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

EMBO J. 1992 Jan;11(1):97-105.

Abstract

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 / genetics*
  • Base Sequence
  • Binding Sites
  • Casein Kinase II
  • Cells, Cultured
  • DNA-Binding Proteins / metabolism*
  • Gene Expression
  • Genes, fos / genetics
  • HeLa Cells
  • Humans
  • Insecta / cytology
  • Insecta / metabolism*
  • Molecular Sequence Data
  • Nuclear Proteins / metabolism*
  • Phosphoproteins / metabolism
  • Phosphorylation
  • Protein-Serine-Threonine Kinases / metabolism*
  • Recombinant Proteins / metabolism
  • Regulatory Sequences, Nucleic Acid / genetics
  • Serum Response Factor
  • Transcription Factors / metabolism*

Substances

  • DNA-Binding Proteins
  • Nuclear Proteins
  • Phosphoproteins
  • Recombinant Proteins
  • Serum Response Factor
  • Transcription Factors
  • Casein Kinase II
  • Protein-Serine-Threonine Kinases