Phosphorylation of lens intrinsic membrane proteins by protein kinase C

Paul D. LAMPE, Mohammed D. BAZZI, Gary L. NELSESTUEN, Ross G. JOHNSON

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Abstract

Two intrinsic proteins of bovine lens membranes with apparent relative molecular masses (Mr,app) of 26000 and 18000 were phosphorylated in intact membranes by protein kinase C prepared from either bovine brain or lens. The kinase preparations exhibited histone H 1 phosphorylation dependent on calcium and phospholipid but not on cAMP. Sodium dodecyl sulfate/polyacrylamide gel electrophoresis of the lens membranes showed a major band at Mr,app= 26000 (identified as MP26, the main intrinsic protein of lens fiber cells), an intermediate band at Mr,app= 18000 and several minor bands. Autoradiography of complete assay mixture containing protein kinase C, calcium, magnesium and [γ‐32P]ATP showed major bands at Mr,app= 18000 and 26000. Several lines of evidence indicated that the label at Mr,app= 26000 was associated with MP26, a protein which has been found in lens junctions and which may form cell‐cell channels. Treatment of the phosphorylated membranes with chymotrypsin and V8 protease cleaved the major band at Mr,app= 26000 to fragments of Mr,app= 22000 and 24000. Label was not detected in the resulting Mr,app= 22000 peptide, but the Mr,app= 24000 peptide was found to be labeled. Phosphoamino acid analysis of MP26 indicated that approximately 75% of the label was on phosphoserine and 25% was on phosphothreonine. No label was found on phosphotyrosine. These results differ from those reported for cAMP‐dependent phosphorylation of lens proteins. Phosphorylation by protein kinase C may account for some of the labeling of MP26 detected in vivo.

Original languageEnglish (US)
Pages (from-to)351-357
Number of pages7
JournalEuropean Journal of Biochemistry
Volume156
Issue number2
DOIs
StatePublished - Apr 1986

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