Phosphorylation of the human ubiquitin-conjugating enzyme, CDC34, by casein kinase 2.
The ubiquitin-conjugating enzyme, CDC34, has been implicated in the ubiquitination of a number of vertebrate substrates, including p27(Kip1), IkappaBalpha, Wee1, and MyoD. We show that mammalian CDC34 is a phosphoprotein that 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 CK2beta in transfected cells. CDC34 is specifically phosphorylated in vitro by recombinant CK2 and HeLa nuclear extract at five sites within the carboxyl-terminal 36 amino acids of CDC34. Importantly, this phosphorylation is inhibited by heparin, a substrate-specific inhibitor of CK2. We have also identified a kinase activity associated with CDC34 in proliferating cells, and we show that this kinase is sensitive to heparin and can utilize GTP, strongly suggesting it is CK2. Phosphorylation of CDC34 by the associated 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, abolishes the phosphorylation of CDC34 observed in vivo and markedly shifts nuclearly localized CDC34 to the cytoplasm. These results suggest a potential role for CK2-mediated phosphorylation in the regulation of CDC34 cell localization and function.
Block
K
K
Department of Molecular Medicine, Institute of Biotechnology, University of Texas Health Science Center at San Antonio, San Antonio, Texas 78245-3207, USA.
Boyer
T G
TG
Yew
P R
PR
D000595:Amino Acid Sequence; D064173:Anaphase-Promoting Complex-Cyclosome; D000818:Animals; D047390:Casein Kinase II; D002455:Cell Division; D002457:Cell Extracts; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006160:Guanosine Triphosphate / Q000378:metabolism; D006367:HeLa Cells; D006493:Heparin / Q000494:pharmacology; D006801:Humans; D008025:Ligases / Q000378:metabolism*; D051379:Mice; D008822:Mice, Transgenic; D008969:Molecular Sequence Data; D010766:Phosphorylation; D017346:Protein Serine-Threonine Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism; D013379:Substrate Specificity; D014162:Transfection; D020798:Two-Hybrid System Techniques; D044763:Ubiquitin-Conjugating Enzymes; D043743:Ubiquitin-Protein Ligase Complexes; D044767:Ubiquitin-Protein Ligases
D002457:Cell Extracts; D004791:Enzyme Inhibitors; D011994:Recombinant Proteins; D006160:Guanosine Triphosphate; D006493:Heparin; C577447:CDC34 protein, human; D044763:Ubiquitin-Conjugating Enzymes; D043743:Ubiquitin-Protein Ligase Complexes; D064173:Anaphase-Promoting Complex-Cyclosome; D044767:Ubiquitin-Protein Ligases; D047390:Casein Kinase II; D017346:Protein Serine-Threonine Kinases; D008025:Ligases