Pseudophosphorylated αB-Crystallin Is a Nuclear Chaperone Imported into the Nucleus with Help of the SMN Complex

Abstract

The human small heat shock protein αB-crystallin (HspB5) is a molecular chaperone which is mainly localized in the cytoplasm. A small fraction can also be found in nuclear speckles, of which the localization is mediated by successional phosphorylation at Ser-59 and Ser-45. αB-crystallin does not contain a canonical nuclear localization signal sequence and the mechanism by which αB-crystallin is imported into the nucleus is not known. Here we show that after heat shock pseudophosphorylated αB-crystallin mutant αB-STD, in which all three phosphorylatable serine residues (Ser-19, Ser-45 and Ser-59) were replaced by negatively charged aspartate residues, is released from the nuclear speckles. This allows αB-crystallin to chaperone proteins in the nucleoplasm, as shown by the ability of αB-STD to restore nuclear firefly luciferase activity after a heat shock. With the help of a yeast two-hybrid screen we found that αB-crystallin can interact with the C-terminal part of Gemin3 and confirmed this interaction by co-immunoprecipitation. Gemin3 is a component of the SMN complex, which is involved in the assembly and nuclear import of U-snRNPs. Knockdown of Gemin3 in an in situ nuclear import assay strongly reduced the accumulation of αB-STD in nuclear speckles. Furthermore, depletion of SMN inhibited nuclear import of fluorescently labeled recombinant αB-STD in an in vitro nuclear import assay, which could be restored by the addition of purified SMN complex. These results show that the SMN-complex facilitates the accumulation of hyperphosphorylated αB-crystallin in nuclear speckles, thereby creating a chaperone depot enabling a rapid chaperone function in the nucleus in response to stress.

Drosophila Small Heat Shock Proteins: An Update on Their Features and Functions

“…Analysis of the CG43851 sequence predicts nuclear or cytoplasmic localization depending on the prediction tool used (Euk-mPLoc 2.0 (Chou and Shen 2010 ), SLP-Local (Matsuda et al 2005 ), CELLO v2.5 (Yu et al 2006 ), Sherloc2 ), MultiLoc2 . In mammals, all sHsps are mainly located in the cytoplasm but some shuttle to the nucleus or to the mitochondria (Bryantsev et al 2007 ;den Engelsman et al 2013 ;Marunouchi et al 2013 ;Nakagawa et al 2001 ;van den IJssel et al 2003 ). Since Drosophila sHsps are localized in different compartments, it has long been thought that their shuttling to other cellular compartments was not needed.…”

Regulation of αA- and αB-crystallins via phosphorylation in cellular homeostasis

“…It is believed that this prevents translocation of Bcl2 into the mitochondria, repressing Bcl2 antiapoptotic ability [38], thereby limiting cell immortality and cancer pathogenesis. Dual phosphorylation at Ser59 in conjunction with Ser45 induced by PAR-2 activation has also been implicated in the protection of astrocytes against cytotoxic stress induced with C2-ceramide and staurosporine [49], and in the formation of nuclear speckles in HeLa cells [47,48]. It was reported that aB phosphorylated at Ser59 interacts with Gemin-3, part of the Survival Motor Neuron complex [48], and is translocated into the nucleoplasm where phosphorylation at Ser45 is involved in speckle formation [47].…”

“…Dual phosphorylation at Ser59 in conjunction with Ser45 induced by PAR-2 activation has also been implicated in the protection of astrocytes against cytotoxic stress induced with C2-ceramide and staurosporine [49], and in the formation of nuclear speckles in HeLa cells [47,48]. It was reported that aB phosphorylated at Ser59 interacts with Gemin-3, part of the Survival Motor Neuron complex [48], and is translocated into the nucleoplasm where phosphorylation at Ser45 is involved in speckle formation [47]. Nuclear speckles act as depots to ensure a readily available source of chaperone in the event of cellular stress, upon which aB is then released from the speckles to prevent cell death [48].…”

“…It was reported that aB phosphorylated at Ser59 interacts with Gemin-3, part of the Survival Motor Neuron complex [48], and is translocated into the nucleoplasm where phosphorylation at Ser45 is involved in speckle formation [47]. Nuclear speckles act as depots to ensure a readily available source of chaperone in the event of cellular stress, upon which aB is then released from the speckles to prevent cell death [48]. Interestingly, the hyperphosphorylated R120G aB mutant is unable to be translocated into the nucleus due to the formation of cytoplasmic inclusions [47].…”

“…In the lenticular environment, the crystallins are susceptible to the accumulation of posttranslational modifications, due largely to their extended life spans. Such modifications, notably phosphorylation, have also been reported in extralenticular aB, often being implicated in the regulation of the protein's activity [3,[36][37][38][39][40][41][42][43][44][45][46][47][48][49][50]. Although such modifications have been extensively reported in the lens in recent years [23-25, 27-31], the implications, particularly for aA and aB functions, have yet to be characterised.…”