Regulation of pokemon 1 activity by sumoylation.
Pokemon 1 is a proto-oncogenic transcriptional regulator that contains a POZ domain at the N-terminus and four Kruppel-like zinc fingers at the C-terminus. Pokemon 1 plays an important role in adipogenesis, osteogenesis, oncogenesis, and transcription of NF-kB responsive genes. Recent reports have shown that biological activities of transcription factors are regulated by sumolylation. We investigated whether Pokemon 1 is post-translationally modified by sumoylation and whether the modification affects Pokemon 1's transcriptional properties. We found that Pokemon 1 is sumoylated in vitro and in vivo. Upon careful analysis of the amino acid sequence of Pokemon 1, we found ten potential sumoylation sites located at lysines 61, 354, 371, 379, 383, 396, 486, 487, 536 and 539. We mutated each of these amino acids into arginine and tested whether the mutation could affect the transcriptional properties of Pokemon 1 on the Pokemon 1 responsive genes, such as ADH5/FDH and pG5-FRE-Luc. Wild-type Pokemon 1 potently represses transcription of ADH5/FDH. Most of the mutants, however, were weaker transcription repressors and repressed transcription 1.3-3.3 fold less effective. Although potential sumoylation sites were located close to the DNA binding domain or the nuclear localization sequence, the mutations did not alter nuclear localization or DNA binding activity. In addition, on the pG5-FRE-Luc test promoter construct, ectopic SUMO-1 repressed transcription in the presence of Pokemon 1. The sumoylation target lysine residue at amino acid 61, which is located in the middle of the POZ-domain, is important because K61R mutation resulted in a much weaker molecular interaction with corepressors. Our data suggest that Pokemon 1's activity as a transcription factor may involve sumoylation, and that sumoylation might be important in the regulation of transcription by Pokemon 1.
Roh
Hee-Eun
HE
Department of Biochemistry and Molecular Biology, Brain Korea 21 Project for Medical Science, Institute of Genetic Science, Yonsei University School of Medicine, Seoul, South Korea.
Lee
Min-Nyung
MN
Jeon
Bu-Nam
BN
Choi
Won-Il
WI
Kim
Yoo-Jin
YJ
Yu
Mi-Young
MY
Hur
Man-Wook
MW
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D001483:Base Sequence; D001665:Binding Sites / Q000235:genetics; D002460:Cell Line; D017931:DNA Primers / Q000235:genetics; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D066298:In Vitro Techniques; D008239:Lysine / Q000737:chemistry; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011499:Protein Processing, Post-Translational; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012097:Repressor Proteins / Q000378:metabolism; D025842:SUMO-1 Protein / Q000235:genetics / Q000378:metabolism; D014157:Transcription Factors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D014158:Transcription, Genetic
D017931:DNA Primers; D004268:DNA-Binding Proteins; D011994:Recombinant Proteins; D012097:Repressor Proteins; D025842:SUMO-1 Protein; D014157:Transcription Factors; C485514:ZBTB7A protein, human; D008239:Lysine