Analysis of HSF-1 phosphorylation in A549 cells treated with a variety of stresses

Abstract

In the absence of stress, heat shock transcription factor-1 (HSF-1) exists as a monomer. After the treatment of cells with variety of stresses, HSF-1 forms a trimer and binds to the heat shock element (HSE), a motif consisting of three consecutive NGAAN sequences located in an inverted orientation upstream of the heat shock genes. HSF-1 is then phosphorylated causing transactivation of heat shock mRNAs. Treatment of cells with some of the stresses has been shown to increase HSF binding to HSE without detectibly increasing the synthesis of heat shock mRNAs. Here we used antibody specific to HSF-1 to detect its phosphorylation status following exposure of A549, a human lung carcinoma cell line to a variety of stresses in order to correlate HSF-1 phosphorylation with its transactivation ability. Our studies show that HSF-1 is phosphorylated following heat shock (43 °C for 1 h), hypoxia (5 h exposure to 0·02% oxygen), 8% ethanol (1 h exposure at 37°C), or 200 μM sodium arsenite (1 h exposure at 37°C). All such stresses have previously been shown to increase the synthesis of heat shock proteins (hsps). However, there are no detectable increases in HSF-1 phosphorylation after the treatment of cells with X-irradiation (2–8 Gy) or 100 μM canavanine, an amino acid analogue (1 h exposure at 37°C). Treatment of cells with X-irradiation increases HSF binding to HSE without increasing the synthesis of hsps, while treatment of cells with canavanine has been shown to increase the synthesis of hsps. These results suggest that with the exception of amino acid analogues, all stresses which cause an increase in HSF-1 phosphorylation also enhance the synthesis of hsps. We also attempted to inhibit phosphorylation of HSF-1 with protein kinase inhibitors. Neither herbimycin A, a general protein tyrosine kinase inhibitor, nor staurosporine, a ser/thr protein kinase inhibitor interfered with the phosphorylation of HSF-1 in A549 cells.