Phosphorylation of HMG-I by protein kinase C attenuates its binding affinity to the promoter regions of protein kinase C gamma and neurogranin/RC3 genes

A 20-kDa DNA-binding protein that binds the AT-rich sequences within the pr omoters of the brain-specific protein kinase C (PKC) gamma and neurogranin/ RC3 genes has been characterized as chromosomal nonhistone high-mobility-gr oup protein (HMG)-I, This protein is a substrate of PKC alpha, beta, gamma, and delta but is poorly phosphorylated by PKC epsilon and zeta. Two major (Ser(44) and Ser(64)) and four minor phosphorylation sites have been identi fied. The extents of phosphorylation of Ser(44) and Ser(64) were 1:1, where as those of the four minor sites all together were <30% of the major one. T hese PKC phosphorylation sites are distinct from those phosphorylated by cd c2 kinase, which phosphorylates Thr(53) and Thr(78). Phosphorylation of HMG -I by PKC resulted in a reduction of DNA-binding affinity by 28-fold as com pared with 12-fold caused by the phosphorylation with cdc2 kinase, HMG-I co uld be additively phosphorylated by cdc2 kinase and PKC, and the resulting doubly phosphorylated protein exhibited a >100-fold reduction in binding af finity. The two cdc2 kinase phosphorylation sites of HMG-I are adjacent to the N terminus of two of the three predicted DNA-binding domains. In compar ison, one of the major PKC phosphorylation sites, Ser(64), is adjacent to t he C terminus of the second DNA-binding domain, whereas Ser(44) is located within the spanning region between the first and second DNA-binding domains . The current results suggest that phosphorylation of the mammalian HMG-I b y PKC alone or in combination with cdc2 kinase provides an effective mechan ism for the regulation of HMG-I function.