Phosphorylation-induced Change of the Oligomerization state of alpha B-crystallin

alphaB-crystallin in cells can be phosphorylated at three serine residues i n 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. Bio l. Chem. 273, 28346-28354). In the present study, we determined effects of phosphorylation of alphaB-crystallin on its oligomerization state, mainly b y using site-directed mutagenesis, in which all three phosphorylation sites were substituted with aspartate to mimic the phosphorylation state (3D-alp haB). From results of sucrose density gradient centrifugation, we found tha t wild type alphaB-crystallin (wt-alphaB) and 3D-alphaB sedimented in fract ions corresponding to apparent molecular masses of about 500 and 300 kDa, r espectively. 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 cell s, alphaB-crystallin was observed as large oligomers with apparent molecula r masses about 500 kDa and the oligomerization size was reduced after phosp horylation induced by phorbol 12-myristate 13-acetate and okadaic acid. Coe xpression 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-crystal lin causes dissociation of large oligomers to smaller sizes molecules and r eduction of chaperone-like activity, like in the case of HSP27.