Differential Regulation of Sentrinized Proteins by a Novel Sentrin-specific Protease*

Sentrin-1, also called SUMO-1, is a protein of 101 residues that is distantly related to ubiquitin and another ubiquitin-like protein, NEDD8. Here we report the cloning of a novel sentrin-specific protease, SENP1, which has no homology to the known de-ubiquitinating enzymes or ubiquitin C-terminal hydrolases. However, SENP1 is distantly related to the yeast Smt3-specific protease, Ulp1. A COS cell expression system was used to demonstrate the activity of SENP1 in vivo. When HA-tagged sentrin-1 was co-expressed with SENP1, the higher molecular weight sentrin-1 conjugates were completely removed. Surprisingly, the major sentrinized band at 90 kDa remained intact. The disappearance of the high molecular weight sentrin-1 conjugates also coincided with an increase in free sentrin-1 monomers. SENP1 is also active against proteins modified by sentrin-2, but not those modified by ubiquitin or NEDD8. In addition, sentrinized PML, a tumor suppressor protein that resides in the nucleus, was selectively affected by SENP1, whereas sentrinized RanGAP1, which is associated with the cytoplasmic fibrils of the nuclear pore complex, remained intact. The inability of SENP1 to process sentrinized RanGAP1in vivo is most likely due to its nuclear localization because SENP1 is active against sentrinized RanGAP1 in vitro. The identification of a nuclear-localized, sentrin-specific protease will provide a unique tool to study the role of sentrinization in the biological function of PML and in the pathogenesis of acute promyelocytic leukemia.

[1]  L. Caskey,et al.  Preferential Interaction of Sentrin with a Ubiquitin-conjugating Enzyme, Ubc9* , 1997, The Journal of Biological Chemistry.

[2]  M. Hochstrasser,et al.  A new protease required for cell-cycle progression in yeast , 1999, Nature.

[3]  A. Dejean,et al.  Conjugation with the ubiquitin‐related modifier SUMO‐1 regulates the partitioning of PML within the nucleus , 1998, The EMBO journal.

[4]  M. Dasso,et al.  Ubc9p and the conjugation of SUMO-1 to RanGAP1 and RanBP2 , 1998, Current Biology.

[5]  G. Blobel,et al.  The ubiquitin‐like protein Smt3p is activated for conjugation to other proteins by an Aos1p/Uba2p heterodimer , 1997, The EMBO journal.

[6]  E. Yeh,et al.  Preferential Modification of Nuclear Proteins by a Novel Ubiquitin-like Molecule* , 1997, The Journal of Biological Chemistry.

[7]  E. Yeh,et al.  Characterization of NEDD8, a Developmentally Down-regulated Ubiquitin-like Protein* , 1997, The Journal of Biological Chemistry.

[8]  D. Koshland,et al.  Evidence that the MIF2 gene of Saccharomyces cerevisiae encodes a centromere protein with homology to the mammalian centromere protein CENP-C. , 1995, Molecular biology of the cell.

[9]  E. Yeh,et al.  Covalent Modification of PML by the Sentrin Family of Ubiquitin-like Proteins* , 1998, The Journal of Biological Chemistry.

[10]  R. Hay,et al.  SUMO-1 modification of IkappaBalpha inhibits NF-kappaB activation. , 1998, Molecular cell.

[11]  E. Yeh,et al.  Identification of Three Major Sentrinization Sites in PML* , 1998, The Journal of Biological Chemistry.

[12]  E. Yeh,et al.  Pml Is Critical for Nd10 Formation and Recruits the Pml-Interacting Protein Daxx to This Nuclear Structure When Modified by Sumo-1 , 1999, The Journal of cell biology.

[13]  A. Ciechanover,et al.  The ubiquitin system. , 1998, Annual review of biochemistry.

[14]  F. Melchior,et al.  A Small Ubiquitin-Related Polypeptide Involved in Targeting RanGAP1 to Nuclear Pore Complex Protein RanBP2 , 1997, Cell.

[15]  E. Yeh,et al.  Protection against Fas/APO-1- and tumor necrosis factor-mediated cell death by a novel protein, sentrin. , 1996, Journal of immunology.

[16]  Den Dunnen Jt.  Cosmid-based exon trapping. , 1999 .

[17]  G. Maul,et al.  The periphery of nuclear domain 10 (ND10) as site of DNA virus deposition , 1996, The Journal of cell biology.

[18]  G. Blobel,et al.  Ubc9p Is the Conjugating Enzyme for the Ubiquitin-like Protein Smt3p* , 1997, The Journal of Biological Chemistry.

[19]  P. Freemont,et al.  PIC 1, a novel ubiquitin-like protein which interacts with the PML component of a multiprotein complex that is disrupted in acute promyelocytic leukaemia. , 1996, Oncogene.

[20]  E. Yeh,et al.  Identification of the Activating and Conjugating Enzymes of the NEDD8 Conjugation Pathway* , 1999, The Journal of Biological Chemistry.

[21]  M. Kozak.  Structural features in eukaryotic mRNAs that modulate the initiation of translation. , 1991, The Journal of biological chemistry.

[22]  G. Blobel,et al.  A novel ubiquitin-like modification modulates the partitioning of the Ran-GTPase-activating protein RanGAP1 between the cytosol and the nuclear pore complex , 1996, The Journal of cell biology.

[23]  F. Melchior,et al.  Molecular Characterization of the SUMO-1 Modification of RanGAP1 and Its Role in Nuclear Envelope Association , 1998, The Journal of cell biology.

[24]  T. Sternsdorf,et al.  Evidence for Covalent Modification of the Nuclear Dot–associated Proteins PML and Sp100 by PIC1/SUMO-1 , 1997, The Journal of cell biology.

[25]  K. Wilkinson.  Regulation of ubiquitin‐dependent processes by deubiquitinating enzymes , 1997, FASEB journal : official publication of the Federation of American Societies for Experimental Biology.

[26]  G. Maul,et al.  Human Cytomegalovirus Immediate Early Interaction with Host Nuclear Structures: Definition of an Immediate Transcript Environment , 1997, The Journal of cell biology.

[27]  G. Blobel,et al.  SUMO-1 Modification and Its Role in Targeting the Ran GTPase-activating Protein, RanGAP1, to the Nuclear Pore Complex , 1998, The Journal of cell biology.

[28]  E. Yeh,et al.  Characterization of a Second Member of the Sentrin Family of Ubiquitin-like Proteins* , 1998, The Journal of Biological Chemistry.

[29]  Jonathan D. Licht,et al.  Deconstructing a Disease: RAR, Its Fusion Partners, and Their Roles in the Pathogenesis of Acute Promyelocytic Leukemia , 1999 .

[30]  E. Yeh,et al.  Molecular cloning and characterization of human AOS1 and UBA2, components of the sentrin‐activating enzyme complex , 1999, FEBS letters.