Involvement of the conserved adaptor protein Alix in actin cytoskeleton assembly.
The conserved adaptor protein Alix, also called AIP1 or Hp95, promotes flattening and alignment of cultured mammalian fibroblasts; however, the mechanism by which Alix regulates fibroblast morphology is not understood. Here we demonstrate that Alix in WI38 cells, which require Alix expression for maintaining typical fibroblast morphology, associates with filamentous actin (F-actin) and F-actin-based structures lamellipodia and stress fibers. Reducing Alix expression by small interfering RNA (siRNA) decreases F-actin content and inhibits stress fiber assembly. In cell-free systems, Alix directly interacts with F-actin at both the N-terminal Bro1 domain and the C-terminal proline-rich domain. In Alix immunoprecipitates from WI38 cell lysates, actin is the most abundant partner protein of Alix. In addition, the N-terminal half of the middle region of Alix binds cortactin, an activator of the ARP2/3 complex-mediated initiation of actin polymerization. Alix is required for lamellipodial localization of cortactin. The C-terminal half of the middle region of Alix interacts with alpha-actinin, a key factor that bundles F-actin in stress fibers. Alix knockdown decreases the amount of alpha-actinin that associates with F-actin. These findings establish crucial involvement of Alix in actin cytoskeleton assembly.
Pan
Shujuan
S
Department of Experimental Therapeutics, University of Texas, M. D. Anderson Cancer Center, Houston, Texas 77030, USA.
Wang
Ruoning
R
Zhou
Xi
X
He
Guangan
G
Koomen
John
J
Kobayashi
Ryuji
R
Sun
Le
L
Corvera
Joe
J
Gallick
Gary E
GE
Kuang
Jian
J
D000199:Actins / Q000378:metabolism*; D002135:Calcium-Binding Proteins / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D002352:Carrier Proteins / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D018797:Cell Cycle Proteins / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D002478:Cells, Cultured; D003599:Cytoskeleton / Q000378:metabolism*; D056827:Endosomal Sorting Complexes Required for Transport; D005347:Fibroblasts / Q000166:cytology / Q000378:metabolism; D005455:Fluorescent Antibody Technique; D005982:Glutathione Transferase / Q000235:genetics / Q000378:metabolism; D006801:Humans; D015151:Immunoblotting; D047468:Immunoprecipitation; D008168:Lung / Q000166:cytology / Q000378:metabolism; D016133:Polymerase Chain Reaction; D034741:RNA, Small Interfering; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D014158:Transcription, Genetic
D000199:Actins; D002135:Calcium-Binding Proteins; D002352:Carrier Proteins; D018797:Cell Cycle Proteins; D056827:Endosomal Sorting Complexes Required for Transport; C117020:PDCD6IP protein, human; D034741:RNA, Small Interfering; D011993:Recombinant Fusion Proteins; D005982:Glutathione Transferase