The mammalian verprolin, WIRE induces filopodia independent of N-WASP through IRSp53.
The mammalian verprolin family of proteins, WIP (WASP Interacting Protein), CR16 (Corticoid Regulated) and WIRE (WIp-RElated) regulate the actin cytoskeleton through WASP/N-WASP (Wiskott Aldrich Syndrome Protein and Neural-WASP). In order to characterize the WASP/N-WASP-independent function of WIRE, we screened and identified IRSp53 (Insulin Receptor Substrate) as a WIRE interacting protein. Expression of IRSp53 with WIRE in N-WASP(-/-) mouse fibroblast cells induced filopodia while co-expression of IRSp53 with WIP did not. The induction of filopodia is dependent on WIRE-IRSp53 interaction as mutation in the SH3 domain of IRSp53 abolished WIRE-IRSp53 interaction as well as the ability to induce filopodia. Similarly, the Verprolin (V)-domain of WIRE is critical for IRSp53-WIRE interaction and for filopodia formation. The interaction between WIRE and IRSp53 is regulated by Cdc42 as mutations which abolish Cdc42-IRSp53 interaction lead to loss of IRSp53-WIRE interaction as shown by pull down assay. The plasma membrane localization of IRSp53 is dependent on Cdc42 and WIRE. Expression of Cdc42(G12V) (active mutant) with WIRE-IRSp53 caused significant increase in the number of filopodia per cell. Thus our results show that Cdc42 regulates the activity of IRSp53 by regulating the IRSp53-WIRE interaction as well as localization of the complex to plasma membrane to generate filopodia.
Misra
Ashish
A
School of Biological Sciences, Nanyang Technological University, Singapore, Republic of Singapore.
Rajmohan
Rajamuthiah
R
Lim
Rina Pei Zhi
RP
Bhattacharyya
Swagata
S
Thanabalu
Thirumaran
T
D000818:Animals; D002352:Carrier Proteins / Q000378:metabolism / Q000502:physiology*; D002460:Cell Line; D005347:Fibroblasts / Q000166:cytology / Q000648:ultrastructure; D055785:Gene Knockdown Techniques; D006801:Humans; D051379:Mice; D008840:Microfilament Proteins; D009419:Nerve Tissue Proteins / Q000378:metabolism*; D011485:Protein Binding / Q000502:physiology; D011554:Pseudopodia; D034741:RNA, Small Interfering / Q000494:pharmacology; D014162:Transfection; D051317:Wiskott-Aldrich Syndrome Protein, Neuronal; D020764:cdc42 GTP-Binding Protein / Q000378:metabolism
C119539:BAIAP2 protein, human; D002352:Carrier Proteins; D008840:Microfilament Proteins; D009419:Nerve Tissue Proteins; D034741:RNA, Small Interfering; C495771:WASL protein, human; C465744:WIPF2 protein, human; D051317:Wiskott-Aldrich Syndrome Protein, Neuronal; D020764:cdc42 GTP-Binding Protein