Casein kinase II phosphorylation increases the rate of serum response factor-binding site exchange.
Recombinant baculoviruses were used to express wild-type serum response factor (SRF) and a mutant, SRF.CKIIA, which lacks all four serine residues in the major casein kinase II (CKII) site at residues 77-90. Purified recombinant SRF binds DNA with an affinity and specificity indistinguishable from that of HeLa cell SRF, and activates transcription in vitro. Comparative phosphopeptide analysis of the wild-type and mutant proteins demonstrated that the wild-type protein is phosphorylated at the major CKII site in insect cells. Dephosphorylation of recombinant SRF does not affect its affinity for the c-fos SRE, and results in only a 3-fold reduction in binding to the synthetic site ACT.L. However, dephosphorylation does cause a large decrease in the rates of association with and dissociation from either site. These effects are due solely to phosphorylation at the major CKII site: the binding properties of the SRF.CKIIA mutant are identical to those of dephosphorylated wild-type SRF, and CKII phosphorylation in vitro converts dephosphorylated wild-type SRF from a slow-binding to a fast-binding form without significantly changing binding affinity. CKII phosphorylation thus acts to potentiate SRF-DNA exchange rates rather than alter equilibrium binding affinity.
Marais
R M
RM
Imperial Cancer Research Fund, London, UK.
Hsuan
J J
JJ
McGuigan
C
C
Wynne
J
J
Treisman
R
R
D000818:Animals; D016367:Baculoviridae / Q000235:genetics*; D001483:Base Sequence; D001665:Binding Sites; D047390:Casein Kinase II; D002478:Cells, Cultured; D004268:DNA-Binding Proteins / Q000378:metabolism*; D015870:Gene Expression; D016762:Genes, fos / Q000235:genetics; D006367:HeLa Cells; D006801:Humans; D007313:Insecta / Q000166:cytology / Q000378:metabolism*; D008969:Molecular Sequence Data; D009687:Nuclear Proteins / Q000378:metabolism*; D010750:Phosphoproteins / Q000378:metabolism; D010766:Phosphorylation; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism; D012045:Regulatory Sequences, Nucleic Acid / Q000235:genetics; D026362:Serum Response Factor; D014157:Transcription Factors / Q000378:metabolism*
D004268:DNA-Binding Proteins; D009687:Nuclear Proteins; D010750:Phosphoproteins; D011994:Recombinant Proteins; D026362:Serum Response Factor; D014157:Transcription Factors; D047390:Casein Kinase II; D017346:Protein Serine-Threonine Kinases