Biochemical Journal

Research article

A novel RCE1 isoform is required for H-Ras plasma membrane localization and is regulated by USP17

Jakub Jaworski, Ureshnie Govender, Cheryl McFarlane, Michelle de la Vega, Michelle K. Greene, Neil D. Rawlings, James A. Johnston, Christopher J. Scott, James F. Burrows

Abstract

Processing of the ‘CaaX’ motif found on the C-termini of many proteins, including the proto-oncogene Ras, requires the ER (endoplasmic reticulum)-resident protease RCE1 (Ras-converting enzyme 1) and is necessary for the proper localization and function of many of these ‘CaaX’ proteins. In the present paper, we report that several mammalian species have a novel isoform (isoform 2) of RCE1 resulting from an alternate splice site and producing an N-terminally truncated protein. We demonstrate that both RCE1 isoform 1 and the newly identified isoform 2 are required to reinstate proper H-Ras processing and thus plasma membrane localization in RCE1-null cells. In addition, we show that the deubiquitinating enzyme USP17 (ubiquitin-specific protease 17), previously shown to modulate RCE1 activity, can regulate the abundance and localization of isoform 2. Furthermore, we show that isoform 2 is ubiquitinated on Lys43 and deubiquitinated by USP17. Collectively, the findings of the present study indicate that RCE1 isoform 2 is required for proper ‘CaaX’ processing and that USP17 can regulate this via its modulation of RCE1 isoform 2 ubiquitination.

  • deubiquitination
  • protease
  • Ras
  • Ras-converting enzyme 1 (RCE1)
  • ubiquitin
  • ubiquitin-specific protease 17 (USP17)

Abbreviations: DUB, deubiquitinating enzyme; ER, endoplasmic reticulum; FTase, farnesyltransferase; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; HA, haemagglutinin; HEK, human embryonic kidney; HRP, horseradish peroxidase; IMAC, immobilized metal-ion-affinity chromatography; MEF, mouse embryonic fibroblast; RCE1, Ras-converting enzyme 1; RCE1-ISO1–FLAG, RCE1 isoform 1 with a C-terminal FLAG tag; RCE1-ISO2–FLAG, RCE1 isoform 2 with a C-terminal FLAG tag; RCE1-ISO1–His6, RCE1 isoform 1 with a C-terminal His6 tag; RCE1-ISO2–His6, RCE1 isoform 2 with a C-terminal His6 tag; RIPA, radio-immunoprecipitation assay; RT-PCR, reverse transcription–PCR; WT, wild-type

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