Inducible clustering of membrane-targeted SH3 domains of the adaptor protein Nck triggers localized actin polymerization.
BACKGROUND
SH2/SH3 adaptor proteins play a critical role in tyrosine kinase signaling pathways, regulating essential cell functions by increasing the local concentration or altering the subcellular localization of downstream effectors. The SH2 domain of the Nck adaptor can bind tyrosine-phosphorylated proteins, while its SH3 domains can modulate actin polymerization by interacting with effectors such as WASp/Scar family proteins. Although several studies have implicated Nck in regulating actin polymerization, its role in living cells is not well understood.


RESULTS
We used an antibody-based system to experimentally modulate the local concentration of Nck SH3 domains on the plasma membrane of living cells. Clustering of fusion proteins containing all three Nck SH3 domains induced localized polymerization of actin, including the formation of actin tails and spots, accompanied by general cytoskeletal rearrangements. All three Nck SH3 domains were required, as clustering of individual SH3 domains or a combination of the two N-terminal Nck SH3 domains failed to promote significant local polymerization of actin in vivo. Changes in actin dynamics induced by Nck SH3 domain clustering required the recruitment of N-WASp, but not WAVE1, and were unaffected by downregulation of Cdc42.


CONCLUSIONS
We show that high local concentrations of Nck SH3 domains are sufficient to stimulate localized, Cdc42-independent actin polymerization in living cells. This study provides strong evidence of a pivotal role for Nck in directly coupling ligand-induced tyrosine phosphorylation at the plasma membrane to localized changes in organization of the actin cytoskeleton through a signaling pathway that requires N-WASp.
Rivera
Gonzalo M
GM
Department of Genetics and Developmental Biology, University of Connecticut Health Center, 263 Farmington Avenue, Farmington, CT 06030, USA.
Briceño
César A
CA
Takeshima
Fuminao
F
Snapper
Scott B
SB
Mayer
Bruce J
BJ
D000199:Actins / Q000378:metabolism*; D048868:Adaptor Proteins, Signal Transducing; D000818:Animals; D002462:Cell Membrane / Q000378:metabolism*; D005455:Fluorescent Antibody Technique; D006801:Humans; D015151:Immunoblotting; D051379:Mice; D041681:NIH 3T3 Cells; D009419:Nerve Tissue Proteins / Q000378:metabolism; D015513:Oncogene Proteins / Q000378:metabolism*; D015398:Signal Transduction; D014162:Transfection; D014443:Tyrosine / Q000378:metabolism; D051317:Wiskott-Aldrich Syndrome Protein, Neuronal; D018909:src Homology Domains
D000199:Actins; D048868:Adaptor Proteins, Signal Transducing; C078332:Nck protein; D009419:Nerve Tissue Proteins; D015513:Oncogene Proteins; C495771:WASL protein, human; C495772:Wasl protein, mouse; D051317:Wiskott-Aldrich Syndrome Protein, Neuronal; D014443:Tyrosine