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.
Highlights
Noonan syndrome (NS) belongs to the RASopathies, a group of developmental diseases caused by mutations in genes encoding RAS-MAPK pathway components
We show that RIT1 binds p21-activated kinase 1 (PAK1), an effector of the RHO GTPases RAC1 and CDC42, which are important regulators of cytoskeletal dynamics
RIT1 belongs to the RAS superfamily of low molecular weight GTP-binding proteins that function as guanine nucleotide-regulated molecular switches in the cell by changing between an active GTP-bound and an inactive GDP-bound state [1, 2]
Summary
RIT1 belongs to the RAS superfamily of low molecular weight GTP-binding proteins that function as guanine nucleotide-regulated molecular switches in the cell by changing between an active GTP-bound and an inactive GDP-bound state [1, 2]. NS-associated RIT1 mutations reported to date affect codons 57, 82 and 95 and result in amino acid changes in the switch I and II regions (Fig 1A) [14, 16,17,18,19,20,21,22,23,24,25,26,27,28,29,30] These two protein motifs are involved in nucleotide, effector and regulator binding and thereby ensure molecular functioning of RIT1 [31, 32]. Almost all causative genes for NS and clinically overlapping diseases encode components or regulators of RAS-mediated signaling, and dysregulated RAS-MAPK signaling has been postulated to be the pathogenic mechanism shared among these disorders, summarized as RASopathies [33,34,35]
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