Constitutive Nuclear Localization of an Alternatively Spliced Sirtuin-2 Isoform

Abstract

Sirtuin-2 (SIRT2), the cytoplasmic member of the sirtuin family, has been implicated in the deacetylation of nuclear proteins. Although the enzyme has been reported to be located to the nucleus during G2/M phase, its spectrum of targets suggests functions in the nucleus throughout the cell cycle. While a nucleocytoplasmic shuttling mechanism has been proposed for SIRT2, recent studies have indicated the presence of a constitutively nuclear isoform. Here we report the identification of a novel splice variant (isoform 5) of SIRT2 that lacks a nuclear export signal and encodes a predominantly nuclear isoform. This novel isoform 5 fails to show deacetylase activity using several assays, both in vitro and in vivo, and we are led to conclude that this isoform is catalytically inactive. Nevertheless, it retains the ability to interact with p300, a known interaction partner. Moreover, changes in intrinsic tryptophan fluorescence upon denaturation indicate that the protein is properly folded. These data, together with computational analyses, confirm the structural integrity of the catalytic domain. Our results suggest an activity-independent nuclear function of the novel isoform.

SIRT2 inactivation reveals a subset of hyperacetylated perinuclear microtubules inaccessible to HDAC6

“…pcDNA3.1+N-Myc harboring the ORF of human MAP2C (accession number NM_031845), cloned in through the BamHI and EcoRV restriction sites, was ordered from Genscript. pcDNA3.1/V5-His harboring the ORF of human SIRT2 isoform 1 is described in Rack et al (2014). The vectors were used for transient transfection.…”

Human tRNA synthetase catalytic nulls with diverse functions

“…Members of other enzyme families, though perhaps to a lesser extent, likely also gain new functions through splice variants. The recently reported catalytically impaired natural splice variants of several oncogenic kinases (31) and of the SIRT2 histone deacetylase (32) suggest that other enzyme families have undergone similar, though perhaps less extensive variation.…”

Photo Cross‐Linking Probes Containing ϵ‐N‐Thioacyllysine and ϵ‐N‐Acyl‐(δ‐aza)lysine Residues

“…With the myristoylated probe ( 3B‐Kmyr ) we enriched two isoforms of SIRT2 and the labeling was successfully abolished by competition using 3 C B‐Kmyr for both bands. This constitutes the first demonstration of a versatile photo cross‐linking probe that is able to enrich two different endogenous and physiologically relevant splice variants of SIRT2 [i.e., isoform 1 (43 kDa) and isoform 2 (39 kDa], which underscores the utility of our new probe design (Figure A). Affinity purification of both splice variants of SIRT2 from HL60 cell lysate has previously been demonstrated.…”