mDia2 shuttles between the nucleus and the cytoplasm through the importin-{alpha}/{beta}- and CRM1-mediated nuclear transport mechanism.
Mammalian homolog of Drosophila diaphanous (mDia) consisting of three isoforms, mDia1, mDia2, and mDia3, is an effector of Rho GTPases that catalyzes actin nucleation and polymerization. Although the mDia actions on actin dynamics in the cytoplasm have been well studied, whether mDia accumulates and functions in the nucleus remains largely unknown. Given the presence of actin and actin-associated proteins in the nucleus, we have examined nuclear localization of mDia isoforms. We expressed each of mDia isoforms as a green fluorescent protein fusion protein and examined their localization. Although all the mDia isoforms were localized predominantly in the cytoplasm under the steady-state conditions, mDia2 and not mDia1 or mDia3 accumulated extensively in the nucleus upon treatment with leptomycin B (LMB), an inhibitor of CRM1-dependent nuclear export. The LMB-induced nuclear accumulation was confirmed for endogenous mDia2 by using an antibody specific to mDia2. Studies using green fluorescent protein fusions of various truncation mDia2 mutants and point mutants of some of these proteins identified a functional nuclear localization signal in the N terminus of mDia2 and at least one functional nuclear export signal in the C terminus. The nuclear localization signal of mDia2 bound to importin-alpha and was imported into the nucleus by importin-alpha/beta complex in an in vitro transport assay. Consistently, depletion of importin-beta with RNA interference suppressed the LMB-induced nuclear localization of endogenous mDia2. These results suggest that mDia2 continuously shuttles between the nucleus and the cytoplasm using specific nuclear transport machinery composing of importin-alpha/beta and CRM1.
Miki
Takashi
T
Department of Pharmacology and Frontier Technology Center, Kyoto University Faculty of Medicine, Kyoto 606-8501, Japan.
Okawa
Katsuya
K
Sekimoto
Toshihiro
T
Yoneda
Yoshihiro
Y
Watanabe
Sadanori
S
Ishizaki
Toshimasa
T
Narumiya
Shuh
S
D021581:Active Transport, Cell Nucleus; D048868:Adaptor Proteins, Signal Transducing / Q000235:genetics / Q000378:metabolism; D000935:Antifungal Agents / Q000494:pharmacology; D002352:Carrier Proteins / Q000235:genetics / Q000378:metabolism*; D002467:Cell Nucleus / Q000235:genetics / Q000378:metabolism*; D003593:Cytoplasm / Q000235:genetics / Q000378:metabolism*; D005231:Fatty Acids, Unsaturated / Q000494:pharmacology; D005455:Fluorescent Antibody Technique; D000080865:Formins; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D028884:Karyopherins / Q000235:genetics / Q000378:metabolism*; D019913:Nuclear Localization Signals / Q000235:genetics / Q000378:metabolism; D020033:Protein Isoforms; D034741:RNA, Small Interfering / Q000494:pharmacology; D018160:Receptors, Cytoplasmic and Nuclear / Q000235:genetics / Q000378:metabolism*; D028901:alpha Karyopherins / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D028961:beta Karyopherins / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D000097606:Exportin 1 Protein
D048868:Adaptor Proteins, Signal Transducing; D000935:Antifungal Agents; D002352:Carrier Proteins; D005231:Fatty Acids, Unsaturated; D000080865:Formins; D049452:Green Fluorescent Proteins; D028884:Karyopherins; D019913:Nuclear Localization Signals; D020033:Protein Isoforms; D034741:RNA, Small Interfering; D018160:Receptors, Cytoplasmic and Nuclear; D028901:alpha Karyopherins; D028961:beta Karyopherins; D000097606:Exportin 1 Protein; C108962:DIAPH1 protein, human; C491462:DIAPH2 protein, human; C485184:enhanced green fluorescent protein; C038753:leptomycin B