IRSp53 is colocalised with WAVE2 at the tips of protruding lamellipodia and filopodia independently of Mena.
The insulin receptor tyrosine kinase substrate p53 (IRSp53) links Rac and WAVE2 and has been implicated in lamellipodia protrusion. Recently, however, IRSp53 has been reported to bind to both Cdc42 and Mena to induce filopodia. To shed independent light on IRSp53 function we determined the localisations and dynamics of IRSp53 and WAVE2 in B16 melanoma cells. In cells spread well on a laminin substrate, IRSp53 was localised by antibody labelling at the tips of both lamellipodia and filopodia. The same localisation was observed in living cells with IRSp53 tagged with enhanced green florescence protein (EGFP-IRSp53), but only during protrusion. From the transfection of deletion mutants the N-terminal region of IRSp53, which binds active Rac, was shown to be responsible for its localisation. Although IRSp53 has been reported to regulate filopodia formation with Mena, EGFP-IRSp53 showed the same localisation in MVD7 Ena/VASP (vasodilator stimulated phosphoprotein) family deficient cells. WAVE2 tagged with DsRed1 colocalised with EGFP-IRSp53 at the tips of protruding lamellipodia and filopodia and, in double-transfected cells, the IRSp53 signal in filopodia decreased before that of WAVE2 during retraction. These results suggest an alternative modulatory role for IRSp53 in the extension of both filopodia and lamellipodia, through WAVE2.
Department of Cell Biology, Institute of Molecular Biology, Austrian Academy of Sciences, Billrothstrasse 11, Salzburg A-5020, Austria.
Hiroyuki
H
Nakagawa
Hiroaki
H
Miki
Motohiro
M
Nozumi
Tadaomi
T
Takenawa
Shigeaki
S
Miyamoto
Jürgen
J
Wehland
J Victor
JV
Small
D000818:Animals; D002352:Carrier Proteins / Q000378:metabolism*; D015815:Cell Adhesion Molecules / Q000172:deficiency / Q000235:genetics; D045744:Cell Line, Tumor; D002465:Cell Movement / Q000502:physiology; D003598:Cytoskeletal Proteins; D049452:Green Fluorescent Proteins; D008164:Luminescent Proteins; D051379:Mice; D008840:Microfilament Proteins / Q000378:metabolism*; D018715:Microscopy, Video; D009154:Mutation / Q000235:genetics; D010750:Phosphoproteins / Q000172:deficiency / Q000235:genetics; D017434:Protein Structure, Tertiary / Q000235:genetics; D021381:Protein Transport / Q000502:physiology; D011554:Pseudopodia / Q000378:metabolism* / Q000648:ultrastructure; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D051300:Wiskott-Aldrich Syndrome Protein Family; D000098662:Vasodilator-Stimulated Phosphoprotein
D002352:Carrier Proteins; D015815:Cell Adhesion Molecules; D003598:Cytoskeletal Proteins; C102176:Enah protein, mouse; D008164:Luminescent Proteins; D008840:Microfilament Proteins; D010750:Phosphoproteins; D011993:Recombinant Fusion Proteins; C492303:Wasf2 protein, mouse; D051300:Wiskott-Aldrich Syndrome Protein Family; D000098662:Vasodilator-Stimulated Phosphoprotein; D049452:Green Fluorescent Proteins