TY - JOUR
T1 - Mechanism of IRSp53 inhibition and combinatorial activation by Cdc42 and downstream effectors
AU - Kast, David J.
AU - Yang, Changsong
AU - Disanza, Andrea
AU - Boczkowska, Malgorzata
AU - Madasu, Yadaiah
AU - Scita, Giorgio
AU - Svitkina, Tatyana
AU - Dominguez, Roberto
N1 - Funding Information:
This work was supported by the US National Institutes of Health (NIH) grant R01 MH087950 to R.D. D.J.K. was supported by NIH grant T32 AR053461 and American Cancer Society grant PF-13-033-01-DMC. T.S. and C.Y. were supported by NIH grant GM095977. G.S. and A.D. were supported by the Associazione Italiana per la Ricerca sul Cancro grant IG-2013-14104. Use of IMCA-CAT beamline 17-ID was supported by the Industrial Macromolecular Crystallography Association through a contract with the Hauptman-Woodward Medical Research Institute. The Advanced Photon Source was supported by the US Department of Energy Contract DE-AC02-06CH11357. We thank P. Leavis (Tufts University) for the synthesis of the CRIB–PR peptide.
PY - 2014/4
Y1 - 2014/4
N2 - The Rho family GTPase effector IRSp53 has essential roles in filopodia formation and neuronal development, but its regulatory mechanism is poorly understood. IRSp53 contains a membrane-binding BAR domain followed by an unconventional CRIB motif that overlaps with a proline-rich region (CRIB-PR) and an SH3 domain that recruits actin cytoskeleton effectors. Using a fluorescence reporter assay, we show that human IRSp53 adopts a closed inactive conformation that opens synergistically with the binding of human Cdc42 to the CRIB-PR and effector proteins, such as the tumor-promoting factor Eps8, to the SH3 domain. The crystal structure of Cdc42 bound to the CRIB-PR reveals a new mode of effector binding to Rho family GTPases. Structure-inspired mutations disrupt autoinhibition and Cdc42 binding in vitro and decouple Cdc42- and IRSp53-dependent filopodia formation in cells. The data support a combinatorial mechanism of IRSp53 activation.
AB - The Rho family GTPase effector IRSp53 has essential roles in filopodia formation and neuronal development, but its regulatory mechanism is poorly understood. IRSp53 contains a membrane-binding BAR domain followed by an unconventional CRIB motif that overlaps with a proline-rich region (CRIB-PR) and an SH3 domain that recruits actin cytoskeleton effectors. Using a fluorescence reporter assay, we show that human IRSp53 adopts a closed inactive conformation that opens synergistically with the binding of human Cdc42 to the CRIB-PR and effector proteins, such as the tumor-promoting factor Eps8, to the SH3 domain. The crystal structure of Cdc42 bound to the CRIB-PR reveals a new mode of effector binding to Rho family GTPases. Structure-inspired mutations disrupt autoinhibition and Cdc42 binding in vitro and decouple Cdc42- and IRSp53-dependent filopodia formation in cells. The data support a combinatorial mechanism of IRSp53 activation.
UR - http://www.scopus.com/inward/record.url?scp=84897996949&partnerID=8YFLogxK
U2 - 10.1038/nsmb.2781
DO - 10.1038/nsmb.2781
M3 - Article
C2 - 24584464
AN - SCOPUS:84897996949
SN - 1545-9993
VL - 21
SP - 413
EP - 422
JO - Nature Structural and Molecular Biology
JF - Nature Structural and Molecular Biology
IS - 4
ER -