Cutting the Brakes on Ras-Cytoplasmic GAPs as Targets of Inactivation in Cancer

Abstract

The Ras pathway is frequently deregulated in cancer, actively contributing to tumor development and progression. Oncogenic activation of the Ras pathway is commonly due to point mutation of one of the three Ras genes, which occurs in almost one third of human cancers. In the absence of Ras mutation, the pathway is frequently activated by alternative means, including the loss of function of Ras inhibitors. Among Ras inhibitors, the GTPase-Activating Proteins (RasGAPs) are major players, given their ability to modulate multiple cancer-related pathways. In fact, most RasGAPs also have a multi-domain structure that allows them to act as scaffold or adaptor proteins, affecting additional oncogenic cascades. In cancer cells, various mechanisms can cause the loss of function of Ras inhibitors; here, we review the available evidence of RasGAP inactivation in cancer, with a specific focus on the mechanisms. We also consider extracellular inputs that can affect RasGAP levels and functions, implicating that specific conditions in the tumor microenvironment can foster or counteract Ras signaling through negative or positive modulation of RasGAPs. A better understanding of these conditions might have relevant clinical repercussions, since treatments to restore or enhance the function of RasGAPs in cancer would help circumvent the intrinsic difficulty of directly targeting the Ras protein.

Keywords: GTPase-Activating Proteins; RAS oncogene; cell signaling; mechanisms of transformation; signal transduction; tumor suppressor genes.

Figure 1

Domain organization of RasGAPs (Ras GTPase-Activating Proteins). In addition to the GAP domain, different RasGAP proteins are characterized by a specific array of functional domains, orchestrating their subcellular localization and regulation (SH2 = Src homology 2 domain; SH3 = Src homology 3 domain; PH = plekstrin homology domain; C2 = calcium-dependent phospholipid-binding motif; CSR = Cys/Ser-rich region; SEC14 = CRAL-TRIO lipid-binding domain; CTD = C-terminal domain; Z = Btk-type zinc finger motif; PER = period-like domain; PR = proline-rich region). Representative proteins are drawn based on UniProtKB entries (2020_05 release, https://www.uniprot.org/). Note that NF1 is not drawn to scale.

Figure 2

Schematic overview of current evidence for post-transcriptional regulation of RasGAPs. (A) MicroRNA-dependent regulation. Both miRNAs and long non-coding RNAs (lncRNA and circ-RNA) are involved in modulating RasGAP mRNA translation both directly and indirectly. (B) Control of mRNA stability. Stability of RASA1 mRNA can be fostered by direct binding with the RNA-binding protein Quaking-5 (QKI-5); loss of QKI-5 can reduce RASA1 expression in cancer. (C) Control of alternative splicing. Expression of missense p53 mutant proteins enhances the inclusion of cytosine-rich exons in the mRNA of RasGAPs; the encoded proteins show defects in membrane association and reduced Ras inhibitory function.

Figure 3

Post-translational regulation of RasGAPs. Examples are shown of RasGAPs that are regulated by protein modification, cellular localization, interaction with other proteins, and degradation via proteasome. (A) The activity and stability of some RasGAPs can be regulated by phosphorylation. NF1 is inhibited by protein kinase A (PKA)-mediated phosphorylation at the N-terminus, marking the protein for binding with 14-3-3η. NF1 also undergoes activating phosphorylation by protein kinase C-alpha (PKC-α) in cells treated with epidermal growth factor (EGF). DAB2IP can be inhibited by Akt-mediated phosphorylation on a proline-rich domain or stimulated by receptor interacting protein-1 (RIP-1)-mediated phosphorylation on the PER domain. (B) The activity of RasGAPs is enhanced by membrane recruitment, increasing local protein concentration and facilitating interaction with active Ras. NF1 interaction with SPRED-1 (sprout related EVH1 domain containing 1), and RASA1 interaction with Annexin A6 stimulate their membrane localization and Ras inhibitory activity. RasGAPs can also be recruited to the membrane by interaction with specific lipids, including phosphatidic acid, phosphatidylinositol phosphates, arachidonic acid, and eicosanoids (not shown). (C) The binding with interacting proteins can affect RasGAP cellular localization and activity. HNL1 can bind RASA4, and missense mutant p53 proteins can bind DAB2IP, interfering with their ability to bind their physiological targets. (D) RasGAPs can be sequestered in the nucleus, reducing their functional interaction with active Ras. When phosphorylated in the C-terminal domain, NF1 traslocates to the nucleus, where it interacts with lamin A/C. NF1 can also be delocalized by binding to tubulin (see text for more detail). RASAL2 is a cargo for importin 5 (IPO5). (E) Some RasGAPs undergo proteasomal degradation by specific ubiquitin-ligases. NF1 is polyubiquitinated upon PKC-mediated phosphorylation by a Cullin3/KBTBD7 (kelch repeat and BTB domain containing 7) complex. DAB2IP can be ubiquitinated in response to different conditions by at least three different E3: the FBW7 (F-box and WD repeat domain-containing 7)/SCF (skip1-Cul1-F-box protein) complex, the Skp2 (S-phase kinase-associated protein2)/SCF complex, and Smurf1 (SMAD-specific E3 ubiquitin-ligase 1).

Figure 3

Post-translational regulation of RasGAPs. Examples are shown of RasGAPs that are regulated by protein modification, cellular localization, interaction with other proteins, and degradation via proteasome. (A) The activity and stability of some RasGAPs can be regulated by phosphorylation. NF1 is inhibited by protein kinase A (PKA)-mediated phosphorylation at the N-terminus, marking the protein for binding with 14-3-3η. NF1 also undergoes activating phosphorylation by protein kinase C-alpha (PKC-α) in cells treated with epidermal growth factor (EGF). DAB2IP can be inhibited by Akt-mediated phosphorylation on a proline-rich domain or stimulated by receptor interacting protein-1 (RIP-1)-mediated phosphorylation on the PER domain. (B) The activity of RasGAPs is enhanced by membrane recruitment, increasing local protein concentration and facilitating interaction with active Ras. NF1 interaction with SPRED-1 (sprout related EVH1 domain containing 1), and RASA1 interaction with Annexin A6 stimulate their membrane localization and Ras inhibitory activity. RasGAPs can also be recruited to the membrane by interaction with specific lipids, including phosphatidic acid, phosphatidylinositol phosphates, arachidonic acid, and eicosanoids (not shown). (C) The binding with interacting proteins can affect RasGAP cellular localization and activity. HNL1 can bind RASA4, and missense mutant p53 proteins can bind DAB2IP, interfering with their ability to bind their physiological targets. (D) RasGAPs can be sequestered in the nucleus, reducing their functional interaction with active Ras. When phosphorylated in the C-terminal domain, NF1 traslocates to the nucleus, where it interacts with lamin A/C. NF1 can also be delocalized by binding to tubulin (see text for more detail). RASAL2 is a cargo for importin 5 (IPO5). (E) Some RasGAPs undergo proteasomal degradation by specific ubiquitin-ligases. NF1 is polyubiquitinated upon PKC-mediated phosphorylation by a Cullin3/KBTBD7 (kelch repeat and BTB domain containing 7) complex. DAB2IP can be ubiquitinated in response to different conditions by at least three different E3: the FBW7 (F-box and WD repeat domain-containing 7)/SCF (skip1-Cul1-F-box protein) complex, the Skp2 (S-phase kinase-associated protein2)/SCF complex, and Smurf1 (SMAD-specific E3 ubiquitin-ligase 1).

Figure 4

Multiple extracellular inputs can affect intracellular RasGAPs levels and activities in cancer cells. Schematic representation of extracellular signals that inhibit (red) or stimulate (green) expression and/or activity of RasGAPs, thus potentially affecting Ras signaling and oncogenic cell behaviors (see text for details).