Protein scaffolds play an important role in signal transduction, functioning to facilitate protein interactions and localize key pathway components to specific signaling sites. Connector enhancer of ...KSR-2 (CNK2) is a neuronally expressed scaffold recently implicated in nonsyndromic, X-linked intellectual disability (NS-XLID) 1–3. NS-XLID patients have deficits in cognitive function and their neurons often exhibit dendritic spine abnormalities 4, suggesting a role for CNK2 in synaptic signaling and/or spine formation. To gain insight regarding how CNK2 might contribute to these processes, we used mass spectrometry to identify proteins that interact with the endogenous CNK2 scaffold. Here, we report that the major binding partner of CNK2 is Vilse/ARHGAP39 and that CNK2 complexes are enriched for proteins involved in Rac/Cdc42 signaling, including Rac1 itself, α-PIX and β-PIX, GIT1 and GIT2, PAK3 and PAK4, and members of the cytohesin family. Binding between CNK2 and Vilse was found to be constitutive, mediated by the WW domains of Vilse and a proline motif in CNK2. Through mutant analysis, protein depletion and rescue experiments, we identify CNK2 as a spatial modulator of Rac cycling during spine morphogenesis and find that the interaction with Vilse is critical for maintaining RacGDP/GTP levels at a balance required for spine formation.
•The CNK2 scaffold interacts with components of Rac/Cdc42 pathway signaling•The primary binding partner of endogenous CNK2 is Vilse/ARHGAP39•CNK2 is required for proper dendritic spine morphogenesis in hippocampal neurons•CNK2 is a spatial modulator of RacGDP/GTP cycling during spine morphogenesis
Using mass spectrometry to identify proteins that interact with the CNK2 scaffold, Lim et al. find that CNK2 associates with numerous components of Rac/Cdc42 pathway signaling, including Vilse/ARHGAP39. Through these interactions, CNK2 functions in the spatial regulation of RacGDP/GTP cycling during the morphogenesis of dendritic spines.
BRAF is frequently activated via mutation in human cancer and the RASopathy syndromes; however, for BRAF activation to occur, autoinhibitory interactions between the regulatory and catalytic domains ...must be relieved. Here, we present a proximity-based NanotBRET (bioluminescence resonance energy transfer) assay for real-time measurement of BRAF autoinhibition in live cells. We describe steps for seeding, transfecting, and replating cells. We then detail procedures for reading the NanoBRET emissions and confirming protein expression.
For complete details on the use and execution of this protocol, please refer to Spencer-Smith et al. (2022).1
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•NanoBRET assay for the real-time measurement of BRAF autoinhibition in live cells•Monitors the autoinhibitory interactions of the BRAF regulatory and catalytic domains•Evaluate the effects of internal mutations or external stimuli on BRAF autoinhibition•Relief of autoinhibition is an essential step in BRAF activation
Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.
BRAF is frequently activated via mutation in human cancer and the RASopathy syndromes; however, for BRAF activation to occur, autoinhibitory interactions between the regulatory and catalytic domains must be relieved. Here, we present a proximity-based NanotBRET (bioluminescence resonance energy transfer) assay for real-time measurement of BRAF autoinhibition in live cells. We describe steps for seeding, transfecting, and replating cells. We then detail procedures for reading the NanoBRET emissions and confirming protein expression.
Collective cell migration is required for normal embryonic development and contributes to various biological processes, including wound healing and cancer cell invasion. The M-Ras GTPase and its ...effector, the Shoc2 scaffold, are proteins mutated in the developmental RASopathy Noonan syndrome, and, here, we report that activated M-Ras recruits Shoc2 to cell surface junctions where M-Ras/Shoc2 signaling contributes to the dynamic regulation of cell–cell junction turnover required for collective cell migration. MCF10A cells expressing the dominant-inhibitory M-RasS27N variant or those lacking Shoc2 exhibited reduced junction turnover and were unable to migrate effectively as a group. Through further depletion/reconstitution studies, we found that M-Ras/Shoc2 signaling contributes to junction turnover by modulating the E-cadherin/p120-catenin interaction and, in turn, the junctional expression of E-cadherin. The regulatory effect of the M-Ras/Shoc2 complex was mediated at least in part through the phosphoregulation of p120-catenin and required downstream ERK cascade activation. Strikingly, cells rescued with the Noonan-associated, myristoylated-Shoc2 mutant (Myr-Shoc2) displayed a gain-of-function (GOF) phenotype, with the cells exhibiting increased junction turnover and reduced E-cadherin/p120-catenin binding and migrating as a faster but less cohesive group. Consistent with these results, Noonan-associated C-Raf mutants that bypass the need for M-Ras/Shoc2 signaling exhibited a similar GOF phenotype when expressed in Shoc2-depleted MCF10A cells. Finally, expression of the Noonan-associated Myr-Shoc2 or C-Raf mutants, but not their WT counterparts, induced gastrulation defects indicative of aberrant cell migration in zebrafish embryos, further demonstrating the function of the M-Ras/Shoc2/ERK cascade signaling axis in the dynamic control of coordinated cell movement.
Unlocking the code of 14-3-3 Dougherty, Michele K; Morrison, Deborah K
Journal of cell science,
2004-Apr-15, 2004-04-15, 20040415, Letnik:
117, Številka:
Pt 10
Journal Article
Recenzirano
Odprti dostop
One of the most striking 'rags to riches' stories in the protein world is that of 14-3-3, originally identified in 1967 as merely an abundant brain protein. The first clues that 14-3-3 would play an ...important role in cell biology came almost 25 years later when it was found to interact with various proto-oncogene proteins and signaling proteins. The subsequent identification of 14-3-3 as a phosphoserine/phosphothreonine-binding protein firmly established its importance in cell signaling. 14-3-3 family members are found in all eukaryotes - from plants to mammals - and more than 100 binding partners have been identified to date. The targets of 14-3-3 are found in all subcellular compartments and their functional diversity is overwhelming - they include transcription factors, biosynthetic enzymes, cytoskeletal proteins, signaling molecules, apoptosis factors and tumor suppressors. 14-3-3 binding can alter the localization, stability, phosphorylation state, activity and/or molecular interactions of a target protein. Recent studies now indicate that the serine/threonine protein phosphatases PP1 and PP2A are important regulators of 14-3-3 binding interactions, and demonstrate a role for 14-3-3 in controlling the translocation of certain proteins from the cytoplasmic and endoplasmic reticulum to the plasma membrane. New reports also link 14-3-3 to several neoplastic and neurological disorders, where it might contribute to the pathogenesis and progression of these diseases.
RAF kinase inhibitors can induce ERK cascade signaling by promoting dimerization of RAF family members in the presence of oncogenic or normally activated RAS 1–3. This interaction is mediated by a ...dimer interface region in the RAF kinase domain that is conserved in members of the ERK cascade scaffold family, kinase suppressor of RAS (KSR) 4, 5. In this study, we find that most RAF inhibitors also induce the binding of KSR1 to wild-type and oncogenic B-RAF proteins, including V600E B-RAF, but promote little complex formation between KSR1 and C-RAF. The inhibitor-induced KSR1/B-RAF interaction requires direct binding of the drug to B-RAF and is dependent on conserved dimer interface residues in each protein, but, unexpectedly, is not dependent on binding of B-RAF to activated RAS. Inhibitor-induced KSR/B-RAF complex formation can occur in the cytosol and is observed in normal mouse fibroblasts, as well as a variety of human cancer cell lines. Strikingly, we find that KSR1 competes with C-RAF for inhibitor-induced binding to B-RAF and, as a result, alters the effect of the inhibitors on ERK cascade signaling.
► Select RAF inhibitors promote KSR1/B-RAF binding but little KSR1/C-RAF binding ► Inhibitor-induced KSR1/B-RAF binding is RAS independent and occurs in the cytoplasm ► KSR1 can compete with C-RAF for inhibitor-induced binding to B-RAF ► The presence of KSR1 can alter the effects of RAF inhibitors on ERK cascade signaling
Signaling dynamics of the KSR1 scaffold complex McKay, Melissa M; Ritt, Daniel A; Morrison, Deborah K
Proceedings of the National Academy of Sciences - PNAS,
07/2009, Letnik:
106, Številka:
27
Journal Article
Recenzirano
Odprti dostop
Scaffold proteins contribute to the spatiotemporal control of MAPK signaling and KSR1 is an ERK cascade scaffold that localizes to the plasma membrane in response to growth factor treatment. To ...better understand the molecular mechanisms of KSR1 function, we examined the interaction of KSR1 with each of the ERK cascade components, Raf, MEK, and ERK. Here, we identify a hydrophobic motif within the proline-rich sequence (PRS) of MEK1 and MEK2 that is required for constitutive binding to KSR1 and find that MEK binding and residues in the KSR1 CA1 region enable KSR1 to form a ternary complex with B-Raf and MEK following growth factor treatment that enhances MEK activation. We also find that docking of active ERK to the KSR1 scaffold allows ERK to phosphorylate KSR1 and B-Raf on feedback S/TP sites. Strikingly, feedback phosphorylation of KSR1 and B-Raf promote their dissociation and result in the release of KSR1 from the plasma membrane. Together, these findings provide unique insight into the signaling dynamics of the KSR1 scaffold and reveal that through regulated interactions with Raf and ERK, KSR1 acts to both potentiate and attenuate ERK cascade activation, thus regulating the intensity and duration of ERK cascade signaling emanating from the plasma membrane during growth factor signaling.
A distinct profile of NRAS mutants is observed in each tumor type. It is unclear whether these profiles are determined by mutagenic events or functional differences between NRAS oncoproteins. Here, ...we establish functional hallmarks of NRAS mutants enriched in human melanoma. We generate eight conditional, knock-in mouse models and show that rare melanoma mutants (NRAS G12D, G13D, G13R, Q61H, and Q61P) are poor drivers of spontaneous melanoma formation, whereas common melanoma mutants (NRAS Q61R, Q61K, or Q61L) induce rapid tumor onset with high penetrance. Molecular dynamics simulations, combined with cell-based protein-protein interaction studies, reveal that melanomagenic NRAS mutants form intramolecular contacts that enhance BRAF binding affinity, BRAF-CRAF heterodimer formation, and MAPK > ERK signaling. Along with the allelic series of conditional mouse models we describe, these results establish a mechanistic basis for the enrichment of specific NRAS mutants in human melanoma.
Current BRAF inhibitors block signaling from monomeric BRAFV600E, but not from oncogenic RAS, which requires RAF dimerization. In this issue of Cancer Cell, Yao and colleagues investigate why current ...drugs are ineffective against RAF dimers, while Peng and colleagues describe a pan-RAF inhibitor targeting both monomeric and dimeric RAF.
Current BRAF inhibitors block signaling from monomeric BRAFV600E, but not from oncogenic RAS, which requires RAF dimerization. In this issue of Cancer Cell, Yao and colleagues elucidate why current drugs are ineffective against RAF dimers, while Peng and colleagues describe a pan-RAF inhibitor targeting both monomeric and dimeric RAF.
Lung squamous cell carcinoma (LSCC) is the second most prevalent type of lung cancer. Despite extensive genomic characterization, no targeted therapies are approved for the treatment of LSCC. Distal ...amplification of the 3q chromosome is the most frequent genomic alteration in LSCC, and there is an urgent need to identify efficacious druggable targets within this amplicon. We identify the protein kinase TNIK as a therapeutic target in LSCC.
is amplified in approximately 50% of LSCC cases. TNIK genetic depletion or pharmacologic inhibition reduces the growth of LSCC cells
and
. In addition, TNIK inhibition showed antitumor activity and increased apoptosis in established LSCC patient-derived xenografts. Mechanistically, we identified the tumor suppressor Merlin/
as a novel TNIK substrate and showed that TNIK and Merlin are required for the activation of focal adhesion kinase. In conclusion, our data identify targeting TNIK as a potential therapeutic strategy in LSCC. SIGNIFICANCE: Targeted therapies have not yet been approved for the treatment of LSCC, due to lack of identification of actionable cancer drivers. We define TNIK catalytic activity as essential for maintaining LSCC viability and validate the antitumor efficacy of TNIK inhibition in preclinical models of LSCC.
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The Raf protein kinases are key intermediates in cellular signal transduction, functioning as direct effectors of the Ras GTPases and as the initiating kinases in the ERK cascade. In human cancer, ...Raf activity is frequently dysregulated due to mutations in the Raf family member B-Raf or to alterations in upstream Raf regulators, including Ras and receptor tyrosine kinases. First-generation Raf inhibitors, such as vemurafenib and dabrafenib, have yielded dramatic responses in malignant melanomas containing B-Raf mutations; however, their overall usefulness has been limited by both intrinsic and acquired drug resistance. In particular, issues related to the dimerisation of the Raf kinases can impact the efficacy of these compounds and are a primary cause of drug resistance. Here, we will review the importance of Raf dimerisation in cell signalling as well as its effects on Raf inhibitor therapy, and we will present the new strategies that are being pursued to overcome the 'Raf Dimer Dilemma'.
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