Activating transcription factor-2 DNA-binding activity is stimulated by phosphorylation catalyzed by p42 and p54 microtubule-associated protein kinases.
Recent studies have detailed the ability of activating transcription factor-2 (ATF-2) to mediate adenoviral E1a stimulation of gene expression; however, an endogenous regulator for the transcriptional activity of this protein has not been described. To characterize the regulation of ATF-2 activity, we have expressed full-length and truncated peptides corresponding to various regions of the ATF-2 protein in bacteria and the baculovirus insect cell system. Bacterially expressed truncated (350-505) but not full-length ATF-2, was able to bind a consensus cAMP response element-containing oligonucleotide, suggesting the N-terminal moiety may serve as a negative regulator of DNA-binding activity. In contrast, the full-length ATF-2 protein expressed in Spodoptera frugiperda (Sf9) cells using a recombinant baculovirus was fully competent to bind DNA. Protein phosphatase 2A reversed the DNA-binding activity by dephosphorylating the ATF-2 polypeptide. Microtubule-associated protein kinase catalyzed the phosphorylation and stimulated the DNA-binding activity of bacterially expressed full-length ATF-2. Phosphopeptide mapping of phosphorylated ATF-2 proteins identified a single peptide in the N-terminal moiety of ATF-2 phosphorylated by p42 or p54 microtubule-associated protein kinase. Therefore, we propose that phosphorylation of this regulatory site is sufficient to induce an allosteric structural change in the ATF-2 protein, which allows dimerization and subsequent DNA binding.
Abdel-Hafiz
H A
HA
Department of Medicine, University of Colorado School of Medicine, Denver 80262.
Heasley
L E
LE
Kyriakis
J M
JM
Avruch
J
J
Kroll
D J
DJ
Johnson
G L
GL
Hoeffler
J P
JP
D000595:Amino Acid Sequence; D000818:Animals; D016367:Baculoviridae / Q000235:genetics; D001483:Base Sequence; D001665:Binding Sites; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D002478:Cells, Cultured; D004247:DNA / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics; D005822:Genetic Vectors; D008969:Molecular Sequence Data; D009036:Moths; D010446:Peptide Fragments / Q000378:metabolism*; D010766:Phosphorylation; D011494:Protein Kinases / Q000378:metabolism*; D011499:Protein Processing, Post-Translational; D011993:Recombinant Fusion Proteins / Q000378:metabolism*; D014157:Transcription Factors / Q000378:metabolism*; D016335:Zinc Fingers
D010446:Peptide Fragments; D011993:Recombinant Fusion Proteins; D014157:Transcription Factors; D004247:DNA; D011494:Protein Kinases; D017871:Calcium-Calmodulin-Dependent Protein Kinases