C-terminal domain phosphorylation of ERK3 controlled by Cdk1 and Cdc14 regulates its stability in mitosis

Area of Science:

• Cell Biology
• Molecular Biology
• Biochemistry

Background:

• Extracellular-signal-regulated kinase 3 (ERK3), an atypical MAPK, is implicated in cell-cycle progression and differentiation.
• The cell-cycle-dependent regulation of ERK3 function remains largely uncharacterized.

Purpose of the Study:

• To investigate the regulation of ERK3 during the cell cycle.
• To identify the mechanisms controlling ERK3 phosphorylation and stability.

Main Methods:

• Stoichiometric phosphorylation analysis of ERK3 across the cell cycle.
• In vitro kinase assays using ERK3 deletion mutants and mitotic cell extracts.
• Mass spectrometry to identify novel phosphorylation sites.
• In vitro and in vivo studies of cyclin B-Cdk1 and Cdc14A/B phosphatases.
• Site-directed mutagenesis to assess the role of C-terminal phosphorylation sites.

Main Results:

• ERK3 undergoes stoichiometric hyperphosphorylation upon entry into mitosis and dephosphorylation at the M-G1 transition.
• Novel phosphorylation sites (Ser684, Ser688, Thr698, Ser705) were identified at the C-terminus of ERK3.
• Cyclin B-Cdk1 phosphorylates these C-terminal sites in vitro, with Cdk1 identified as a major Thr698 kinase in vivo.
• Cdc14A and Cdc14B phosphatases bind ERK3 and reverse its C-terminal phosphorylation.
• Mutation of these four C-terminal sites significantly reduced ERK3 half-life during mitosis.

Conclusions:

• ERK3 phosphorylation is a cell-cycle-dependent process, occurring during mitosis.
• The C-terminal region of ERK3 is a key site for mitotic phosphorylation.
• Phosphorylation by cyclin B-Cdk1 and subsequent dephosphorylation by Cdc14A/B phosphatases regulate ERK3 levels.
• Mitotic phosphorylation of ERK3 is crucial for its stabilization during mitosis, revealing a novel regulatory mechanism.