Abstract
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 α5 subunit of the proteasome. The modified residue, α5-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 α5-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. α5-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 α5, including PLK1, a key regulator of mitosis. α5–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 α5-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.
Highlights
The cell cycle is essential for all cellular life and is tightly regulated throughout the entire living history of an organism [1]
These findings are consistent with the notion that S16A mutation does not interfere with α5 incorporation into the proteasome and indicate that α5-TBHA and endogenous α5 are interchangeable with regard to proteasome assembly
Polo-like kinase 1 (PLK1) is required for full activation of CDK1 upon mitotic entry, which coincides with the appearance of α5-S16 phosphorylation
Summary
The cell cycle is essential for all cellular life and is tightly regulated throughout the entire living history of an organism [1]. Several other kinases (e.g. PLK1, Aurora kinases) and phosphatases (e.g. PP1, PP2A, and Cdc25s) play critical roles in the phosphoregulation of mitosis [4] Another quintessential component of the vast signaling network of cell cycle control is the ubiquitin–proteasome system (UPS), which degrades almost all cell cycle-related proteins in a highly regulated manner with extraordinary spatiotemporal preciseness [6]. We investigated the function and regulation of a highly conserved SP site on the 20S subunit α5 that is phosphorylated in yeast and mammalian cells. This site, α5-Ser, is only phosphorylated in mitotic cells, probably by CDK1/cyclin B. S16 phosphorylation occurred only on unassembled α5 outside the proteasome, suggesting a degradationindependent function
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