Casein kinase II phosphorylation increases the rate of serum response factor-binding site exchange.

AUTOR(ES)

Marais, R M

RESUMO

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.

Documentos Relacionados

• Mutation of serum response factor phosphorylation sites and the mechanism by which its DNA-binding activity is increased by casein kinase II.
• Mutations in an integration host factor-binding site: effect on lambda site-specific recombination and regulatory implications.
• The upstream factor-binding site is not essential for activation of transcription from the adenovirus major late promoter.
• DNA Replication Efficiency Depends on Transcription Factor-Binding Sites
• Identification of a fibroblast growth factor-binding protein in Drosophila melanogaster.