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. 2018:1844:309-319.
doi: 10.1007/978-1-4939-8706-1_20.

Exploring the Regulation of Proteasome Function by Subunit Phosphorylation

Jordan J S VerPlank  1 , Alfred L Goldberg  2
Affiliations

Exploring the Regulation of Proteasome Function by Subunit Phosphorylation

Jordan J S VerPlank et al. Methods Mol Biol. 2018.

Abstract

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.

Keywords: DYRK2; Proteasome activation; Proteasome phosphorylation; Protein degradation; Protein homeostasis; Protein kinase; Protein kinase A; Ubiquitin.

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Figures

Figure 1:
Figure 1:
26S proteasomes are not phosphorylated by PKA on Rpn6 when ‘phosphomimetic’ or ‘phosphodead’ mutations at serine 14 on Rpn6 are expressed in HEK293 cells.
Figure 2:
Figure 2:
26S proteasomes purified from HEK293 cells after kinase overexpression exhibit many phospho-serine positive bands by western analysis. The ovexpression of diffferent kinases results in distinct phospho-serine banding patterns on the 26S proteasomes.
See this image and copyright information in PMC

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