thereby linking phosphorylation to the stabilization of the kinase. The results of the present study identify a novel regulatory mechanism of ERK3 that operates in a cell-cycle-dependent manner.
Institut de Recherche en Immunologie et Cancérologie, Université de Montréal, Montreal, Quebec, Canada H3C 3J7.
Pierre-Luc
PL
Tanguay
Geneviève
G
Rodier
Sylvain
S
Meloche
D016203:CDC2 Protein Kinase / Q000378:metabolism*; D006367:HeLa Cells; D006801:Humans; D048053:Mitogen-Activated Protein Kinase 6 / Q000378:metabolism*; D008938:Mitosis; D010749:Phosphoprotein Phosphatases / Q000378:metabolism*; D010766:Phosphorylation; D014162:Transfection
D016203:CDC2 Protein Kinase; D048053:Mitogen-Activated Protein Kinase 6; D010749:Phosphoprotein Phosphatases