Conserved Mitotic Phosphorylation of a Proteasome Subunit Regulates Cell Proliferation

Reversible phosphorylation has emerged as an important mechanism for regulating proteasome function in various physiological processes. Essentially all proteasome phosphorylations characterized thus far occur on proteasome holoenzyme or subcomplexes to regulate substrate degradation. Here, we report a highly conserved phosphorylation that only exists on the unassembled alpha 5 subunit of the proteasome. The modified residue, alpha 5-Ser16, is within a SP motif typically recognized by cyclin-dependent kinases (CDKs). Using a phospho-specific antibody generated against this site, we found that alpha 5-S16 phosphorylation is mitosis-specific in both yeast and mammalian cells. Blocking this site with a S16A mutation caused growth defect and G2/M arrest of the cell cycle. alpha 5-S16 phosphorylation depends on CDK1 activity and is highly abundant in some but not all mitotic cells. Immunoprecipitation and mass spectrometry (IP-MS) studies identified numerous proteins that could interact with phosphorylated alpha 5, including PLK1, a key regulator of mitosis. alpha 5-PLK1 interaction increased upon mitosis and could be facilitated by S16 phosphorylation. CDK1 activation downstream of PLK1 activity was delayed in S16A mutant cells, suggesting an important role of alpha 5-S16 phosphorylation in regulating PLK1 and mitosis. These data have revealed an unappreciated function of exo-proteasome phosphorylation of a proteasome subunit and may bring new insights to our understanding of cell cycle control.

Automated summary

A highly conserved phosphorylation was identified on the unassembled alpha 5 subunit of the proteasome, specifically during mitosis. Blocking this phosphorylation site resulted in growth defects and cell cycle arrest. The phosphorylation of alpha 5-Ser16 plays an important role in regulating PLK1 and mitosis.