Phosphorylation of lens intrinsic membrane proteins by protein kinase C.
Two intrinsic proteins of bovine lens membranes with apparent relative molecular masses (Mr, app) of 26,000 and 18,000 were phosphorylated in intact membranes by protein kinase C prepared from either bovine brain or lens. The kinase preparations exhibited histone H1 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 = 26,000 (identified as MP26, the main intrinsic protein of lens fiber cells), an intermediate band at Mr, app = 18,000 and several minor bands. Autoradiography of complete assay mixture containing protein kinase C, calcium, magnesium and [gamma-32P]ATP showed major bands at Mr, app = 18,000 and 26,000. Several lines of evidence indicated that the label at Mr, app = 26,000 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 = 26,000 to fragments of Mr, app .= 22,000 and 24,000. Label was not detected in the resulting Mr, app = 22,000 peptide, but the Mr, app = 24,000 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.
P D
PD
Lampe
M D
MD
Bazzi
G L
GL
Nelsestuen
R G
RG
Johnson
D000596:Amino Acids / Q000032:analysis; D000818:Animals; D020346:Aquaporins; D002417:Cattle; D004591:Electrophoresis, Polyacrylamide Gel; D005136:Eye Proteins / Q000378:metabolism*; D006868:Hydrolysis; D007908:Lens, Crystalline / Q000378:metabolism*; D008562:Membrane Glycoproteins; D010447:Peptide Hydrolases; D010766:Phosphorylation; D011493:Protein Kinase C / Q000378:metabolism*; D013194:Staining and Labeling
D000596:Amino Acids; D020346:Aquaporins; D005136:Eye Proteins; D008562:Membrane Glycoproteins; C030328:aquaporin 0; D011493:Protein Kinase C; D010447:Peptide Hydrolases