Phosphorylation of the human ubiquitin-conjugating enzyme, CDC34, by casein kinase 2

The ubiquitin-conjugating enzyme, CDC34, has been implicated in the ubiquit ination of a number of vertebrate substrates, including p27(Kipl), I kappaB alpha, Weel, and MyoD. We show that mammalian CDC34 is a phosphoprotein th at is phosphorylated in proliferating cells. By yeast two-hybrid screening, we identified the regulatory (beta) subunit of human casein kinase 2 (CK2) as a CDC34-interacting protein and show that human CDC34 interacts in vivo with CK2 beta in transfected cells. CDC34 is specifically phosphorylated i n vitro by recombinant CK2 and HeLa nuclear extract at five sites within th e carboxyl-terminal 36 amino acids of CDC34. Importantly, this phosphorylat ion is inhibited by heparin, a substrate-specific inhibitor of CK2. We have also identified a kinase activity associated with CDC34 in proliferating c ells, and we show that this kinase is sensitive to heparin and can utilize GTP, strongly suggesting it is CK2. Phosphorylation of CDC34 by the associa ted kinase maps predominantly to residues 203 and 222. Mutation of CDC34 at CK2-targeted residues, Ser-203, Ser-222, Ser-231, Thr-233, and Ser-236, ab olishes the phosphorylation of CDC34 observed in vivo and markedly shifts n uclearly localized CDC34 to the cytoplasm. These results suggest a potentia l role for CK2-mediated phosphorylation in the regulation of CDC34 cell loc alization and function.