PHOSPHORYLATION OF ALPHA-BETA-CRYSTALLIN IN MITOTIC CELLS AND IDENTIFICATION ENZYMATIC-ACTIVITIES RESPONSIBLE FOR PHOSPHORYLATION

The immunofluorescence localization of alpha B-crystallin in U373 MG h uman glioma cells with an antibody specific for alpha B-crystallin tha t had been phosphorylated at Ser-45 revealed an intense staining of ce lls in the mitotic phase of the cell cycle. Phosphorylated forms of al pha B-crystallin in mitotic cells were detected in all cell lines exam ined and in tissue sections of mouse embryos. Increases in the levels of alpha B-crystallin that had been phosphorylated at Ser-45 and Ser-1 9, but not at Ser-59, were detected biochemically by isoelectric focus ing or SDS-polyacrylamide gel electrophoresis and a subsequent Western blot analysis of extracts of cells collected at the mitotic phase. Wh en we estimated the phosphorylation activity specific for alpha B-crys tallin in extracts of mitotic U373 MG cells, using the amino-terminal 72-amino acid peptide derived from unphosphorylated alpha B2-crystalli n as the substrate, we found that the activities responsible for the p hosphorylation of Ser-45 and Ser-19 were markedly enhanced but that th e activity responsible for the phosphorylation of Ser-59 was suppresse d. The protein kinases responsible for the phosphorylation of Ser-45 a nd Ser-59 in the amino-terminal 72-amino acid peptide were partially p urified from extracts of cells that had been stimulated by exposure to H2O2 in the presence of calyculin A. The activities responsible for t he phosphorylation of Ser-45 and Ser-59 were eluted separately from a column of Superdex 200 at fractions corresponding to about 40 and 60 k Da, respectively, while the kinase for Ser-19 was unstable. p44/42 mit ogen-activated protein (MAP) kinase and MAP kinase-activated protein ( MAPKAP) kinase-2 were concentrated in the Ser-45 kinase fraction and S er-59 kinase fraction, respectively. Recombinant human p44 MAP kinase and MAPKAP kinase-l purified from rabbit muscle selectively phosphoryl ated Ser-45 and -59, respectively. The Ser-45 kinase fraction and Ser- 59 kinase fraction phosphorylated myelin basic protein and hsp27, resp ectively. These results suggest that the phosphorylations of Ser-45 an d Ser-59 in alpha B-crystallin are catalyzed by p44/42 MAP kinase and MAPKAP kinase-a, respectively, in cells and that the phosphorylation o f Ser-45 by p44/42 MAP kinase is enhanced while the phosphorylation of Ser-59 by MAPKAP kinase-a is suppressed during cell division.