Hsp70 translocates into a cytoplasmic aggregate during lymphocyte activation.
The percentage of T and B lymphocytes expressing a distinct cytoplasmic aggregate enriched in spectrin, ankyrin, and in several other proteins including protein kinase C greatly increases following various activation protocols. Members of the 70 kDa family of heat shock proteins (hsp70) temporarily bind to and stabilize unfolded segments of other proteins, a function apparently required for proper protein folding and assembly. Considering the multiprotein and dynamic nature of the lymphocyte aggregate, the possibility that hsp70 also might be associated with components of this structure is considered here. Double immunofluorescence analysis indicates that hsp70 is a component of the lymphocyte aggregate and is coincident with spectrin in a subpopulation of freshly isolated, untreated lymphocytes from various murine tissues and in a T-lymphocyte hybridoma. When cell lysates of lymph node T cells are immunoprecipitated using an antibody against hsp70 or spectrin and then analyzed by Western blot utilizing the alternate antibody, it was found that hsp70 and spectrin coprecipitated with one another. Moreover, this coprecipitation could be abolished by addition of ATP. This latter observation was extended to lymphoid cells using a transient permeabilization procedure, and it was shown that addition of exogenous ATP results in the dissipation of the aggregate structure itself. Finally, 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 from a membrane/cytosolic locale in congruence with spectrin. These data suggest that hsp70 is an active component of the aggregate and that it may function in the interactions believed to occur in this unique activation-associated organelle.
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Department of Molecular and Cellular Biology, Roswell Park Cancer Institute, Buffalo, New York 14263, USA.
Repasky
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Laszlo
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Calderwood
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Subjeck
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J
D000255:Adenosine Triphosphate / Q000494:pharmacology; D000818:Animals; D001692:Biological Transport; D015153:Blotting, Western; D003593:Cytoplasm / Q000378:metabolism*; D005260:Female; D018840:HSP70 Heat-Shock Proteins / Q000378:metabolism*; D050884:HSP72 Heat-Shock Proteins; D006360:Heat-Shock Proteins / Q000378:metabolism; D008213:Lymphocyte Activation / Q000502:physiology*; D008214:Lymphocytes / Q000378:metabolism; D051379:Mice; D008807:Mice, Inbred BALB C; D011233:Precipitin Tests; D013049:Spectrin / Q000378:metabolism; D013601:T-Lymphocytes / Q000378:metabolism; D014018:Tissue Distribution
D018840:HSP70 Heat-Shock Proteins; D050884:HSP72 Heat-Shock Proteins; D006360:Heat-Shock Proteins; D013049:Spectrin; D000255:Adenosine Triphosphate