The role of small ubiquitin-like modifier-interacting motif in the assembly and regulation of metal-responsive transcription factor 1.
Metal-responsive transcription factor 1 (MTF-1) is an essential protein required for mouse embryonic development. We report here the occurrence of sumoylation on MTF-1. Mutational studies demonstrated that sumoylation occurs on the lysine residue at position 627 (Lys(627)) of mouse MTF-1. Small ubiquitin-like modifier (SUMO)-1 was fused to the C terminus of MTF-1 to mimic the sumoylated form of the protein and it suppressed the transcriptional activity of MTF-1. The nuclear translocation activity, DNA-binding activity, and protein stability of SUMO-fused MTF-1 are similar to that of wild type MTF-1. The level of sumoylation was reduced by metal in a dose- and time-dependent manner. The fact that zinc reduces MTF-1 sumoylation makes the suppressive role of sumoylated MTF-1 in transcription physiologically less significant because the SUMO moiety of MTF-1 is removed when MTF-1 translocates into nucleus. We further identified a SUMO-interacting motif (SIM) on MTF-1. Remarkably, MTF-1 binds sumoylated MTF-1 and/or other cellular factors in a SIM-dependent manner. This interaction was disrupted by treating cells with zinc. Gel permeation chromatography demonstrated that MTF-1 forms SIM-dependent complexes. This cross-interaction transpires in the cytoplasm and markedly reduces upon nuclear translocation. It can therefore be concluded that SUMO conjugation and the SIM on MTF-1 do not play a critical role in suppressing transcriptional activity. Instead, MTF-1 forms complexes with cellular factors through SIM and SUMO moiety in the cytoplasm. The result explores a new understanding for the mode of MTF-1 assembly and regulation in cells.
Institute of Molecular and Cellular Biology, and Department of Life Science, National Tsing Hua University, Hsinchu, Taiwan.
Ya-Chuan
YC
Liu
Meng-Chieh
MC
Lin
Hsiang-Chi
HC
Chen
Ming F
MF
Tam
Lih-Yuan
LY
Lin
D020816:Amino Acid Motifs; D000818:Animals; D016466:CHO Cells; D002104:Cadmium / Q000494:pharmacology; D002460:Cell Line; D047369:Chromatin Immunoprecipitation; D002846:Chromatography, Affinity; D006224:Cricetinae; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D024202:Electrophoretic Mobility Shift Assay; D006801:Humans; D047468:Immunoprecipitation; D011485:Protein Binding / Q000187:drug effects; D060888:Real-Time Polymerase Chain Reaction; D025842:SUMO-1 Protein / Q000235:genetics / Q000378:metabolism*; D013329:Structure-Activity Relationship; D058207:Sumoylation / Q000187:drug effects; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism*; D015032:Zinc / Q000494:pharmacology; D000097809:Transcription Factor MTF-1
D002104:Cadmium; D004268:DNA-Binding Proteins; D025842:SUMO-1 Protein; D014157:Transcription Factors; D015032:Zinc; D000097809:Transcription Factor MTF-1