Altered patterns of phosphorylation in cultured mouse lenses during development of buthionine sulfoximine cataracts.
Buthionine sulfoximine (BSO), a specific inhibitor of glutathione biosynthesis, induces oxidative cataracts following multiple injections into mice at 1 week of age. Cultures of lenses with (35)S-methionine have previously demonstrated altered patterns of protein biosynthesis that precede and accompany these cataracts. To obtain parallel information about changes in protein phosphorylation during cataract development, lenses from BSO-treated or control mouse pups were cultured for 3 hr at 37 degrees C with (32)P(i), homogenized in phosphate buffer, and resolved by centrifugation into water-soluble (WS) and water-insoluble (WI) fractions. These were characterized by 2D-gel electrophoresis, Coomassie blue staining, phosphorimaging, immunoblotting, and tandem mass spectrometry. Heaviest labelling was in the WI fraction. The labelled 2D-gel spots included: (1) a series of phosphorylated filensins at 95 kDa; (2) a major radioactive spot at 45-50 kDa, slightly anodic to actin and the beaded filament protein, phakinin (CP 49); (3) a phosphorylated betaB1-crystallin, considerably anodic to parent betaB1; (4) an acidic cluster of labelled alphaA-crystallins, phosphorylated in part at serine-148, and (5) a labelled trace alpha crystallin, slightly anodic to alphaB-crystallin. The results confirm previously reported phosphorylations of actin, phakinin, alphaA- and alphaB-crystallin, demonstrate previously unrecognized phosphorylations of filensin and betaB1-crystallin, and provide unequivocal evidence for phosphorylation of alphaA-crystallin at serine-148. The earliest changes in phosphorylation detected after BSO treatment were increased labelling of alphaA- and alphaB-crystallin during cataract stages 1-3, coupled with a general decrease in protein labelling. In stage 5 cataracts, phosphorylated alpha crystallins persisted as the dominant labelled species. However, the major modifications of alphaA-crystallin in advanced BSO cataracts were unlabelled and partially degraded, in contrast to phosphorylated alphaA. It is therefore proposed that phosphorylation of alphaA-crystallin may confer resistance to proteolytic degradation.
Li
Wenjie
W
Department of Biochemistry and Molecular Biology, UMDNJ-New Jersey Medical School, Newark, NJ 07103, USA.
Calvin
Harold I
HI
David
Larry L
LL
Wu
Kaili
K
McCormack
Ashley L
AL
Zhu
Guan-Ping
GP
Fu
S-C Joseph
SC
D000818:Animals; D015153:Blotting, Western; D019328:Buthionine Sulfoximine; D002386:Cataract / Q000139:chemically induced / Q000378:metabolism*; D003459:Crystallins / Q000378:metabolism*; D046508:Culture Techniques; D018450:Disease Progression; D015180:Electrophoresis, Gel, Two-Dimensional; D005136:Eye Proteins / Q000378:metabolism; D005260:Female; D007381:Intermediate Filament Proteins / Q000378:metabolism; D007908:Lens, Crystalline / Q000378:metabolism*; D013058:Mass Spectrometry; D051379:Mice; D008815:Mice, Inbred Strains; D010766:Phosphorylation; D038202:alpha-Crystallin A Chain / Q000378:metabolism
D003459:Crystallins; D005136:Eye Proteins; D007381:Intermediate Filament Proteins; D038202:alpha-Crystallin A Chain; C079098:filensin; D019328:Buthionine Sulfoximine