SUMO-1 Conjugation in Vivo Requires Both a Consensus Modification Motif and Nuclear Targeting

Abstract

SUMO-1 is a small ubiquitin-related modifier that is covalently linked to many cellular protein targets. Proteins modified by SUMO-1 and the SUMO-1-activating and -conjugating enzymes are located predominantly in the nucleus. Here we define a transferable sequence containing the ⌿KXE motif, where ⌿ represents a large hydrophobic amino acid, that confers the ability to be SUMO-1-modified on proteins to which it is linked. Whereas addition of short sequences from p53 and IB␣, containing the ⌿KXE motif, to a carrier protein is sufficient for modification in vitro, modification in vivo requires the additional presence of a nuclear localization signal. Thus, protein substrates must be targeted to the nucleus to undergo SUMO-1 conjugation. SUMO-11 is a small ubiquitin-related modifier (also known as sentrin, GMP1, UBL1, PIC1, or SMT3 in yeast) that has been found covalently conjugated to various cellular proteins (for reviews see Refs. 1-3). Several substrates for SUMO-1 have been reported: the RanGTPase-activating protein (Ran-GAP1) (4, 5) and Ran-binding protein 2 (6) implicated in nucleocytoplasmic trafficking; the promyelocytic leukemia protein (PML) and Sp100 (7) found in subnuclear structures known as PML oncogenic domains or PODs; the IB␣ inhibitor of the transcription factor nuclear factor B, implicated in the control of immune and inflammatory responses (8); and the tumor suppressor protein p53 (9, 10). Consequently, "SUMOylation" plays a role in multiple vital cellular processes such as oncogenesis, cell cycle control, apoptosis, and response to virus infection.SUMO-1 is conjugated to a target protein by a pathway that is distinct from but analogous to ubiquitin conjugation. Like ubiquitin, SUMO-1 is proteolytically processed to expose its mature C terminus by recently described SUMO-1-specific proteases variously called Ulp1 and Ulp2 in yeast (11,12) or SENP1 and SUSP-1 in vertebrates (13-15). Ulp1, Ulp2, and SENP1, but not SUSP-1, are capable of both deconjugating SUMO-1 from modified proteins and removing four amino acids from the C terminus of the 101-amino acid SUMO-1 precursor to generate the mature 97-amino acid form. SUMO-1 addition is accomplished by a thioester cascade, with SUMO-1 first being activated by a heterodimeric SUMO-1-activating enzyme (SAE) that adenylates the C-terminal glycine of before catalyzing the formation of a thioester bond between the C terminus of SUMO-1 and a cysteine residue in SAE. In a transesterification reaction SUMO-1 is transferred from the SAE to the SUMO-1-conjugating enzyme Ubc9, which catalyzes the formation of an isopeptide bond between the C terminus of SUMO-1 and the ⑀-amino group of a lysine residue of the target protein (6, 20 -23). Ubc9 is required for cell cycle progression in yeast (24). Unlike ubiquitin conjugation, SUMO-1 modification of target proteins in vitro is not dependent on the equivalent of an E3 protein ligase (17,19).Here, we demonstrate that a short sequence containing the consensus ⌿KXE, where ⌿ represents a large hydrophobic amino acid, co...

Sumoylation of MEL1S at lysine 568 and its interaction with CtBP facilitates its repressor activity and the blockade of G-CSF-induced myeloid differentiation

“…We searched for the SM cKxE (where c is a large hydrophobic residue, K is the lysine to which the SUMO is conjugated, x is any amino acid and E is glutamic acid) (Rodriguez et al, 2001) in MEL1S using the SUMOplot analysis program (ABGENT, http://www.abgent.com/ tools/sumoplot). There are seven high-probability putative SUMO consensus sites (scores over 0.69) in MEL1S (Supplementary Table 1).…”

Sumoylation of ING2 regulates the transcription mediated by Sin3A

“…This result indicates that the predominantly sumoylated lysine residue of ING2 may be located between the residues 165 and 204. Lysines subjected to sumoylation are commonly found in a sumoylation consensus motif, cKxE (where c is a hydrophobic residue, K the sumoylated lysine, x is any residue and E a glutamic acid) (Rodriguez et al, 2001).…”

Chemical shift perturbation mapping of the Ubc9-CRMP2 interface identifies a pocket in CRMP2 amenable for allosteric modulation of Nav1.7 channels

“…Atomic-level details of Ubc9’s interaction with a few SUMO substrate proteins have been mapped in studies using X-ray crystallography and NMR [12–16]. Within target protein(s), Ubc9 recognizes a SUMOylation motif – “Ψ-K-x-D/E” – where Ψ represents a hydrophobic residue and K is the SUMO acceptor lysine [17]. In addition to this consensus sequence, some SUMO substrate proteins, like the mammalian guanosine triphosphate (GTP)ase-activating protein (RanGAP1), exhibit a second contact surface with Ubc9, which is believed to promote higher SUMOylation efficiency [18].…”