Implication of a small GTPase Rac1 in the activation of c-Jun N-terminal kinase and heat shock factor in response to heat shock

Heat shock induces c-Jun N-terminal kinase (JNK) activation as well as heat shock protein (HSP) expression through activation of the heat shock factor (HSF), but its signal pathway is not clearly understood. Since a small GTPase Rac1 has been suggested to participate in the cellular response to stresses, we examined whether Rac1 is involved in the heat shock response. Here we show that moderate heat shock (39-41 degrees C) induces membrane translocation of Rac1 and membrane ruffling in a Rac1-dependent manner. In addition, Rac1N17, a dominant negative mutant of Rac1, significantly inhibited JNK activation by heat shock. Since Rac1V12 was able to activate JNK, it is suggested that heat shock may activate JNK via Rac1. Similar inhibition by Rac1N17 of HSF activation in response to heat shock was observed. However, inhibitory effects of Rac1N17 on heat shock-induced JNK and HSF activation were reduced as the heat shock temperature increased. Rac1N17 also inhibited HSF activation by l-azetidine-2-carboxylic acid, a proline analog, and heavy metals (CdCl)), suggesting that Rac1 may be linked to HSF activation by denaturation of polypeptides in response to various proteotoxic stresses. However, Rac1N17 did not prevent phosphorylation of HSF1 in response to these proteotoxic stresses. Interestingly, a constitutively active mutant Rac1V12 did not activate the HSF. Therefore, Rac1 activation may be necessary, but not sufficient, for heat shock-inducible HSF activation and HSP expression, or otherwise a signal pathway(s) involving Rac1 may be indirectly involved in the HSF activation. In sum, we suggest that Rac1 may play a critical role(s) in several aspects of the heat shock response.     

An alteration in molecular form associated with activation of human heat shock factor

In higher eucaryotes, heat shock factor (HSF) exists in a cryptic form in unstressed cells. We investigated molecular forms of human HSF before and after activation by sucrose density gradient centrifugation and by gel mobility shift assay using a 32P-labeled heat shock element (HSE). We found that the in vivo or in vitro activated HSF, which is capable of binding to HSE, and its inactive form present in unstressed cells have different sedimentation coefficient; the former is 8 S whereas the latter is 4-5 S. Both the 8 S and 4-5 S forms contain the HSF polypeptide which has the ability to bind to HSE upon activation. The inactive 4-5 S form acquires HSE-binding ability when activated by heat shock or other stimuli. This HSF activity was greatly reduced, however, during recentrifugation in sucrose density gradient and, in addition, the residual activity was not recovered in 8 S fractions. Transformation of the inactive 4-5 S form of HSF to the stable, active 8 S form was achieved when the inactive form was activated and mixed with cytosols of unstressed cells.     

Stat1 mediates an auto-regulation of hsp90β gene in heat shock response

We have reported earlier that a heat shock element in the first intron of human hsp90β gene (iHSE) acts as an intronic enhancer to bind the heat shock factor (HSF1) and activates hsp90β gene under heat shock. Here, we show that, in addition to the HSF1, Stat1 phosphorylation is indispensable in the event. We show that Jak2, a Janus kinase specifically associated with the β subunit of IFNγ receptor, and PKCε? an isoform of the atypical PKC family, are the two dominant kinases responsible for the heat shock induced phosphorylation on Y701 and S727 of Stat1. However, the activation of these kinases under heat shock requires the association of chaperone proteins of the Hsp90 family, in particular, the Hsp90β under heat shock. Furthermore, Brg1, an ATPase subunit of the SWI/SNF chromatin remodeling complex is likely recruited by HSF1 and Stat1 at the iHSE under heat shock. Brg1 further confers an open chromatin conformation at the promoter region that is pivotal to the heat shock induced fully activation of the hsp90β gene in Jurkat cells. This is a novel example of how multiple activation steps occur under heat shock, first on the kinases and then the Stat1 and the SWI/SNF chromatin remodeling complex that follows to conduct an auto-regulation based fully activation of the gene.