Consecutive steps of phosphorylation affect conformation and DNA binding of the Chironomus high mobility group A protein

The high mobility group (HMG) proteins of the AT-hook family (HMGA) lie dow nstream in regulatory networks with protein kinase CI Cdc2 kinase, MAP kina se, and casein kinase 2 (CK2) as final effecters. In the cells of the midge Chironomus, almost all of the HMGA protein (cHMGA) is phosphorylated by CK 2 at two adjacent sites. 40% of the protein population is additionally modi fied by MAP kinase, Using spectroscopic and protein footprinting techniques , we analyzed how individual and consecutive steps of phosphorylation chang e the conformation of an HMGA protein and affect its contacts with poly(dA- dT). poly(dA-dT) and a fragment of the interferon-beta promoter. We demonst rate that phosphorylation of cHMGA by CK2 alters its conformation and modul ates its DNA binding properties such that a subsequent phosphorylation by C dc2 kinase changes the organization of the protein-DNA complex. In contrast , consecutive phosphorylation by MAP kinase, which results in a dramatic ch ange in cHMGA conformation, has no direct effect on the complex. Because th e phosphorylation of the HMGA proteins attenuates binding affinity and redu ces the extent of contacts between the DNA and protein, it is likely that t his process mirrors the dynamics and diversity of regulatory processes in c hromatin.