Overexpression of HSP‐70 inhibits the phosphorylation of HSF1 by activating protein phosphatase and inhibiting protein kinase C activity

This laboratory reported previously that overexpressed heat shock protein 70 kDa (HSP‐70) inhibited the activation of its transcriptional factor, HSF1. We had conducted experiments to understand the mechanisms whereby HSP‐70 down‐regulated the activation of HSF1. Genetically overexpressed HSP‐70 had no effects on the HSF1 level in cytosol, but significantly inhibited phosphorylation of HSF1 in the nucleus. Transfection of cells with HSF1 cDNA resulted in increases in the unphosphorylated, but not phosphorylated, HSF1 levels in both the cytosol and nucleus. Because serine phosphorylation of various proteins was reduced in HSP‐70 cDNA‐transfected cells, we measured the activity of enzymes involved in serine phosphorylation. Overexpressed HSP‐70 significantly inhibited the enzymatic activities of protein kinase A (PKA by 73 and 62% in the cytosol and membrane‐bound fraction, respectively) and protein kinase C (PKC by 61% in membrane‐bound fraction), whereas it activated that of protein phosphatase (PP by 33 and 86% in the cytosol and the membrane‐bound fraction, respectively). Forskolin (a PKA stimulator), PMA (a PKC stimulator), and okadaic acid (an inhibitor of PP) were used to investigate whether HSP‐70‐induced changes in PKA, PKC, and PP were responsible for the HSF1 dephosphorylation. For‐skolin did not change nuclear HSF1 phosphorylation, suggesting that decreases in PKA activity in HSP‐70 overexpressing cells is not associated with HSF1 phosphorylation. PMA and okadaic acid induced an increase in HSF1 phosphorylation in both vector‐and HSP‐70 cDNA‐transfected cells, although levels of phosphorylated HSF1 in HSP‐70 cDNA‐transfected cells were lower than those in vector‐transfected cells. The PMA‐induced increase in HSF1 phosphorylation in HSP‐70 cDNA‐transfected cells was blocked by pretreatment with stau‐rosporine, a PKC inhibitor. These results suggest that overexpression of HSP‐70 inhibits phosphorylation of HSF1 at serine residues by activating PP and inhibiting PKC activity.—Ding, X. Z., Tsokos, G. C., Kiang, J. G. Overexpression of HSP‐70 inhibits the phosphorylation of HSF1 by activating protein phosphatase and inhibiting protein kinase C activity FASEB J. 12, 451–459 (1998)

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