GA-binding protein regulates KIS gene expression, cell migration, and cell cycle progression.
The cyclin-dependent kinase inhibitor p27(Kip1) arrests cell cycle progression through G1/S phases and is regulated by phosphorylation of serine/threonine residues. Recently, we identified the serine/threonine kinase, KIS, which phosphorylates p27(Kip1) on serine 10 leading to nuclear export of p27(Kip1) and protein degradation. However, the molecular mechanisms of transcriptional activation of the human KIS gene and its biological activity are not known. We mapped the transcription initiation site approximately 116 bp 5' to the translation start site, and sequences extending to -141 were sufficient for maximal promoter activity. Mutation in either of two Ets-binding sites in this region resulted in an approximately 75-80% decrease in promoter activity. These sites form at least 3 specific complexes, which contained GA-binding protein (GABP). Knocking down GABPalpha by siRNA in vascular smooth muscle cells (VSMCs) diminished KIS gene expression and reduced cell migration. Correspondingly, in serum stimulated GABPalpha-deficient mouse embryonic fibroblasts (MEFs), KIS gene expression was also significantly reduced, which was associated with an increase in p27(Kip1) protein levels and a decreased percentage of cells in S-phase. Consistent with these findings, following vascular injury in vivo, GABPalpha-heterozygous mice demonstrated reduced KIS gene expression within arterial lesions and these lesions were significantly smaller compared to GABP+/+ mice. In summary, serum-responsive GABP binding to Ets-binding sites activates the KIS promoter, leading to KIS gene expression, cell migration, and cell cycle progression.
Crook
Martin F
MF
Cardiovascular Branch, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, Maryland 20892, USA.
Olive
Michelle
M
Xue
Hai-Hui
HH
Langenickel
Thomas H
TH
Boehm
Manfred
M
Leonard
Warren J
WJ
Nabel
Elizabeth G
EG
D001483:Base Sequence; D002453:Cell Cycle / Q000502:physiology*; D002465:Cell Movement / Q000502:physiology*; D002478:Cells, Cultured; D050760:Cyclin-Dependent Kinase Inhibitor p27; D017931:DNA Primers; D024202:Electrophoretic Mobility Shift Assay; D051268:GA-Binding Protein Transcription Factor / Q000235:genetics / Q000502:physiology*; D015971:Gene Expression Regulation, Enzymologic / Q000502:physiology*; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins / Q000235:genetics* / Q000378:metabolism; D010766:Phosphorylation; D016133:Polymerase Chain Reaction; D017346:Protein Serine-Threonine Kinases / Q000235:genetics* / Q000378:metabolism; D034741:RNA, Small Interfering
C436291:CDKN1B protein, human; D017931:DNA Primers; D051268:GA-Binding Protein Transcription Factor; D047908:Intracellular Signaling Peptides and Proteins; D034741:RNA, Small Interfering; D050760:Cyclin-Dependent Kinase Inhibitor p27; D017346:Protein Serine-Threonine Kinases; C530658:UHMK1 protein, human; C530659:Uhmk1 protein, mouse