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.
Mesh Terms:
Animals, Baculoviridae, Base Sequence, Binding Sites, Casein Kinase II, Cells, Cultured, DNA-Binding Proteins, Gene Expression, Genes, fos, Hela Cells, Humans, Insects, Molecular Sequence Data, Nuclear Proteins, Phosphoproteins, Phosphorylation, Protein-Serine-Threonine Kinases, Recombinant Proteins, Regulatory Sequences, Nucleic Acid, Serum Response Factor, Transcription Factors
Animals, Baculoviridae, Base Sequence, Binding Sites, Casein Kinase II, Cells, Cultured, DNA-Binding Proteins, Gene Expression, Genes, fos, Hela Cells, Humans, Insects, Molecular Sequence Data, Nuclear Proteins, Phosphoproteins, Phosphorylation, Protein-Serine-Threonine Kinases, Recombinant Proteins, Regulatory Sequences, Nucleic Acid, Serum Response Factor, Transcription Factors
EMBO J.
Date: Jan. 01, 1992
PubMed ID: 1740119
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