Phosphorylation of αB-crystallin in Mitotic Cells and Identification of Enzymatic Activities Responsible for Phosphorylation

Kanefusa Kato,Hidenori Ito,Keiko Kamei,Yutaka Inaguma,Ikuko Iwamoto,Shinsuke Saga

doi:10.1074/jbc.273.43.28346

Abstract

The immunofluorescence localization of alphaB-crystallin in U373 MG human glioma cells with an antibody specific for alphaB-crystallin that had been phosphorylated at Ser-45 revealed an intense staining of cells in the mitotic phase of the cell cycle. Phosphorylated forms of alphaB-crystallin in mitotic cells were detected in all cell lines examined and in tissue sections of mouse embryos. Increases in the levels of alphaB-crystallin that had been phosphorylated at Ser-45 and Ser-19, but not at Ser-59, were detected biochemically by isoelectric focusing or SDS-polyacrylamide gel electrophoresis and a subsequent Western blot analysis of extracts of cells collected at the mitotic phase. When we estimated the phosphorylation activity specific for alphaB-crystallin in extracts of mitotic U373 MG cells, using the amino-terminal 72-amino acid peptide derived from unphosphorylated alphaB2-crystallin as the substrate, we found that the activities responsible for the phosphorylation of Ser-45 and Ser-19 were markedly enhanced but that the activity responsible for the phosphorylation of Ser-59 was suppressed. The protein kinases responsible for the phosphorylation of Ser-45 and Ser-59 in the amino-terminal 72-amino acid peptide were partially purified from extracts of cells that had been stimulated by exposure to H2O2 in the presence of calyculin A. The activities responsible for the phosphorylation of Ser-45 and Ser-59 were eluted separately from a column of Superdex 200 at fractions corresponding to about 40 and 60 kDa, respectively, while the kinase for Ser-19 was unstable. p44/42 mitogen-activated protein (MAP) kinase and MAP kinase-activated protein (MAPKAP) kinase-2 were concentrated in the Ser-45 kinase fraction and Ser-59 kinase fraction, respectively. Recombinant human p44 MAP kinase and MAPKAP kinase-2 purified from rabbit muscle selectively phosphorylated Ser-45 and -59, respectively. The Ser-45 kinase fraction and Ser-59 kinase fraction phosphorylated myelin basic protein and hsp27, respectively. These results suggest that the phosphorylations of Ser-45 and Ser-59 in alphaB-crystallin are catalyzed by p44/42 MAP kinase and MAPKAP kinase-2, respectively, in cells and that the phosphorylation of Ser-45 by p44/42 MAP kinase is enhanced while the phosphorylation of Ser-59 by MAPKAP kinase-2 is suppressed during cell division.

Highlights

␣-Crystallin, a major structural protein of the vertebrate eye lens, is a polymeric protein with a molecular mass of about 800 kDa
We raised antibodies in rabbits that recognized ␣B-crystallin that had been phosphorylated at each of the three serine residues individually [24]. By using these antibodies, we found that the phosphorylation at each site in ␣B-crystallin is regulated differently during mitosis, and we obtained evidence suggesting that p44/42 mitogen-activated protein (MAP) kinase and MAP kinase-activated protein (MAPKAP) kinase-2 are responsible for phosphorylation of Ser-45 and Ser-59, respectively, in ␣B-crystallin in vivo
Ser-59 kinase and MAPKAP kinase-2 phosphorylated, to a similar extent, hsp27 and the peptides derived from ␣B2-crystallin and hsp27 but barely phosphorylated ␣B2-crystallin. These results suggest that the phosphorylation of ␣B-crystallin is catalyzed by three different protein kinases; that Ser-45 and -59 in the molecule were phosphorylated by p44/42 MAP kinase and MAPKAP kinase-2, respectively; and that the phosphorylation of Ser-45 by p44/42 MAP kinase is enhanced while the phosphorylation of Ser-59 by MAPKAP kinase-2 is suppressed during cell division

Summary

Introduction

␣-Crystallin, a major structural protein of the vertebrate eye lens, is a polymeric protein with a molecular mass of about 800 kDa. Aliquots of 2–15 ␮l of the mixture were subjected to Tricine/ SDS-PAGE and a Western blot analysis with antibodies that recognized each of the phosphorylated serine residues or with antibodies against the amino-terminal peptide of ␣B-crystallin, as described above.

Results

Conclusion