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

doi:10.1074/jbc.273.43.28346

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

From the ^‡Department of Biochemistry, Institute for Developmental Research, Aichi Human Service Center, Kasugai, Aichi 480-0392 and the ^¶Department of Pathology, Aichi Medical University, Nagakute, Aichi 480-11, Japan

Abstract

The immunofluorescence localization of αB-crystallin in U373 MG human glioma cells with an antibody specific for αB-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 αB-crystallin in mitotic cells were detected in all cell lines examined and in tissue sections of mouse embryos. Increases in the levels of αB-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 αB-crystallin in extracts of mitotic U373 MG cells, using the amino-terminal 72-amino acid peptide derived from unphosphorylated αB2-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 αB-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.

Footnotes

↵* This work was supported in part by a Grant-in-Aid for Scientific Research on Priority Areas from the Ministry of Education, Science, Sports and Culture of Japan.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
↵§ To whom correspondence should be addressed. Dept. of Biochemistry, Institute for Developmental Research, Aichi Human Service Center, 713-8 Kamiya, Kasugai, Aichi 480-0392, Japan. Tel.: 81-568-88-0811; Fax: 81-568-88-0829; E-mail: kato{at}inst-hsc.pref.aichi.jp.
Abbreviations:

hsp

heat shock protein

MAP

mitogen-activated protein

MAPKAP

MAP kinase-activated protein

PMA

phorbol 12-myristate 13-acetate

PKA

protein kinase A

N-72K peptide

amino-terminal 72-amino acid peptide derived from αB2-crystallin

p19S

p45S, and p59S, peptides corresponding to internal sequences of human αB-crystallin that contain phosphorylated Ser-19 (residues 17–27), phosphorylated Ser-45 (residues 44–54), and phosphorylated Ser-59 (residues 57–67), respectively

Ser-19 kinase

Ser-45 kinase, and Ser-59 kinase, protein kinases responsible for phosphorylation of Ser-19, Ser-45, and Ser-59, respectively, in αB2-crystallin

PAGE

polyacrylamide gel electrophoresis

IEF

isoelectric focusing

Tricine

N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine.
- Received July 9, 1998.
- Revision received August 12, 1998.
The American Society for Biochemistry and Molecular Biology, Inc.