GELDANAMYCIN DISRUPTS THE P56LCK-HEAT SHOCK PROTEIN-90 COMPLEX IN T-CELLS

Abstract

523 Heat shock protein 90 (hsp90) forms complexes with newly translated proteins including p56lck, a protein tyrosine kinase. We previously reported that Geldanamycin (GA), a benzoquinoid ansamycin, causes a increase in hsp72 and a decrease in p56lck levels in T-cells. We sought to determine the mechanism of GA-mediated p56lck loss. p56lck levels, visualized by western blotting using a anti-p56lck antibody, decrease in Jurkat cells treated with GA. To determine if proteosomal degradation mediated the loss of p56lck from GA-treated T cells, Jurkat cultures were treated with GA and/or the proteosome inhibitor clasto-lactacystin β-lactone(lactacystin). Lactacystin slightly reduced the magnitude of GA-induced depletion of p56lck from total cell lysates and induced the expression of hsp72. Detergent-insoluble fractions were compared to p56lck levels in the detergent-soluble fractions. Treatment with lactacystin alone yielded a nearly quantitative recruitment of p56lck to detergent insoluble cellular structures. The effects of GA on p56lck half-life were assessed by using anti-p56lck antibodies to immunoabsorb [35S]p56lck from cell lysates prepared from radiolabeled cells. In untreated cells radiolabeled by pulse-chase technique, the amounts of [35S]p56lck declined only slightly following chase incubations of up to 80 min. In contrast, [35S]p56lck produced in GA-treated cells was very unstable, with ∼50% of [35S]p56lck lost within 40 min and with [35S]p56lck content continuing to decline thereafter. Thus, in the absence of GA-inhibitable hsp90 function, newly synthesized p56lck molecules were not stable. Conversely, mature [35S]p56lck was equally stable in GA-treated cells as in control cells. When [35S]p56lck was isolated from treated cells by boiling lysis and immunoadsorption, pulse-chase analyses of p56lck half-life suggested that lactacystin provided some protection against GA-induced degradation of nascent [35S]p56lck molecules. In conclusion, GA disrupts the p56lck-hsp90 complex, displacing the incompletely folded nascent p56lck protein. We suspect that the displaced p56lck protein is ubiquinated and proteolytically degraded. Hsp72 could play a role in translocating p56lck to another subcell locale. Reports by other investigators suggest that hsp72 can either refold denatured proteins, thus salvaging the protein, or facilitate lysosomal degradation.