Exploring the Regulation of Proteasome Function by Subunit Phosphorylation

  • Jordan J. S. VerPlank
  • Alfred L. GoldbergEmail author
Part of the Methods in Molecular Biology book series (MIMB, volume 1844)


Rates of degradation by the ubiquitin proteasome system depend not only on rates of ubiquitination, but also on the level of proteasome activity which can be regulated through phosphorylation of proteasome subunits. Many protein kinases have been proposed to influence proteasomal activity. However, for only two is there strong evidence that phosphorylation of a specific 26S subunit enhances the proteasome’s capacity to degrade ubiquitinated proteins and promotes protein breakdown in cells: (1) protein kinase A (PKA), which after a rise in cAMP phosphorylates the 19S subunit Rpn6, and (2) dual tyrosine receptor kinase 2 (DYRK2), which during S through M phases of the cell cycle phosphorylates the 19S ATPase subunit Rpt3. In this chapter, we review and discuss the different methods used to assess the impact of phosphorylation by these two kinases on proteasomal activity and intracellular protein degradation. In addition, we present one method to determine if phosphorylation is responsible for an observed increase in proteasomal activity and another to evaluate by Phos-tag gel electrophoresis whether a specific proteasome subunit is modified by phosphorylation. The methods reviewed and presented here should be useful in clarifying the roles of other kinases and other posttranslational modifications of proteasome subunits.

Key words

Proteasome phosphorylation Protein degradation Protein kinase Proteasome activation Protein homeostasis Protein kinase A DYRK2 Ubiquitin 



The methods described here were developed through research support to our laboratory from the NIH-NIGMS (R01 GM51923), Cure Alzheimer’s Fund, Muscular Dystrophy Association (MDA-419143), and Project A.L.S (2015-06).


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Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2018

Authors and Affiliations

  1. 1.Harvard Medical SchoolBostonUSA

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