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

Abstract

The immunofluorescence localization of ␣B-crystallin in U373 MG human glioma cells with an antibody specific for ␣B-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 ␣B-crystallin in mitotic cells were detected in all cell lines examined and in tissue sections of mouse embryos. Increases in the levels of ␣B-crystallin that had been phosphorylated at Ser-45 and Ser-19, but not at Ser-59, were detected biochemically by isoelectric focusing or SDSpolyacrylamide 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 ␣B-crystallin in extracts of mitotic U373 MG cells, using the amino-terminal 72-amino acid peptide derived from unphosphorylated ␣B2-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 ␣-Crystallin, a major structural protein of the vertebrate eye lens, is a polymeric protein with a molecular mass of about 800 kDa. The ␣-crystallin of the bovine lens is composed predominantly of two types of polypeptide, the A (␣A1 and ␣A2) and B (␣B1 and ␣B2) subunits (1). ␣A1 and ␣B1 are the phosphorylated forms of the primary gene products, ␣A2 and ␣B2 (2, 3). The molecular mass of each subunit is about 20 kDa, and the similarity between the primary structures of the two subunits is greater than 50% (4). The ␣-crystallins also share sequence similarity with the small heat shock proteins (hsps) 1 of numerous species (5). Because of the striking similarities among the primary structures of the carboxyl-terminal half of each molecule (the ␣-crystallin domain), ␣A-crystallin, ␣B-crystallin, hsp27, and p20 (6) are considered to be members of the ␣-crystallin small hsp family in vertebrates (7,8).A common feature of small hsps is their formation of large oligomeric complexes such as ␣A-crystallin and ␣B-crystallin in the lens. In the skeletal muscle, ␣B-crystallin, hsp27, and p20 seem to form a large heteropolymer, because the three proteins were copurified from the extract and coimmunoprecipitated with antibodies against each of the three proteins (6, 9). The ␣-crystallin domain of each small hsp was suggested to be important for this complex formation and the chaperone activity (8). Both ␣A-crystallin (10) and ␣B-crystallin (11) are also present in nonlenticular tissues, and the expression of ␣B-crystallin, but not ␣A-crystallin and p20, is induced in cells under various stressful conditions, as is hsp27 (12, 13).The major posttranscriptional modifications of the ␣-crystallin small hsp family are due to the phosphorylation of serine residues. The phosphorylation of hsp27 by mitogen-activated protein (MAP) kinase-activated protein (MAPKAP) kinase-2 is enhanced when cells are exposed to heat (14, 15) or chemicals (16). p20 in vascular smooth m...

MAPKAPK-2 modulates p38-MAPK localization and small heat shock protein phosphorylation but does not mediate the injury associated with p38-MAPK activation during myocardial ischemia

“…Instead, despite absence of ischemia-induced HSP25 phosphorylation in MK2 −/− hearts, infarct size was similar to that in MK2 +/+ hearts, implying that HSP25 phosphorylation is not critical to outcome in ischemia. Earlier studies had indicated that activation of p38 MAPK and MK2 are necessary for the phosphorylation of αB-crystallin on Ser-59 (Kato et al 1998;Ito et al 1997). In the absence of MK2, phosphor- ylation of αB-crystallin on Ser-59 was reduced in response to ischemia but was not completely abolished, implying that other kinases may act in concert with MK2 to achieve maximal αB-crystallin phosphorylation.…”

“…Activated p38 MAPK phosphorylates nuclear MK2 and forms a complex whereby an MK2 nuclear export signal is unmasked, resulting in its rapid export from the nucleus (Maulik et al 1996;Engel et al 1995;Ben Levy et al 1998). In the cytoplasm, MK2 phosphorylates the small heat shock proteins (HSP) 25/27 (Stokoe et al 1992;Freshney et al 1994;Rouse et al 1994) and αB-crystallin (Hoover et al 2000;Ito et al 2001;Kato et al 1998). The phosphorylation of HSP25/27 induces its dissociation from large aggregates into dimers and monomers (Kato et al 1994).…”

“…It has been proposed that αB-crystallin and HSP25/27 mediate cardioprotection by binding to proteins of the cytoskeleton and the myofilaments to attenuate ischemic injury (Eaton et al 2000). Overexpression of αB-crystallin has been shown to attenuate ischemic injury in cultured cardiac myocytes (Bluhm et al 1998) and in transgenic mice (Ray et al 2001), and the cytoprotection appears dependent on the phosphorylation of the Ser-59 residue, which in turn is dependent on MAPKAPK-2 (Kato et al 1998) and p38 MAPK (Morrison et al 2003).…”

Small heat-shock proteins: important players in regulating cellular proteostasis

“…For example, Bc is phosphorylated at three serine residues, Ser19, Ser45 and Ser59 [118,120,121]; phosphorylation at Ser45 is mediated by p44/p42 MAPK, at Ser59 by MAPKAPK-2 [118,122], whilst the kinase responsible for phosphorylation at Ser19 remains to be identified. Similarly, Hsp27 has three serine residues (Ser15, Ser78 and Ser82) that undergo phosphorylation [123,124].…”

Crystallin proteins and amyloid fibrils

“…Both inside and outside the lens, the major post-translational modification described for αB-crystallin and Hsp25 is phosphorylation at three serine residues; in αB-crystallin these are S19, S45 and S59 (S15, S78 and S82 in Hsp27) [111][112][113][114]. Various types of cellular stress, such as heat, oxidation and increased intracellular calcium levels, stimulate the phosphorylation of sHsps [111].…”