Quantitative GTPase Affinity Purification Identifies Rho Family Protein Interaction Partners

Abstract

Although Rho GTPases are essential molecular switches involved in many cellular processes, an unbiased experimental comparison of their interaction partners was not yet performed. Here, we develop quantitative GTPase affinity purification (qGAP) to systematically identify interaction partners of six Rho GTPases (Cdc42, Rac1, RhoA, RhoB, RhoC, and RhoD), depending on their nucleotide loading state. The method works with cell line or tissuederived protein lysates in combination with SILAC-based or label-free quantification, respectively. We demonstrate that qGAP identifies known and novel binding partners that can be validated in an independent assay. Our interaction network for six Rho GTPases contains many novel binding partners, reveals highly promiscuous interaction of several effectors, and mirrors evolutionary relationships among Rho GTPases. The Ras superfamily of small guanosine triphosphatases (Ras GTPases) consists of more than 150 members in mammals and conserved orthologs in all eukaryotes (1). As molecular switches, they cycle between an active GTP-and an inactive GDP-bound state. GTPase-activating proteins (GAPs) 1 stimulate the slow intrinsic GTPase activity, which inactivates the GTPase. Conversely, guanine nucleotide exchange factors (GEFs) promote release of GDP, which is replaced by GTP, thereby transforming the GTPase into the active state. The GTP-bound form binds to downstream effector proteins to initiate their specific cellular function. In this manner, GTPases control numerous biological processes, including cytoskeletal rearrangements, membrane dynamics, and gene expression. Rho GTPases form a subfamily of the Ras superfamily (2). Of its 22 mammalian members, RhoA, Rac1, and Cdc42 have been studied most intensively and are best known for their role in regulating the actin cytoskeleton (3).Identifying effector proteins is key to understanding Rho GTPase function. More than 70 effector proteins have already been identified for each of the three prototypical family members, RhoA, Rac1, and Cdc42 (4). However, new effector proteins are still being discovered, and little is known about effectors of less well studied family members. Systematic screens for GTPase effectors employed the yeast two-hybrid approach (5) or immobilized GTPases for affinity purification (6, 7). However, these approaches are semiquantitative at best, which makes it difficult to distinguish loading-statespecific binders from constitutive interactors and nonspecific contaminants. Due to these challenges, an unbiased interactor screen for multiple Rho GTPases has not yet been reported.We sought to systematically identify proteins that interact with Rho GTPases in a loading-state-specific manner. Quantitative affinity purification combined with mass spectrometry is a powerful technology that can be used to identify proteinprotein interactions (PPIs) in an unbiased way (8 -11). Here, we develop quantitative GTPase affinity purification (qGAP) as a novel variant of quantitative affinity purification combined with mass spectro...

In vivo identification of GTPase interactors by mitochondrial relocalization and proximity biotinylation

“…Its function is unknown, but its paralog SHKBP1 is also present in the list of hits. In addition some hits are regulators of other members of the Rho family such as the RhoA GEFs ARHGEF11/PDX-RhoGEF, ARHGEF12/LARG, ARHGEF40/Solo and PLEKHG4 reflecting the known cross talk between the Rho family GTPases (Paul et al, 2017).…”

Structural and functional dissection of the DH and PH domains of oncogenic Bcr-Abl tyrosine kinase

“…While the Rho GTPase family comprises up to 25 members, RhoA, Rac1, and Cdc42 represent the most studied members of the Rho family and have also been used in previous studies on RhoGTPase activation in Bcr-Abl-expressing cells 10 , 42 . In addition recent functional proteomics studies on six Rho GTPases (including RhoA, Rac1, and Cdc42), as well as on the Bcr-Abl interactome from others and us, did not indicate that Rho GTPase directly interact with Bcr-Abl 8 , 9 , 43 . We also carefully checked the structural location and possible functional role of the S509A mutation that was previously proposed to modulate leukemogenesis in a mouse model 13 , but found no supporting evidence for these findings, as the DH domain structure or binding remains unaltered (Supplementary Fig.…”

High Throughput strategies Aimed at Closing the GAP in Our Knowledge of Rho GTPase Signaling

“…The emergence of large-scale proteomic approaches has paved the way toward a more global view of these wide protein networks ( Table 1 ). In a first study, Paul et al developed a quantitative GTPase affinity purification (qGAP) combined with mass spectrometry strategy to identify protein partners of CDC42, RAC1, RHOA, RHOB, RHOC, and RHOD [ 61 ]. Briefly, beads loaded with each recombinant Rho GTPases were loaded with GDP or GTPγ and used to pull-down proteins from differentially SILAC (stable isotope labeling by amino acids in cell culture)-labeled HeLa cell lysates.…”