HSP70 TRANSLOCATES INTO A CYTOPLASMIC AGGREGATE DURING LYMPHOCYTE-ACTIVATION

The percentage of T and B lymphocytes expressing a distinct cytoplasmi c aggregate enriched in spectrin, ankyrin, and in several other protei ns including protein kinase C greatly increases following various acti vation protocols. Members of the 70 kDa family of heat shock proteins (hsp70) temporarily bind to and stabilize unfolded segments of other p roteins, a function apparently required for proper protein folding and assembly. Considering the multiprotein and dynamic nature of the lymp hocyte aggregate, the possibility that hsp70 also might be associated with components of this structure is considered here. Double immunoflu orescence analysis indicates that hsp70 is a component of the lymphocy te aggregate and is coincident with spectrin in a subpopulation of fre shly isolated, untreated lymphocytes from various murine tissues and i n a T-lymphocyte hybridoma. When cell lysates of lymph node T cells ar e immunoprecipitated using an antibody against hsp70 or spectrin and t hen analyzed by Western blot utilizing the alternate antibody, it was found that hsp70 and spectrin coprecipitated with one another. Moreove r, this coprecipitation could be abolished by addition of ATP. This la tter observation was extended to lymphoid cells using a transient perm eabilization procedure, and it was shown that addition of exogenous AT P results in the dissipation of the aggregate structure itself. Finall y, conditions that result in T-cell activation and aggregate formation , i.e., treatment with the phorbol ester PMA or T-cell receptor cross- linking, also lead to the repositioning of hsp70 into the aggregate fr om a membrane/cytosolic locale in congruence with spectrin. These data suggest that hsp70 is an active component of the aggregate and that i t may function in the interactions believed to occur in this unique ac tivation-associated organelle. (C) 1995 Wiley-Liss, Inc.