Expression of the human myotonic dystrophy kinase-related Cdc42-binding kinase gamma is regulated by promoter DNA methylation and Sp1 binding.
Myotonic dystrophy kinase-related Cdc42 binding kinases (MRCKs) are family members most related to the myotonic dystrophy kinase (DMPK), RhoA-binding kinase (ROK), and citron kinase. Two highly conserved members, MRCKalpha and -beta, have been previously identified and characterized. We now describe a novel isoform, MRCKgamma, which is functionally and structurally related to members of this kinase family. We show these kinases to have marked similarities in their genomic organization, substrate phosphorylation, and catalytic autoinhibition. Unlike MRCKalpha and -beta, which are expressed ubiquitously, MRCKgamma mRNA was only expressed in heart and skeletal muscle. In cultured cells, MRCKgamma showed differential expression with high levels of expression only in certain cell lines. DNA analysis showed that lack of expression is correlated with promoter DNA methylation. We have mapped the methylation sites in the MRCKgamma promoter. Significantly, agents that suppressed DNA methylation caused increases in the expression of the kinase in low-expressing cells, further supporting the notion that promoter DNA methylation plays an important role in the expression of MRCKgamma. Analysis of the MRCKgamma promoter has also revealed two proximal Sp1 sites that are essential for transcriptional activity. We conclude that both promoter DNA methylation and Sp1 binding are important regulators for MRCKgamma expression.
Ng
Yvonne
Y
GSK-IMCB Group, Institute of Molecular and Cell Biology, 61 Biopolis Drive, Singapore 138673, Singapore.
Tan
Ivan
I
Lim
Louis
L
Leung
Thomas
T
D000595:Amino Acid Sequence; D000818:Animals; D001374:Azacitidine / Q000031:analogs & derivatives* / Q000494:pharmacology; D001483:Base Sequence; D015152:Blotting, Northern; D019556:COS Cells; D002460:Cell Line; D045744:Cell Line, Tumor; D003593:Cytoplasm / Q000378:metabolism; D004247:DNA / Q000378:metabolism; D019175:DNA Methylation; D000077209:Decitabine; D020558:GTP Phosphohydrolases / Q000378:metabolism; D017930:Genes, Reporter; D014644:Genetic Variation; D005822:Genetic Vectors; D005982:Glutathione Transferase / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D006877:Hydroxamic Acids / Q000494:pharmacology; D008156:Luciferases / Q000378:metabolism; D008957:Models, Genetic; D008969:Molecular Sequence Data; D065636:Myotonin-Protein Kinase; D011233:Precipitin Tests; D011401:Promoter Regions, Genetic; D011485:Protein Binding; D020033:Protein Isoforms; D017346:Protein Serine-Threonine Kinases / Q000737:chemistry* / Q000378:metabolism*; D017434:Protein Structure, Tertiary; D011505:Protein-Tyrosine Kinases / Q000378:metabolism; D012333:RNA, Messenger / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid; D016329:Sp1 Transcription Factor / Q000737:chemistry* / Q000378:metabolism; D014158:Transcription, Genetic; D014162:Transfection; D020764:cdc42 GTP-Binding Protein / Q000378:metabolism
C585517:DMPK protein, human; D006877:Hydroxamic Acids; D020033:Protein Isoforms; D012333:RNA, Messenger; D011993:Recombinant Fusion Proteins; D016329:Sp1 Transcription Factor; C012589:trichostatin A; D000077209:Decitabine; D004247:DNA; D008156:Luciferases; D005982:Glutathione Transferase; C105518:CDC42BPA protein, human; C118666:CDC42BPB protein, human; D011505:Protein-Tyrosine Kinases; C490364:CDC42BPG protein, human; D065636:Myotonin-Protein Kinase; D017346:Protein Serine-Threonine Kinases; D020558:GTP Phosphohydrolases; D020764:cdc42 GTP-Binding Protein; D001374:Azacitidine