Transcriptional activity of megakaryoblastic leukemia 1 (MKL1) is repressed by SUMO modification.
Megakaryoblastic leukemia 1 (MKL1) was originally identified as a gene translocated in megakaryoblastic leukemia. It has been shown that MKL1 functions as a RhoA-regulated transcriptional coactivator of serum response factor (SRF). In order to identify a protein that regulates the function of MKL1, we performed yeast two-hybrid screening and isolated cDNA that encodes UBC9, an E2 enzyme of small ubiquitin-related modifier-1 (SUMO-1), as an MKL1-binding protein. UBC9 was found to physically interact with MKL1 by GST pull-down assay, and MKL1 was covalently modified with SUMO-1 in 293T cells and in vitro reconstitution system. MKL1 sumoylation is enhanced by either serum stimulation or co-expression of constitutively active form of RhoA. Mutational analysis showed that lysine residues at 499, 576, and 624 are the major acceptor sites for SUMO-1. In addition, reporter gene analysis revealed that mutation of the three sumoylation sites strongly enhances the transcriptional activity of MKL1. The covalent attachment of SUMO-1 to MKL1 by gene fusion represses MKL1-dependent transcription in a complementary manner. Finally, mutation of the sumoylation sites of MKL1 also enhances SRF-dependent transcription without affecting MKL1-SRF interaction. The combined results demonstrated that MKL1 is sumoylated and this modification represses transcriptional activity of MKL1.
Nakagawa
Koji
K
Division of Cancer Gene Regulation, Research Section of Disease Control, Institute for Genetic Medicine, Hokkaido University, Sapporo 060-0815, Japan. nakagawa@igm.hokudai.ac.jp
Kuzumaki
Noboru
N
D002478:Cells, Cultured; D003513:Cycloheximide / Q000494:pharmacology; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D015536:Down-Regulation; D017930:Genes, Reporter; D006367:HeLa Cells; D006801:Humans; D007947:Leukemia, Megakaryoblastic, Acute / Q000378:metabolism*; D015514:Oncogene Proteins, Fusion / Q000235:genetics / Q000378:metabolism; D025842:SUMO-1 Protein / Q000378:metabolism*; D026362:Serum Response Factor / Q000378:metabolism; D013997:Time Factors; D014158:Transcription, Genetic; D014162:Transfection; D014407:Tumor Cells, Cultured; D020798:Two-Hybrid System Techniques; D044763:Ubiquitin-Conjugating Enzymes / Q000378:metabolism; D020742:rhoA GTP-Binding Protein / Q000378:metabolism; D000098843:Ubiquitin-Conjugating Enzyme UBC9
D004268:DNA-Binding Proteins; D015514:Oncogene Proteins, Fusion; D025842:SUMO-1 Protein; D026362:Serum Response Factor; D003513:Cycloheximide; D044763:Ubiquitin-Conjugating Enzymes; D020742:rhoA GTP-Binding Protein; D000098843:Ubiquitin-Conjugating Enzyme UBC9