F-actin and myosin II binding domains in supervillin.
Detergent-resistant membranes contain signaling and integral membrane proteins that organize cholesterol-rich domains called lipid rafts. A subset of these detergent-resistant membranes (DRM-H) exhibits a higher buoyant density ( approximately 1.16 g/ml) because of association with membrane skeleton proteins, including actin, myosin II, myosin 1G, fodrin, and an actin- and membrane-binding protein called supervillin (Nebl, T., Pestonjamasp, K. N., Leszyk, J. D., Crowley, J. L., Oh, S. W., and Luna, E. J. (2002) J. Biol. Chem. 277, 43399-43409). To characterize interactions among DRM-H cytoskeletal proteins, we investigated the binding partners of the novel supervillin N terminus, specifically amino acids 1-830. We find that the supervillin N terminus binds directly to myosin II, as well as to F-actin. Three F-actin-binding sites were mapped to sequences within amino acids approximately 280-342, approximately 344-422, and approximately 700-830. Sequences with combinations of these sites promote F-actin cross-linking and/or bundling. Supervillin amino acids 1-174 specifically interact with the S2 domain in chicken gizzard myosin and nonmuscle myosin IIA (MYH-9) but exhibit little binding to skeletal muscle myosin II. Direct or indirect binding to filamin also was observed. Overexpression of supervillin amino acids 1-174 in COS7 cells disrupted the localization of myosin IIB without obviously affecting actin filaments. Taken together, these results suggest that supervillin may mediate actin and myosin II filament organization at cholesterol-rich membrane domains.
Chen
Yu
Y
Department of Cell Biology, University of Massachusetts Medical School, Worcester, Massachusetts 01605, USA.
Takizawa
Norio
N
Crowley
Jessica L
JL
Oh
Sang W
SW
Gatto
Cheryl L
CL
Kambara
Taketoshi
T
Sato
Osamu
O
Li
Xiang-Dong
XD
Ikebe
Mitsuo
M
Luna
Elizabeth J
EJ
D000199:Actins / Q000737:chemistry* / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D019556:COS Cells; D002417:Cattle; D002645:Chickens; D002784:Cholesterol / Q000378:metabolism; D003599:Cytoskeleton / Q000378:metabolism; D004247:DNA / Q000378:metabolism; D003902:Detergents / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D005982:Glutathione Transferase / Q000378:metabolism; D049452:Green Fluorescent Proteins; D008051:Lipid Bilayers / Q000378:metabolism; D008164:Luminescent Proteins / Q000378:metabolism; D008565:Membrane Proteins / Q000737:chemistry*; D008840:Microfilament Proteins / Q000737:chemistry*; D008954:Models, Biological; D008969:Molecular Sequence Data; D018482:Muscle, Skeletal / Q000378:metabolism; D009132:Muscles / Q000378:metabolism; D024462:Myosin Type II / Q000737:chemistry*; D009218:Myosins / Q000737:chemistry; D024501:Nonmuscle Myosin Type IIB / Q000737:chemistry; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D011817:Rabbits; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D020798:Two-Hybrid System Techniques
D000199:Actins; D003902:Detergents; D008051:Lipid Bilayers; D008164:Luminescent Proteins; D008565:Membrane Proteins; D008840:Microfilament Proteins; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins; D004247:DNA; D002784:Cholesterol; D005982:Glutathione Transferase; D024462:Myosin Type II; D024501:Nonmuscle Myosin Type IIB; D009218:Myosins