ABSTRACT
The mechanism of the translational thermotolerance provided by the small heat shock proteins (sHsps) alphaB-crystallin or Hsp27 is unknown. We show here that Hsp27, but not alphaB-crystallin, increased the pool of mobile stress granule-associated enhanced green fluorescent protein (EGFP)-eukaryotic translation initiation factor (eIF)4E in heat-shocked cells, as determined by fluorescence recovery after photobleaching. Hsp27 also partially prevented the sharp decrease in the pool of mobile cytoplasmic EGFP-eIF4G. sHsps did not prevent the phosphorylation of eIF2alpha by a heat shock, but promoted dephosphorylation during recovery. Expression of the C-terminal fragment of GADD34, which causes constitutive dephosphorylation of eIF2alpha, fully compensated for the stimulatory effect of alphaB-crystallin on protein synthesis in heat-shocked cells, but only partially for that of Hsp27. Our data show that sHsps do not prevent the inhibition of protein synthesis upon heat shock, but restore translation more rapidly by promoting the dephosphorylation of eIF2alpha and, in the case of Hsp27, the availability of eIF4E and eIF4G.
Subject(s)
Eukaryotic Initiation Factor-4G/metabolism , Heat-Shock Response/physiology , Protein Serine-Threonine Kinases/physiology , alpha-Crystallin B Chain/physiology , Animals , Antigens, Differentiation/biosynthesis , Antigens, Differentiation/genetics , Cell Cycle Proteins/biosynthesis , Cell Cycle Proteins/genetics , Cells, Cultured , Cytoplasmic Granules/metabolism , Eukaryotic Initiation Factor-2/metabolism , Fluorescence Recovery After Photobleaching , Humans , Intracellular Signaling Peptides and Proteins , Phosphorylation , Protein Phosphatase 1 , Protein Serine-Threonine Kinases/genetics , Up-Regulation , alpha-Crystallin B Chain/geneticsABSTRACT
The suggested common regulator of the eye lens crystallin genes is c-Maf. Maf responsive elements have been detected in a number of crystallin promoters including that of the rat betaB2-crystallin gene. The betaB2-crystallin gene is active in the post-natal lens and its mRNA reaches its maximal level in the rat lens 6 months after birth. Yet c-Maf has been reported to be present in the rat lens only up to 3 months of age. This discrepancy prompted an investigation into the role of the Maf responsive element (MARE) in the regulation of activity of the rat betaB2-crystallin promoter in rat lens fiber cells. Although betaB2 promoter activity is enhanced by c-Maf in both in vitro differentiating rat lens fiber cells and CHO cells, deletion of the betaB2 MARE, which was mapped to -143/-123, does not decrease betaB2 promoter activity in lens fiber cells. Furthermore, a dominant negative c-Maf construct did not inhibit activity of the betaB2 promoter in lens fiber cells. The data suggest that the betaB2 MARE does not play a major role in regulating activity of the betaB2 promoter. Rather, a putative Sox binding site at -164/-159 and a positive element at -14/-7 seem to be the prime regulatory elements.
