Abstract
alphaB-crystallin in cells can be phosphorylated at three serine residues in response to stress or during mitosis (Ito, H., Okamoto, K., Nakayama, H., Isobe, T., and Kato, K. (1997) J. Biol. Chem. 272, 29934-29941 and Kato, K., Ito, H., Kamei, K., Inaguma, Y., Iwamoto, I., and Saga, S. (1998) J. Biol. Chem. 273, 28346-28354). In the present study, we determined effects of phosphorylation of alphaB-crystallin on its oligomerization state, mainly by using site-directed mutagenesis, in which all three phosphorylation sites were substituted with aspartate to mimic the phosphorylation state (3D-alphaB). From results of sucrose density gradient centrifugation, we found that wild type alphaB-crystallin (wt-alphaB) and 3D-alphaB sedimented in fractions corresponding to apparent molecular masses of about 500 and 300 kDa, respectively. Chaperone-like activity of 3D-alphaB was significantly weaker than that of wt-alphaB. When wt-alphaB and 3D-alphaB were expressed in COS-m6 cells, they sedimented at positions corresponding to apparent molecular masses of about 500 and 100 kDa, respectively. In U373 MG human glioma cells, alphaB-crystallin was observed as large oligomers with apparent molecular masses about 500 kDa and the oligomerization size was reduced after phosphorylation induced by phorbol 12-myristate 13-acetate and okadaic acid. Coexpression of luciferase and wt-alphaB or 3D-alphaB in Chinese hamster ovary cells caused protection of the enzyme from heat inactivation although the degree of protection with 3D-alphaB was less than that with wt-alphaB. From these observations, it is suggested that phosphorylation of alphaB-crystallin causes dissociation of large oligomers to smaller sizes molecules and reduction of chaperone-like activity, like in the case of HSP27.
Highlights
Phosphorylation is one of the most important post-translational modifications and it is known that there are many protein kinase cascades in various organisms
The oligomerization size of ␣B-crystallin in U373 MG cells, which had been treated with phorbol 12-myristate 13-acetate (PMA) and okadaic acid to induce extensive phosphorylation, became smaller and the majority of phosphorylated forms of ␣B-crystallin were detected in fractions corresponding to a smaller oligomerization size (Figs. 6 and 7)
We and other groups previously reported that the phosphorylation of HSP27 caused similar dissociation of large oligomers [11, 32]
Summary
Phosphorylation is one of the most important post-translational modifications and it is known that there are many protein kinase cascades in various organisms. Assay of in Vitro Chaperone-like Activity—Chaperone-like activity was measured as the ability to protect against heat-induced aggregation of lactate dehydrogenase (LDH, 100 g/ml, purified from rabbit muscle, Roche Molecular Biochemicals), monitored by measuring the turbidity at 360 nm at 50 °C in 25 mM sodium phosphate buffer, pH 7.0, containing 100 mM NaCl and 2 mM EDTA for 60 min in the presence or absence of 5 or 10 g/ml recombinant ␣B-crystallin.
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