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.