Characterization of a Family of Nucleolar SUMO-specific Proteases with Preference for SUMO-2 or SUMO-3

Limin Gong,Edward T.H Yeh

doi:10.1074/jbc.m511658200

Abstract

SUMOylation is a reversible process regulated by a family of sentrin/SUMO-specific proteases (SENPs). Of the six SENP family members, except for SENP1 and SENP2, the substrate specificities of the rest of SENPs are not well defined. Here, we have described SENP5, which has restricted substrate specificity. SENP5 showed SUMO-3 C-terminal hydrolase activity but could not process pro-SUMO-1 in vitro. Furthermore, SENP5 showed more limited isopeptidase activity in vitro. In vivo, SENP5 showed isopeptidase activity against SUMO-2 and SUMO-3 conjugates but not against SUMO-1 conjugates. Native SENP5 localized mainly to the nucleolus but was also found in the nucleus. The N terminus of SENP5 contains a stretch of amino acids responsible for the nucleolar localization of SENP5. N-terminal-truncated SENP5 co-localized with PML, a known SUMO substrate. Using PML SUMOylation mutants as model substrates, we showed that SENP5 can remove poly-SUMO-2 or poly-SUMO-3 from the Lys160 or Lys490 positions of PML. However, SENP5 could not remove SUMO-1 from the Lys160 or Lys490 positions of PML. Nonetheless, SENP5 could remove SUMO-1, -2, and -3 from the Lys65 position of PML. Thus, SENP5 also possesses limited SUMO-1 isopeptidase activity. We were also able to show that SENP3 has substrate specificity similar to that of SENP5. Thus, SENP3 and SENP5 constitute a subfamily of SENPs that regulate the formation of SUMO-2 or SUMO-3 conjugates and, to a less extent, SUMO-1 modification.

Highlights

A broad array of cellular processes, such as cell division, differentiation, signal transduction, trafficking, and quality control, depend on the covalent modification of proteins by ubiquitin (Ub),2 a highly conserved 76-amino acid polypeptide [1]
Characterization of SENP5 Gene and cDNA—Using the full-length SENP1 protein sequence as a query in a tBLASTn sequence search, we detected three positive EST clones (T70794, N42548, and AW167097) that were homologous to the C-terminal of SENP1 [21]
Extension of the cloned cDNA by rapid amplification of the 5Ј-cDNA ends (RACE) resulted in the identification of a 2802-bp cDNA clone from a human placenta cDNA library that contained an open reading frame of 2265 bp encoding a protein of 755 amino acids

Summary

Introduction

A broad array of cellular processes, such as cell division, differentiation, signal transduction, trafficking, and quality control, depend on the covalent modification of proteins by ubiquitin (Ub), a highly conserved 76-amino acid polypeptide [1]. The covalent modification of proteins by SUMO is reversibly regulated by a family of sentrin/SUMO-specific proteases (SENPs) [2]. Much of our knowledge of the behavior of SENPs has come from studies of SENP1 and SENP2 These have shown that SENP1 localizes to the nucleus, excluding the nucleolus [21], and that SENP2, called SUSP1, localizes to the nucleoplasmic face of the nuclear pore complex [25, 26]. Both SENP1 and SENP2 are able to remove SUMO from all SUMO-modified proteins. SENP3 and SENP5 constitute a new subfamily of SENPs that share considerable sequence homology and exhibit similar substrate specificities

Methods

Results

Conclusion