Mutation of amino acids 246-251 alters nuclear accumulation of human heat shock protein (HSP) 72 with stress, but does not reduce viability

The stress response includes up-regulation of heat shock protein (HSP) 72 e xpression and accumulation of the protein in the nucleus. This nuclear accu mulation of HSP72 is seen in many different settings, including the ischemi c heart. The identity of the signal(s) regulating nuclear concentration of HSP72 are unknown. We theorized that nuclear accumulation of HSP72 with str ess contributes to its protective properties in the ischemic heart and othe r tissues. Before Rie call test this hypothesis Mle need to alter accumulat ion of HSP72. Using site-directed mutagenesis we investigated the importanc e of amino acids 246-262 (KRKHKKDISQNKRAVRR). the presumed nuclear localiza tion sequence (NLS), in nuclear accumulation in response to stress. Three m utant constructs of this sequence, 985A(AAAHAADISQNKRAVRR), 97M (KRKHKKDISQ NAAAVAR), and B1 (AAAHAADISQNAAAVAR), were transfected into Cos cells. Anal ysis by exhaustive photon reassignment, which allowed examination of the nu cleus in sections, showed that both 985A and B1 had decreased nuclear conce ntration with stress. A Fusion protein with KRKHKK and EGFP localized to th e nucleus in the absence of stress, with prominent accumulation in the nucl eoli, Only B1, which also altered ATP binding, affected viability after hea t shuck, We conclude that amino acids 246-251 influence nucleolar accumulat ion of HSP72, but that this is not essential for early survival after injur y. (C) 1999 Academic Press.