PATHWAYS OF HEAT-SHOCK PROTEIN-28 PHOSPHORYLATION BY TNF IN HUMAN FIBROBLASTS

Treatment of human diploid FS-4 fibroblasts with TNF or IL-1 led to a rapid increase in the phosphorylation of a similar to 28-kDa protein. Increased phosphorylation was seen after 5 min of TNF treatment, it re ached a plateau between 10 and 30 min, and decreased thereafter. Immun oprecipitation with specific antibodies identified the 28-kDa protein as a member of the family of small heat shock proteins (Hsp28). Treatm ent of cells with different kinase inhibitors (staurosporine, H7, H8, HA-1004, or chelerythrine chloride) failed to inhibit TNF-induced Hsp2 8 phosphorylation, suggesting that neither protein kinase C nor other common protein kinases were involved. Treatment of FS-4 cells with sod ium arsenite led to a very strong increase in the phosphorylation of H sp28 demonstrable after 5 min and persisting for at least 4 h. Tyrosin e phosphorylation of pp42 and pp44 MAP kinases was increased by TNF tr eatment, whereas arsenite produced a modest increase in tyrosine phosp horylation of pp44 while decreasing that of pp42 MAP kinase. The findi ng that sodium arsenite strongly increased Hsp28 phosphorylation, toge ther with the resistance of TNF-induced phosphorylation to kinase inhi bitors, supports the notion that increased serine phosphorylation of H sp28 in this system involves inhibition of protein phosphatase activit y.