Mutation of amino acids 566-572 (KKKVLDK) inhibits nuclear accumulation ofheat 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 wit h stress is part of the protein's protective response. Therefore, further u nderstanding of the regulation of this response would be of interest. The s ignals regulating HSP72's nuclear localization have not been completely def ined. Previously, we observed that mutation of amino acids 246-251 (KRKHKK) reduced nuclear accumulation of HSP72 and that a KRKHKK-EGFP (enhanced gre en fluorescent protein) fusion protein concentrated in the nucleoli. In exa mining HSP72 for other potential nuclear localization signals, we identifie d an additional sequence, KKKVLDK, amino acids 566-572, that might effect n uclear accumulation. We now report that mutation of KKKVLDK inhibited nucle ar concentration of HSP72 following heat shock, and the fusion protein KKKV LDK-EGFP concentrated in the nucleus. Cells overexpressing the KKKVLDK muta nt showed reduced resistance to heat shock. Mutation of KKKVLDK and KRKHKK abolished nuclear accumulation of HSP72 and reduced cell viability followin g heat shock to a greater extent than mutation of either site alone. These Endings suggest that these two sequences, KKKHKK and KKKVLDK, have compleme ntary function(s) in cellular protection after heat shock. (C) 2000 Academi c Press.