cAMP-dependent phosphorylation of bovine lens alpha-crystallin.

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Abstract
The A1 and B1 chains of bovine lens .alpha.-crystallin are phosphorylated. When soluble preparations from lens cortex are incubated with [.gamma.-32P]ATP, a cAMP-dependent labeling of a high molecular weight protein is obtained. After NaDodSO4/PAGE [sodium dodecyl sulfate/polyacrylamide gel electrophoresis], the label is found in 2 bands with MW 22,000 and 20,000, corresponding to the B and A chains of .alpha.-crystallin, respectively. Isoelectric focusing indicates that the radioactivity is almost exclusively in bands with pI [isoelectric point] values of 5.58 and 6.70, corresponding to the A1 and B1 chains, respectively. Similar results are obtained in experiments of [32P]orthophosphate incorporation in lens organ culture. Analyses of the digested protein indicate the label is exclusively in phosphoserine. 31P NMR analyses of native, proteolytically digested, and urea-treated .alpha.-crystallin gives a chemical shift of 4.6 ppm relative to 85% H3PO4 at pH 7.4, suggesting that the phosphate is covalently bound to a serine in the protein. An abundance of .apprx. 1 phosphate per 4 or 5 monomer units was found. Similar results were obtained by chemical analyses of independently prepared .alpha.-crystallin samples. The A1 and B1 chains arise as a result of the phosphorylation of directly synthesized A2 and B2 polypeptides. This metabolically controlled phosphorylation may be associated with the terminal differentiation of the lens epithelial cell and the intracellular organization of the lens fiber cell.