PDSM, a motif for phosphorylation-dependent SUMO modification Ville Hietakangas*†‡§, Julius Anckar*†‡, Henri A. Blomster*†, Mitsuaki Fujimoto¶, Jorma J. Palvimoʈ**, Akira Nakai¶, and Lea Sistonen*†,†† *Turku Centre for Biotechnology, University of Turku and Åbo Akademi University, FI-20521, Turku, Finland; †Department of Biology, Åbo Akademi University, FI-20520, Turku, Finland; ʈDepartment of Medical Biochemistry, University of Kuopio, FI-70211, Kuopio, Finland; **Biomedicum Helsinki, Institute of Biomedicine, University of Helsinki, FI-00014, Helsinki, Finland; and ¶Department of Biochemistry and Molecular Biology, Yamaguchi University School of Medicine, Ube 755-8505, Japan Edited by Peter Walter, University of California School of Medicine, San Francisco, CA, and approved November 3, 2005 (received for review May 4, 2005) SUMO (small ubiquitin-like modifier) modification regulates many HSF4 differs functionally from HSF1 (10). HSF4 is constitutively cellular processes, including transcription. Although sumoylation trimeric and exists in two alternatively spliced isoforms; HSF4b often occurs on specific lysines within the consensus tetrapeptide displays transactivation capacity, and HSF4a does not (13, 14). ⌿KxE, other modifications, such as phosphorylation, may regulate Recently, HSF4 was shown to be critical for lens development the sumoylation of a substrate. We have discovered PDSM (phos- (15, 16). In lens fiber cells, HSF4b is needed for proper expres- phorylation-dependent sumoylation motif), composed of a SUMO sion of ␥-crystallins and small heat-shock proteins (Hsps), consensus site and an adjacent proline-directed phosphorylation whereas in lens epithelial cells HSF4b seems to repress expres- site (⌿KxExxSP). The highly conserved motif regulates phospho- sion of fibroblast growth factors FGF-1, FGF-4, and FGF-7 (16). rylation-dependent sumoylation of multiple substrates, such as Thus, HSF4b may function as both a transcriptional activator and heat-shock factors (HSFs), GATA-1, and myocyte enhancer factor 2. repressor in a context-dependent manner. Because the post- In fact, the majority of the PDSM-containing proteins are transcrip- translational regulation of HSF4 activity is unexplored, the tional regulators. Within the HSF family, PDSM is conserved be- reasons behind this specificity are not known. tween two functionally distinct members, HSF1 and HSF4b, whose Here we describe a recurrent motif, ⌿KxExxSP, involved in transactivation capacities are repressed through the phosphoryla- phosphorylation-dependent sumoylation of numerous mutually tion-dependent sumoylation. As the first recurrent sumoylation unrelated transcriptional regulators, such as erythroid transcrip- determinant beyond the consensus tetrapeptide, the PDSM pro- tion factor GATA-1 and myocyte-specific enhancer factor 2A vides a valuable tool in predicting new SUMO substrates. (MEF2A), as well as two functionally distinct members of the heat-shock factor family, HSF1 and HSF4b. The bipartite motif, heat-shock factor ͉ heat-shock protein ͉ transcription named PDSM (phosphorylation-dependent sumoylation motif), comprises a SUMO consensus site and a proline-directed phos- UMO (small ubiquitin-like modifier) is covalently linked by an phorylation site, separated by two amino acids. We also dem- BIOCHEMISTRY Sisopeptide bond to lysine residues in their substrates (1). SUMO onstrate that this motif can be used to predict novel SUMO conjugation often requires a consensus sequence ⌿KxE (⌿, large substrates. Our functional analyses revealed that sumoylation of hydrophobic residue; x, any amino acid) around the target lysine (2). the PDSM efficiently represses transactivation capacity of both Because not all available SUMO consensus sites are modified, other HSF1 and HSF4b. factors must also affect the target site specificity. For example, Materials and Methods phosphorylation of the substrate has been shown to regulate the sumoylation both positively and negatively (3–5). However, apart Plasmid Constructs. For plasmid construction, see Supporting from the ⌿KxE tetrapeptide, no common regulatory motifs in- Materials and Methods, which is published as supporting infor- volved in SUMO conjugation are known (1). The SUMO family mation on the PNAS web site. consists of four members, SUMO-1, -2, -3, and -4, of which SUMO-1 is best studied; SUMO-4 has been so far detected only at Cell Culture and Transient Transfection. Cell culture and transfec- the RNA level (1, 6). SUMO-1 and SUMO-2͞3 have, at least tions were done as described in ref. 4 (see Supporting Materials partially, distinct substrates and regulation. For example, the gen- and Methods). eral pattern of conjugates and the subnuclear distribution are different between SUMO-1 and SUMO-2͞3, and the amount of Immunoprecipitation and Western Blotting. In vivo immunoprecipi- conjugated SUMO-2͞3, but not SUMO-1, is increased upon pro- tation experiments were performed as described in ref. 4 (see tein-damaging stresses (7, 8). Beyond this, the functional differences Supporting Materials and Methods). For standard immunoblots, cleared cell lysates (25–35 ␮g of protein) in lysis buffer were between the SUMO paralogs are largely unknown. ͞ The mammalian HSF family is composed of three members, resolved on SDS PAGE, transferred to nitrocellulose, and blot- HSF1, HSF2, and HSF4. HSF1 is the mammalian counterpart of ted with anti-Myc (Sigma), anti-FLAG (Sigma), anti-HSF1 (17), the single invertebrate HSF and is indispensable for the heat- shock response (9). Upon stress, HSF1 is activated by trimer- Conflict of interest statement: No conflicts declared. ization-induced DNA binding and hyperphosphorylation (10, This paper was submitted directly (Track II) to the PNAS office. 11). Moreover, HSF1 is sumoylated on a conserved lysine, Abbreviations: SUMO, small ubiquitin-like modifier; HSF, heat-shock factor; PDSM, phos- lysine-298 (4, 12). We have demonstrated that HSF1 sumoylation phorylation-dependent sumoylation motif; MEF, myocyte-specific enhancer factor; ERR, depends on phosphorylation of an adjacent site, serine-303 (4). estrogen-related receptor; SENP, SUMO͞sentrin-specific protease. Sumoylation was originally suggested to induce HSF1 DNA ‡V.H. and J.A. contributed equally to this work. binding and to be needed for HSF1-mediated transcription (12). §Present address: European Molecular Biology Laboratory, Heidelberg, Meyerhofstrasse 1, This hypothesis was, however, questioned by our previous study, 69117 Heidelberg, Germany. showing that HSF1 could be fully activated also when the ††To whom correspondence should be addressed at: Turku Centre for Biotechnology, sumoylation was prevented (4). Therefore, the biological con- P.O. Box 123, FI-20521, Turku, Finland. E-mail: lea.sistonen@btk.fi. sequences of HSF1 sumoylation require further clarification. © 2005 by The National Academy of Sciences of the USA www.pnas.org͞cgi͞doi͞10.1073͞pnas.0503698102 PNAS ͉ January 3, 2006 ͉ vol. 103 ͉ no. 1 ͉ 45–50 Downloaded by guest on October 1, 2021 anti-HSF4 (14), or anti-Hsc70 (SPA-815; StressGen Biotechnol- Table 1. Many mutually unrelated transcriptional regulators ogies, Victoria, Canada) antibodies. contain a conserved ⌿KxExxSP motif Protein Accession Motif (aa) EMSA. Whole-cell extracts (15 ␮g) were incubated with a 32P- labeled oligonucleotide representing the proximal heat-shock Transcriptional regulators element of the human hsp70 promoter (18) or the GAL4-binding ATF6-␤ Q99941 87–94; 198–205 site (19). Samples were resolved on 4% native PAGE and Bcl-2-associated transcription Q9NYF8 490–497 visualized by autoradiography as described in ref. 18. factor 1 BTB͞POZ domain-containing Q86UZ6 228–235 In Vivo 32P-Labeling and Phosphopeptide Mapping. Plasmids encod- protein 4 ing HSF4b WT or S299A, GATA-1 WT or S142A, and MEF2A c-Myb P10242 526–533 ETS translocation variant 1 (ER81) P50549 88–95 WT or S408A were transfected into Cos-7 cells 24 h before the ETS translocation variant 4 (E1A-F) P43268 162–169 labeling. The labeling and subsequent analyses were performed ETS translocation variant 5 (ERM) P41161 88–95 as described in ref. 20 with slight modifications (see Supporting Forkhead box protein C2 (FKHL14) Q99958 213–220 Materials and Methods). GATA-1 P15976 136–143 H3 K4-specific MLL3 Q8NEZ4 2822–2829 In Vitro Sumoylation. Reticulocyte-lysate translated GATA-1 was HSF1 Q00613 297–304 incubated at 30°C for2hin20mMHepes (pH 7.4), 110 mM HSF4b Q9ULV5 293–300 potassium acetate, 2 mM magnesium acetate, and 5 mM MgCl2 Jumonji͞ARID domain-containing P41229 281–288 together with 0.1 ␮g of SAE1͞2, 0.4 ␮g of Ubc9, 0.1 ␮g of PIASy, protein 1C and 1 ␮g of SUMO-1 (21). Samples were resolved by SDS͞ MEF2A Q02078 402–409 PAGE and visualized by fluorography. For more details see MEF2C Q06413 390–397 Supporting Materials and Methods. MEF2D Q14814 438–445 Nuclear receptor coactivator 2 Q15596 730–737 Luciferase Reporter Assays. Transiently transfected cells were (NCoA-2) snap-frozen and lysed in passive lysis buffer (Promega) accord- Nuclear receptor corepressor 1 O75376 1105–1112 ing to the manufacturer’s instructions. The cell lysates were (N-CoR1) cleared by centrifugation (15,000 ϫ g for 2 min), and the firefly NFAT5 O94916 555–562 luciferase activity produced by the pG5luc reporter plasmid PPAR-gamma P37231 106–113 Smad nuclear interacting protein 1 Q8TAD8 29–36 (Promega) was measured by a Luminoskan Luminometer (Lab- (SNIP-1) systems) by using luciferase assay reagent (Promega) as a Nuclear receptor ERRalpha P11474 110–117 substrate. The luciferase