Extensive rewiring of the EGFR network in colorectal cancer cells expressing transforming levels of KRASG13D

Abstract

Protein-protein-interaction networks (PPINs) organize fundamental biological processes, but how oncogenic mutations impact these interactions and their functions at a network-level scale is poorly understood. Here, we analyze how a common oncogenic KRAS mutation (KRAS G13D) affects PPIN structure and function of the Epidermal Growth Factor Receptor (EGFR) network in colorectal cancer (CRC) cells. Mapping >6000 PPIs shows that this network is extensively rewired in cells expressing transforming levels of KRAS G13D (mtKRAS). The factors driving PPIN rewiring are multifactorial including changes in protein expression and phosphorylation. Mathematical modelling also suggests that the binding dynamics of low and high affinity KRAS interactors contribute to rewiring. PPIN rewiring substantially alters the composition of protein complexes, signal flow, transcriptional regulation, and cellular phenotype. These changes are validated by targeted and global experimental analysis. Importantly, genetic alterations in the most extensively rewired PPIN nodes occur frequently in CRC and are prognostic of poor patient outcomes.

The Ins and Outs of RAS Effector Complexes

“…Because all effector RBDs recognize the same switch regions of RAS•GTP, binding of effectors is mutually exclusive. Hence, if the concentration of RAS•GTP is limiting, effectors compete for binding to RAS [24][25][26]. Indeed, we have shown previously that increasing the concentration of the effector RIN1 in cultured cells decreased the phosphorylation of CRAF and its downstream targets, which was enhanced even further when RIN1 was artificially localized to the plasma membrane (PM) using a CAAX box [24].…”

The Ins and Outs of RAS Effector Complexes

“…Because all effector RBDs recognize the same switch regions of RAS•GTP, binding of effectors is mutually exclusive. Hence, if the concentration of RAS•GTP is limiting, effectors compete for binding to RAS [21][22][23]. Indeed, we have shown earlier that increasing the concentration of the effector RIN1 in cultured cells decreased the phosphorylation of CRAF and its downstream targets, which was enhanced even further when RIN1 was artificially localized to the plasma membrane (PM) using a CAAX box [21].…”

“…Indeed, we have shown earlier that increasing the concentration of the effector RIN1 in cultured cells decreased the phosphorylation of CRAF and its downstream targets, which was enhanced even further when RIN1 was artificially localized to the plasma membrane (PM) using a CAAX box [21]. Another consequence of the competition of effectors for binding to RAS is that increasing the levels of active RAS•GTP, such as in the case of cancer mutants that prevent efficient hydrolysis of RAS•GTP, is predicted to not only increase the overall amount of effectors in complex with RAS (quantitative change), but also qualitatively change the binding profile, where low affinity effectors proportionally engage more with the additional amount of RAS•GTP available [15,22]. Thus, understanding RAS-effector signaling requires studying all effectors present in a biological system as the cellular output depends on the concentrations (and affinities) of all players present.…”

Mass spectrometry‐based protein–protein interaction networks for the study of human diseases

“…Understanding the molecular functions of RAS interactors may help develop effective therapeutics. Kennedy et al (2020) investigated how a common KRAS mutation, KRASG13D, affects EGFR signaling in colorectal cancer (CRC) cells by utilizing AP-MS to compare the network of 95 bait proteins involved in this pathway in WT and KRAS-mutated cells. Significant differences in PPIs suggest that the majority of rewiring in the EGFR network results from the gain or loss of interacting proteins, linking KRAS activity to a myriad of adaptive network alterations spanning from core interactions throughout the network periphery.…”