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 expression and accumulation of the protein in the nucleus. This nuclear accumulation of HSP72 is seen in many different settings, including the ischemic heart. The identity of the signal(s) regulating nuclear concentration of HSP72 are unknown. We theorized that nuclear accumulation of HSP72 with stress contributes to its protective properties in the ischemic heart and other tissues. Before we can test this hypothesis we need to alter accumulation of HSP72. Using site-directed mutagenesis we investigated the importance of amino acids 246-262 (KRKHKKDISQNKRAVRR), the presumed nuclear localization sequence (NLS), in nuclear accumulation in response to stress. Three mutant constructs of this sequence, 985A(AAAHAADISQNKRAVRR), 97M (KRKHKKDISQNAAAVAR), and B1 (AAAHAADISQNAAAVAR), were transfected into Cos cells. Analysis by exhaustive photon reassignment, which allowed examination of the nucleus in sections, showed that both 985A and B1 had decreased nuclear concentration with stress. A fusion protein with KRKHKK and EGFP localized to the nucleus in the absence of stress, with prominent accumulation in the nucleoli. Only B1, which also altered ATP binding, affected viability after heat shock. We conclude that amino acids 246-251 influence nucleolar accumulation of HSP72, but that this is not essential for early survival after injury.
Knowlton
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AA
VA Medical Center, Houston, TX, USA. annek@bcm.tmc.edu
D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D019556:COS Cells; D002466:Cell Nucleolus / Q000378:metabolism; D002467:Cell Nucleus / Q000378:metabolism*; D002470:Cell Survival; D017353:Gene Deletion; D050884:HSP72 Heat-Shock Proteins; D006360:Heat-Shock Proteins / Q000378:metabolism*; D006801:Humans; D008856:Microscopy, Fluorescence; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D019913:Nuclear Localization Signals; D016133:Polymerase Chain Reaction; D011993:Recombinant Fusion Proteins; D013312:Stress, Physiological / Q000378:metabolism*
D050884:HSP72 Heat-Shock Proteins; D006360:Heat-Shock Proteins; D019913:Nuclear Localization Signals; D011993:Recombinant Fusion Proteins; D000255:Adenosine Triphosphate