Altered patterns of phosphorylation in cultured mouse lenses during development of buthionine sulfoximine cataracts

Wenjie Li, Harold I. Calvin, Larry David, Kaili Wu, Ashley L. McCormack, Guan Ping Zhu, S. C. Joseph Fu

Research output: Contribution to journalArticle

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Abstract

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 35S-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°C with 32Pi, 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.

Original languageEnglish (US)
Pages (from-to)335-346
Number of pages12
JournalExperimental Eye Research
Volume75
Issue number3
DOIs
Publication statusPublished - 2002

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Keywords

  • 2D-gel electrophoresis
  • Actin
  • Alpha crystallin
  • Beta crystallin
  • Buthionine sulfoximine
  • Cataract
  • Filensin
  • Immunoblot
  • Lens
  • Mouse
  • Phakinin
  • Phosphorimage
  • Phosphorylation
  • Tandem mass spectrometry

ASJC Scopus subject areas

  • Ophthalmology
  • Sensory Systems

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