Nucleocytoplasmic shuttling of Pak5 regulates its antiapoptotic properties.
p21-activated kinase 5 (Pak5) is an effector for the small GTPase Cdc42, known to activate cell survival signaling pathways. Previously, we have shown that Pak5 localizes primarily to mitochondria. To study the relationship between Pak5 localization and its effects on apoptosis, we identified three N-terminal regions that regulate the localization of this kinase: a mitochondrial targeting sequence, a nuclear export sequence, and a nuclear localization sequence. When the first two sequences are deleted, Pak5 is retained in the nucleus and no longer protects cells from apoptosis. Moreover, blockade of nuclear export with leptomycin B causes endogenous Pak5 to accumulate in the nucleus. Additionally, the removal of the N-terminal nuclear localization sequence abolishes Pak5 translocation to the nucleus. Finally, we show that reduction of endogenous Pak5 expression in neuroblastoma and neural stem cells increases their sensitivity to apoptosis and that this effect is reversed upon reexpression of wild-type Pak5 but not of a mutant form of Pak5 that cannot localize to mitochondria. These results show that Pak5 shuttles from mitochondria to the nucleus and that the mitochondrial localization of Pak5 is vital to its effects on cell survival.
Fox Chase Cancer Center, 333 Cottman Avenue, Philadelphia, PA 19111-2497, USA. J_Chernoff@fccc.edu
Sophie
S
Cotteret
Jonathan
J
Chernoff
D021581:Active Transport, Cell Nucleus / Q000187:drug effects; D000595:Amino Acid Sequence; D000818:Animals; D017209:Apoptosis; D016466:CHO Cells; D045744:Cell Line, Tumor; D002467:Cell Nucleus / Q000378:metabolism*; D002874:Chromosome Mapping; D016384:Consensus Sequence; D006224:Cricetinae; D003593:Cytoplasm / Q000378:metabolism*; D005231:Fatty Acids, Unsaturated / Q000494:pharmacology; D005434:Flow Cytometry; D005982:Glutathione Transferase / Q000378:metabolism; D049452:Green Fluorescent Proteins / Q000378:metabolism; D006801:Humans; D015151:Immunoblotting; D018613:Microscopy, Confocal; D008928:Mitochondria / Q000737:chemistry / Q000378:metabolism; D008969:Molecular Sequence Data; D009447:Neuroblastoma / Q000473:pathology; D019913:Nuclear Localization Signals; D010766:Phosphorylation; D011233:Precipitin Tests; D011494:Protein Kinases / Q000032:analysis; D017346:Protein Serine-Threonine Kinases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D017434:Protein Structure, Tertiary; D034741:RNA, Small Interfering / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D017384:Sequence Deletion; D051025:bcl-Associated Death Protein / Q000378:metabolism; D054462:p21-Activated Kinases
D005231:Fatty Acids, Unsaturated; D019913:Nuclear Localization Signals; D034741:RNA, Small Interfering; D011993:Recombinant Fusion Proteins; D051025:bcl-Associated Death Protein; D049452:Green Fluorescent Proteins; D005982:Glutathione Transferase; D011494:Protein Kinases; C443802:PAK5 protein, human; D017346:Protein Serine-Threonine Kinases; D054462:p21-Activated Kinases; C038753:leptomycin B