Nuclear export of LIM-kinase 1, mediated by two leucine-rich nuclear-export signals within the PDZ domain.
LIM-kinase 1 (LIMK1) is a serine/threonine kinase that phosphorylates cofilin and regulates actin-filament dynamics. LIMK1, which contains two LIM domains and a single PDZ domain, localizes predominantly in the cytoplasm, but its mutant, deleted with the PDZ domain, localizes mainly in the nucleus, thereby indicating that the PDZ domain plays a role in the cytoplasmic localization of LIMK1. Here we provide evidence that the PDZ domain of LIMK1 contains two functional leucine-rich nuclear-export signals (NESs). The PDZ domain of LIMK1 fused with glutathione S-transferase (GST-PDZ), when injected into the nucleus, was rapidly excluded from the nucleus, but its mutant with replacements of conserved hydrophobic residues in two putative NESs by alanines remained in the nucleus. The nuclear export of GST-PDZ was sensitive to leptomycin B (LMB), a specific inhibitor of nuclear export mediated by leucine-rich NESs. Malfunctional mutation of two NESs or LMB treatment prevented the nuclear export of full-length LIMK1 and induced its nuclear accumulation. These results suggest that the predominant localization of LIMK1 in the cytoplasm is supported by two NESs within the PDZ domain and that LIMK1 normally shuttles between the cytoplasm and the nucleus. We also provide evidence that a short basic cluster sequence within the protein-kinase domain is involved in the nuclear import of LIMK1.
Yang
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Department of Biology, Faculty of Science, Kyushu University, Hakozaki, Higashi-ku, Fukuoka 812-8581, Japan.
Mizuno
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K
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001692:Biological Transport; D019556:COS Cells; D002467:Cell Nucleus / Q000201:enzymology*; D017931:DNA Primers; D006367:HeLa Cells; D006801:Humans; D007930:Leucine / Q000378:metabolism*; D054461:Lim Kinases; D011494:Protein Kinases / Q000737:chemistry / Q000378:metabolism*; D021382:Protein Sorting Signals / Q000737:chemistry / Q000378:metabolism*; D017384:Sequence Deletion; D017386:Sequence Homology, Amino Acid
D017931:DNA Primers; D021382:Protein Sorting Signals; D011494:Protein Kinases; C515892:LIMK1 protein, human; D054461:Lim Kinases; D007930:Leucine