Analysis of a sub-proteome which co-purifies with and is phosphorylated by the Golgi casein kinase.
In an attempt to gain information about the identity of the Golgi apparatus casein kinase(s) (G-CK), responsible for the phosphorylation of caseins in lactating mammary gland, the proteins present in fractions enriched in G-CK activity eluted from DEAE-Sepharose and heparin-Sepharose columns were resolved by two-dimensional electrophoresis and analyzed by mass spectrometry. This led to the identification of 47 proteins altogether, none of which is a bona fide protein kinase. At least 9 of the identified proteins however, are readily phosphorylated by co-purifying G-CK activity, and 7 are physically associated with it to give supramolecular complex(es) of about 500 kDa as judged from Superdex S200 gel fitration and glycerol gradient ultracentrifugation experiments. In contrast, the apparent molecular weight of G-CK estimated from an in gel activity assay after SDSPAGE and renaturation is about 41 kDa. Many of the proteins phosphorylated by and/or associated with G-CK belong to the category of chaperonines, including HSP90, GRP-94 and -78, and various isoforms of protein disulfide isomerases, suggesting a global role of this kinase in the modulation of protein folding.
Tibaldi
E
E
Department of Biochemistry, University of Padua, Viale G. Colombo 3, 35121 Padua, Italy.
Arrigoni
G
G
Brunati
A M
AM
James
P
P
Pinna
L A
LA
D000595:Amino Acid Sequence; D000818:Animals; D047388:Casein Kinases / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D002364:Caseins / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D005260:Female; D006056:Golgi Apparatus / Q000201:enzymology*; D066298:In Vitro Techniques; D007774:Lactation; D008321:Mammary Glands, Animal / Q000201:enzymology*; D008969:Molecular Sequence Data; D010766:Phosphorylation; D011494:Protein Kinases / Q000737:chemistry / Q000302:isolation & purification*; D020543:Proteome / Q000737:chemistry; D051381:Rats
D002364:Caseins; D020543:Proteome; D011494:Protein Kinases; D047388:Casein Kinases