RIT1 controls actin dynamics via complex formation with RAC1/CDC42 and PAK1

PLoS Genet. 2018 May 7;14(5):e1007370. doi: 10.1371/journal.pgen.1007370. eCollection 2018 May.

Abstract

RIT1 belongs to the RAS family of small GTPases. Germline and somatic RIT1 mutations have been identified in Noonan syndrome (NS) and cancer, respectively. By using heterologous expression systems and purified recombinant proteins, we identified the p21-activated kinase 1 (PAK1) as novel direct effector of RIT1. We found RIT1 also to directly interact with the RHO GTPases CDC42 and RAC1, both of which are crucial regulators of actin dynamics upstream of PAK1. These interactions are independent of the guanine nucleotide bound to RIT1. Disease-causing RIT1 mutations enhance protein-protein interaction between RIT1 and PAK1, CDC42 or RAC1 and uncouple complex formation from serum and growth factors. We show that the RIT1-PAK1 complex regulates cytoskeletal rearrangements as expression of wild-type RIT1 and its mutant forms resulted in dissolution of stress fibers and reduction of mature paxillin-containing focal adhesions in COS7 cells. This effect was prevented by co-expression of RIT1 with dominant-negative CDC42 or RAC1 and kinase-dead PAK1. By using a transwell migration assay, we show that RIT1 wildtype and the disease-associated variants enhance cell motility. Our work demonstrates a new function for RIT1 in controlling actin dynamics via acting in a signaling module containing PAK1 and RAC1/CDC42, and highlights defects in cell adhesion and migration as possible disease mechanism underlying NS.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Actins / metabolism*
  • Animals
  • COS Cells
  • Cell Movement
  • Chlorocebus aethiops
  • Cytoskeleton / metabolism
  • HEK293 Cells
  • Humans
  • Multiprotein Complexes / metabolism
  • Mutation
  • Protein Binding
  • cdc42 GTP-Binding Protein / genetics
  • cdc42 GTP-Binding Protein / metabolism*
  • p21-Activated Kinases / genetics
  • p21-Activated Kinases / metabolism*
  • rac1 GTP-Binding Protein / genetics
  • rac1 GTP-Binding Protein / metabolism*
  • ras Proteins / genetics
  • ras Proteins / metabolism*

Substances

  • Actins
  • Multiprotein Complexes
  • PAK1 protein, human
  • p21-Activated Kinases
  • RIT1 protein, human
  • cdc42 GTP-Binding Protein
  • rac1 GTP-Binding Protein
  • ras Proteins

Grant support

This work was supported by grants from the Deutsche Forschungsgemeinschaft (DFG) (KU 1240/9-1 to KK and ZE 524/10-1 to MZ) and the Bundesminesterium für Bildung und Forschung (BMBF) (GeNeRARe, TP6 to GR and KK, funding code 01GM1519E, and TP1 to MZ, funding code 01GM1519A). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.