Mutation of amino acids 566-572 (KKKVLDK) inhibits nuclear accumulation of heat shock protein 72 after heat shock.
Heat shock protein (HSP) 72 translocates from the cytoplasm to the nucleus in response to a wide variety of stresses, including heat shock and tissue ischemia. It is thought that this concentration of HSP72 in the nucleus with stress is part of the protein's protective response. Therefore, further understanding of the regulation of this response would be of interest. The signals regulating HSP72's nuclear localization have not been completely defined. Previously, we observed that mutation of amino acids 246-251 (KRKHKK) reduced nuclear accumulation of HSP72 and that a KRKHKK-EGFP (enhanced green fluorescent protein) fusion protein concentrated in the nucleoli. In examining HSP72 for other potential nuclear localization signals, we identified an additional sequence, KKKVLDK, amino acids 566-572, that might effect nuclear accumulation. We now report that mutation of KKKVLDK inhibited nuclear concentration of HSP72 following heat shock, and the fusion protein KKKVLDK-EGFP concentrated in the nucleus. Cells overexpressing the KKKVLDK mutant showed reduced resistance to heat shock. Mutation of KKKVLDK and KRKHKK abolished nuclear accumulation of HSP72 and reduced cell viability following heat shock to a greater extent than mutation of either site alone. These findings suggest that these two sequences, KRKHKK and KKKVLDK, have complementary function(s) in cellular protection after heat shock.
Knowlton
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AA
Baylor College of Medicine and the V.A. Medical Center, Houston, Texas, USA. annek@bcm.tmc.edu
D000255:Adenosine Triphosphate / Q000378:metabolism; D000494:Allosteric Regulation; D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000818:Animals; D015153:Blotting, Western; D019556:COS Cells; D002458:Cell Fractionation; D002467:Cell Nucleus / Q000378:metabolism*; D002470:Cell Survival; D002522:Chlorocebus aethiops; D003593:Cytoplasm / Q000378:metabolism; D004578:Electron Spin Resonance Spectroscopy; D004591:Electrophoresis, Polyacrylamide Gel; D019143:Evolution, Molecular; D050884:HSP72 Heat-Shock Proteins; D006360:Heat-Shock Proteins / Q000737:chemistry / Q000378:metabolism*; D006358:Hot Temperature; D006801:Humans; D016297:Mutagenesis, Site-Directed; D017510:Protein Folding; D021381:Protein Transport; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D013045:Species Specificity; D013312:Stress, Physiological / Q000378:metabolism
D050884:HSP72 Heat-Shock Proteins; D006360:Heat-Shock Proteins; D011993:Recombinant Fusion Proteins; D000255:Adenosine Triphosphate