Phosphorylation of C8 and C9 subunits of the multicatalytic proteinase by casein kinase II and identification of the C8 phosphorylation sites by direct mutagenesis.
Two 29 kDa subunits of the multicatalytic proteinase (proteasome) complex, the C8 and C9 components, are phosphorylated in vivo and can be phosphorylated in vitro by casein kinase II (CKII). The major phosphate acceptor is the C8 subunit being phosphorylated in serine, both in vivo and in vitro. The phosphopeptides generated by Glu-C endoprotease digestion from the in vivo 29 kDa labeled subunit and from the in vitro phosphorylation of the recombinant C8 subunit with CKII are identical, suggesting that CKII is likely responsible for the in vivo phosphorylation of the C8 subunit. The in vitro stoichiometry of phosphorylation of the proteasome complex and the recombinant C9 and C8 subunits by CKII is 2-2.5, 0.2, and 2 mol of phosphate per mole, respectively. Several C8 protein constructs allow the location of the CKII phosphorylation sites to be the COOH terminal portion of the protein, and direct mutational analyses show that Ser-243 and Ser-250 are the residues of the C8 subunit phosphorylated by CKII. The in vitro phosphorylation of the proteasome by CKII does not affect its proteolytic activity (on proteins or fluorogenic synthetic peptides), therefore suggesting its involvement in the interaction of the proteasome with other cellular proteins, i.e. in the formation of the 26S complex and/or in the interaction with the nuclear translocation machinery.
Castaño
J G
JG
Departamento de Bioquímica e Instituto de Investigaciones Biomédicas del Consejo Superior de Investigaciones Científicas, Facultad de Medicine de la UAM, Madrid, Spain.
Mahillo
E
E
Arizti
P
P
Arribas
J
J
D000595:Amino Acid Sequence; D001483:Base Sequence; D001665:Binding Sites; D047390:Casein Kinase II; D002478:Cells, Cultured; D003001:Cloning, Molecular; D003546:Cysteine Endopeptidases / Q000737:chemistry / Q000378:metabolism*; D017931:DNA Primers; D015180:Electrophoresis, Gel, Two-Dimensional; D008099:Liver / Q000201:enzymology; D008969:Molecular Sequence Data; D009097:Multienzyme Complexes / Q000737:chemistry / Q000378:metabolism*; D016296:Mutagenesis; D010449:Peptide Mapping; D010766:Phosphorylation; D017354:Point Mutation; D011233:Precipitin Tests; D046988:Proteasome Endopeptidase Complex; D017346:Protein Serine-Threonine Kinases / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D012694:Serine / Q000378:metabolism; D012697:Serine Endopeptidases / Q000378:metabolism
D017931:DNA Primers; D009097:Multienzyme Complexes; D011994:Recombinant Proteins; D012694:Serine; D047390:Casein Kinase II; D017346:Protein Serine-Threonine Kinases; D012697:Serine Endopeptidases; C028344:glutamyl endopeptidase; D003546:Cysteine Endopeptidases; D046988:Proteasome Endopeptidase Complex