Regulation of IRSp53-dependent filopodial dynamics by antagonism between 14-3-3 binding and SH3-mediated localization.
Filopodia are dynamic structures found at the leading edges of most migrating cells. IRSp53 plays a role in filopodium dynamics by coupling actin elongation with membrane protrusion. IRSp53 is a Cdc42 effector protein that contains an N-terminal inverse-BAR (Bin-amphipysin-Rvs) domain (IRSp53/MIM homology domain [IMD]) and an internal SH3 domain that associates with actin regulatory proteins, including Eps8. We demonstrate that the SH3 domain functions to localize IRSp53 to lamellipodia and that IRSp53 mutated in its SH3 domain fails to induce filopodia. Through SH3 domain-swapping experiments, we show that the related IRTKS SH3 domain is not functional in lamellipodial localization. IRSp53 binds to 14-3-3 after phosphorylation in a region that lies between the CRIB and SH3 domains. This association inhibits binding of the IRSp53 SH3 domain to proteins such as WAVE2 and Eps8 and also prevents Cdc42-GTP interaction. The antagonism is achieved by phosphorylation of two related 14-3-3 binding sites at T340 and T360. In the absence of phosphorylation at these sites, filopodium lifetimes in cells expressing exogenous IRSp53 are extended. Our work does not conform to current views that the inverse-BAR domain or Cdc42 controls IRSp53 localization but provides an alternative model of how IRSp53 is recruited (and released) to carry out its functions at lamellipodia and filopodia.
Robens
Jeffrey M
JM
RGS Group, Institute of Medical Biology,Singapore 138673, Singapore.
Yeow-Fong
Lee
L
Ng
Elsa
E
Hall
Christine
C
Manser
Ed
E
D048948:14-3-3 Proteins / Q000378:metabolism*; D048868:Adaptor Proteins, Signal Transducing; D000818:Animals; D001665:Binding Sites / Q000502:physiology; D019556:COS Cells; D002465:Cell Movement / Q000502:physiology*; D002522:Chlorocebus aethiops; D006367:HeLa Cells; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins / Q000378:metabolism; D009154:Mutation; D009419:Nerve Tissue Proteins / Q000378:metabolism*; D010766:Phosphorylation; D011485:Protein Binding / Q000502:physiology; D011554:Pseudopodia / Q000378:metabolism* / Q000648:ultrastructure; D051300:Wiskott-Aldrich Syndrome Protein Family / Q000378:metabolism; D020764:cdc42 GTP-Binding Protein / Q000378:metabolism*; D018909:src Homology Domains / Q000502:physiology
D048948:14-3-3 Proteins; D048868:Adaptor Proteins, Signal Transducing; C119539:BAIAP2 protein, human; C489989:EPS8 protein, human; D047908:Intracellular Signaling Peptides and Proteins; D009419:Nerve Tissue Proteins; C120152:WASF2 protein, human; D051300:Wiskott-Aldrich Syndrome Protein Family; D020764:cdc42 GTP-Binding Protein