The RhoGEF GEF-H1 Is Required for Oncogenic RAS Signaling via KSR-1

Cullis, Jane; Meiri, David; Sandí, María José; Radulovich, Nikolina; Kent, Oliver A.; Medrano, Mauricio; Mokady, Daphna; Normand, Josee; LaRose, Jose; Marcotte, Richard; Marshall, Christopher B.; Ikura, Mitsuhiko; Ketela, Troy; Moffat, Jason; Neel, Benjamin G.; Gingras, Anne‐Claude; Tsao, Ming‐Sound; Rottapel, Robert

doi:10.1016/j.ccr.2014.01.025

Cited by 78 publications

(84 citation statements)

References 57 publications

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“…Although the data presented in Fig. 5 C are certainly not complete (for instance, KSR and Akt share an additional regulatory subunit (63,64)), PP2A clearly does not have a distinct regulator for every substrate with which it interacts. Instead, these regulators have clearly evolved to interact with a specific subset of proteins, possibly coupling their responses in functionally meaningful ways (Fig.…”

Section: Discussionmentioning

confidence: 90%

Phosphatase Specificity and Pathway Insulation in Signaling Networks

Rowland

Harrison

Deeds

2015

Biophysical Journal

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Phosphatases play an important role in cellular signaling networks by regulating the phosphorylation state of proteins. Phosphatases are classically considered to be promiscuous, acting on tens to hundreds of different substrates. We recently demonstrated that a shared phosphatase can couple the responses of two proteins to incoming signals, even if those two substrates are from otherwise isolated areas of the network. This finding raises a potential paradox: if phosphatases are indeed highly promiscuous, how do cells insulate themselves against unwanted crosstalk? Here, we use mathematical models to explore three possible insulation mechanisms. One approach involves evolving phosphatase KM values that are large enough to prevent saturation by the phosphatase's substrates. Although this is an effective method for generating isolation, the phosphatase becomes a highly inefficient enzyme, which prevents the system from achieving switch-like responses and can result in slow response kinetics. We also explore the idea that substrate degradation can serve as an effective phosphatase. Assuming that degradation is unsaturatable, this mechanism could insulate substrates from crosstalk, but it would also preclude ultrasensitive responses and would require very high substrate turnover to achieve rapid dephosphorylation kinetics. Finally, we show that adaptor subunits, such as those found on phosphatases like PP2A, can provide effective insulation against phosphatase crosstalk, but only if their binding to substrates is uncoupled from their binding to the catalytic core. Analysis of the interaction network of PP2A's adaptor domains reveals that although its adaptors may isolate subsets of targets from one another, there is still a strong potential for phosphatase crosstalk within those subsets. Understanding how phosphatase crosstalk and the insulation mechanisms described here impact the function and evolution of signaling networks represents a major challenge for experimental and computational systems biology.

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Section: Discussionmentioning

confidence: 90%

Phosphatase Specificity and Pathway Insulation in Signaling Networks

Rowland

Harrison

Deeds

2015

Biophysical Journal

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“…Furthermore, we found that selumetinib increased phosphorylation of KSR-1 (Thr268), a scaffolding protein required for assembly of MAPK signaling complex (34) and altered phosphorylations on GEF-H1 (Ser122/Ser151). GEF-H1 is been known as a guanine nucleotide exchange factor (GEF) for the Rho family small GTPase proteins (35); however, recently GEF-H1 was shown to promote RAS-driven MAPK via modulating KSR-1 phosphorylation (36). KSR-1 and BRAF or CRAF interaction is enhanced by pharmacological RAF inhibition, regulating drug-induced RAF dimer formation (37–39).…”

Section: Resultsmentioning

confidence: 99%

Phosphoproteomics Reveals MAPK Inhibitors Enhance MET- and EGFR-Driven AKT Signaling in KRAS-Mutant Lung Cancer

Kim

Welsh

Fang

et al. 2016

Molecular Cancer Research

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Pathway inhibition of the RAS-driven MAPK pathway using small-molecule kinase inhibitors has been a key focus for treating cancers driven by oncogenic RAS, yet significant clinical responses are lacking. Feedback reactivation of ERK driven by drug-induced RAF activity has been suggested as one of the major drug resistance mechanisms, especially in the context of oncogenic RAS. To determine if additional adaptive resistance mechanisms may co-exist, we characterized global phosphoproteomic changes after MEK inhibitor selumetinib (AZD6244) treatment in KRAS-mutant A427 and A549 lung adenocarcinoma cell lines employing mass spectrometry-based phosphoproteomics. We identified 9,075 quantifiable unique phosphosites (corresponding to 3,346 unique phosphoproteins), of which 567 phosphosites were more abundant and 512 phosphosites were less abundant after MEK inhibition. Selumetinib increased phosphorylation of KSR-1, a scaffolding protein required for assembly of MAPK signaling complex, as well as altered phosphorylation of GEF-H1, a novel regulator of KSR-1 and implicated in RAS-driven MAPK activation. Moreover, selumetinib reduced inhibitory serine phosphorylation of MET at Ser985 and potentiated HGF- and EGF-induced AKT phosphorylation. These results were recapitulated by pan-RAF (LY3009120), MEK (GDC0623), and ERK (SCH772984) inhibitors, which are currently under early-phase clinical development against RAS-mutant cancers. Our results highlight the unique adaptive changes in MAPK scaffolding proteins (KSR-1, GEF-H1) and in RTK signaling, leading to enhanced PI3K-AKT signaling when the MAPK pathway is inhibited.

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“…MC3T3 cells infected with shGFP or shABL, with or without a retroviral vector expressing TAZ (S89A), were replated in regular growth medium and placed into the INCUCYTE Kinetic Imaging System (Essen Bioscience) to monitor cell growth and the percentage of cell confluence as described previously (55).…”

Section: Discussionmentioning

confidence: 99%

Reciprocal stabilization of ABL and TAZ regulates osteoblastogenesis through transcription factor RUNX2

Matsumoto

Rose

Kent

et al. 2016

Journal of Clinical Investigation

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Figure 2. Active ABL assembles the RUNX2-TAZ transcription factor complex required for osteoblast differentiation. (A) Primary murine osteoblasts were cultured in osteogenic medium for 14 days, and phosphotyrosine immune complexes were probed with an anti-ABL antibody. WCLs were probed with the indicated antibodies for Western blot analysis. (B) HEK293T cells were cotransfected with Flag-TAZ and RUNX2, with or without ABL (PP or KD). Flag-TAZ immune complexes were probed with an anti-RUNX2 antibody. (C) Luciferase activity from a BGLAP reporter assay in HEK293T cells cotransfected with the indicated constructs. n = 3. (D and E) HEK293T cells were cotransfected with the indicated constructs, and RUNX2 (D) or ABL (E) immune complexes were probed with an anti-ABL (D) or anti-Flag (E) antibody. (F) HEK293T cells infected with shGFP or shABL were cotransfected with RUNX2 and Flag-TAZ. The nuclear compartment was extracted from the cells, and Flag-TAZ immune complexes were probed with an anti-RUNX2 antibody. (G and H) HEK293T cells were cotransfected with WT or YF RUNX2 (G) or Flag-TAZ (H), with or without ABL (PP). RUNX2 (G) or Flag-TAZ (H) immune complexes were probed with an anti-p-Tyr antibody. (I) Primary murine osteoblasts infected with shGFP or shABL were cultured in osteogenic medium. p-Tyr or RUNX2 immune complexes were probed with an anti-RUNX2 or anti-TAZ antibody. (J) Luciferase activity from a Bglap reporter assay in primary murine osteoblasts in the presence of shGFP or shABL. n = 3. (K and L) Saos-2 cells infected with an empty vector control or an FKBP-ABL-expressing retroviral vector in the presence of shGFP (K and L), shRUNX2 (K), or shTAZ (L) were cultured in growth medium containing 50 nM FK1012. Cells were stained with alizarin red S solution. n = 3. *P < 0.05, by ANOVA with a Tukey-Kramer post-hoc test. Data represent the mean ± SEM. YAP has previously been reported to be stabilized following tyrosine phosphorylation by ABL (25). In distinction, we observed that all-tyrosine-to-phenylalanine-mutant TAZ (YF) was also stabilized by active ABL (PP) (Supplemental Figure 4F), suggesting that TAZ stabilization by ABL is not mediated by tyrosine phosphorylation. TAZ is degraded following the phosphorylation of Ser311 by LATS, which primes for the phosphorylation of Ser314 by CK1ε (26). Phosphorylated Ser314 lies in the TAZ phosphodegron and triggers binding to and subsequent ubiquitylation of TAZ by the E3-ubiquitin ligase β-TrCP (26). We hypothesized that ABL suppressed TAZ ubiquitylation through disruption of the interaction between TAZ and β-TrCP and observed that ABL (PP), but not ABL (KD), diminished the interaction between these proteins ( Figure 4F). These data demonstrate that ABL stabilizes TAZ through the suppression of its ubiquitin-mediated degradation pathway initiated by β-TrCP. The Journal of Clinical Investigation R E S E A R C H A R T I C L EABL stabilizes the TAZ-TEAD complex required for osteoblast expansion. We have shown that ABL mediated stabilization of TAZ protein and asked whet...

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The RhoGEF GEF-H1 Is Required for Oncogenic RAS Signaling via KSR-1

Cited by 78 publications

References 57 publications

Phosphatase Specificity and Pathway Insulation in Signaling Networks

Phosphatase Specificity and Pathway Insulation in Signaling Networks

Phosphoproteomics Reveals MAPK Inhibitors Enhance MET- and EGFR-Driven AKT Signaling in KRAS-Mutant Lung Cancer

Reciprocal stabilization of ABL and TAZ regulates osteoblastogenesis through transcription factor RUNX2

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