The KKRKK sequence is involved in heat shock-induced nuclear translocation of the 18-kDa actin-binding protein, cofilin.
The exposure of cultured mammalian cells to elevated temperatures induces the translocation of actin and cofilin into the nuclei and the formation of intranuclear bundles of actin filaments decorated by cofilin (actin/cofilin rods). Cofilin has a stretch of five basic amino acids, KKRKK, which was assumed to be the sequence involved in the heat shock-dependent accumulation of cofilin in nuclei. To examine this possibility, the site-directed mutagenesis technique was employed to alter the KKRKK sequence of cofilin to KTLKK and the mutated cofilin was expressed under the human beta-actin promoter in transfectants of mouse C3H-2K cell line. All the recombinants derived from porcine cofilin cDNA were constructed so as to possess an extra-nonapeptide at their N-termini when expressed; their intracellular distribution could, therefore, be discriminated from that of endogenous cofilin using the indirect immunofluorescence method with polyclonal antibodies directed against the extra-peptide. The results clearly showed that the mutated cofilin possessing KTLKK instead of KKRKK did not translocate into the nuclei in response to heat shock whereas a recombinant cofilin with the unaltered sequence of KKRKK responded to heat shock and formed intranuclear rods together with actin. Although in vitro actin binding experiments showed that KTLKK-cofilin has a weaker affinity to actin filaments than KKRKK-cofilin, KTLKK-cofilin was found to form cytoplasmic actin/cofilin rods when transformants were incubated in NaCl buffer. Furthermore, we have noted that endogenous cofilin present in cells expressing KTLKK-cofilin behaved normally, translocated into nuclei and formed intranuclear actin/cofilin rods upon heat shock. These results suggest that the KKRKK sequence of cofilin functions as a nuclear location signal upon heat shock.
Iida
K
K
Department of Cell Biology, Tokyo Metropolitan Institute of Medical Science, Japan.
Matsumoto
S
S
Yahara
I
I
D051339:Actin Depolymerizing Factors; D000199:Actins / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D001692:Biological Transport; D001704:Biopolymers; D002460:Cell Line; D006358:Hot Temperature; D008840:Microfilament Proteins / Q000378:metabolism*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009419:Nerve Tissue Proteins / Q000378:metabolism*; D021382:Protein Sorting Signals / Q000502:physiology*
D051339:Actin Depolymerizing Factors; D000199:Actins; D001704:Biopolymers; D008840:Microfilament Proteins; D009419:Nerve Tissue Proteins; D021382:Protein Sorting Signals