Phosphorylation-induced Change of the Oligomerization State of αB-crystallin

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.

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

“…[135]). Similar discrepancies exist in the literature with regards to the effect of phosphorylation on Bc's chaperone activity [133,134,148,150]. Various factors may account for these apparent differences including the target protein used to assess the chaperone activity, the final concentration of sHsp in the assays (wild-type Hsp27 dissociates at low concentrations and therefore may be predominately dimeric, like the phosphorylated form, in some assays [136,138]), and buffer and temperature conditions of the assay, all of which have been shown to influence sHsp chaperone activity [133].…”

“…The main advantage of phosphomimicking forms is that they afford a single homogeneous isoform of the protein. Significantly, phosphomimics of Bc and Hsp27 have similar attributes to the phosphorylated forms of the protein with regards to their oligomeric distribution, chaperone activity, subcellular localisation and cellular trafficking [136,143,150,151]. For example, the translocation of αBc to the nucleus and its association with nuclear speckles during mitosis is phosphorylation-dependent and this is replicated by the S19D/S45D/S59D αBc phosphomimic [143] and both in vitro phosphorylated Hsp27 and the S15D/S78D/S82D 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 Hsp27 phosphomimic have comparable abilities to prevent the aggregation of target proteins in vitro [136].…”

Redistribution of GFAP and αB-crystallin after thermal stress in C6 glioma cell line

“…We expected this might be due to the phosphorylation of the protein. This could be supported by a previous study in which the phsphorylation of aB-crystallin was enhanced in U373MG cells after heat shock [36]. An association of aB-crystallin with the vimentin filaments in NIH 3T3 cells was also found after a 43°C heat shock challenge [37].…”

Crystallin proteins and amyloid fibrils

“…This method has been employed previously for αB-crystallin and led to similar effects as endogenously phosphorylated αB-crystallin with regards to its oligomeric distribution [122], subcellular localization [123] and cellular trafficking [124]. Biophysical characterisation studies of these phosphomimics have shown that an increase in the number of sites that are phosphorylated decreases the oligomerization of the protein [122] by disrupting the dimeric substructure within the oligomer [125]. Studies using these phosphomimics against both amorphous and fibrilforming target proteins have shown that the effect of phosphorylation of αB-crystallin on its chaperone activity is target protein specific [95].…”