CNKSR1 serves as a scaffold to activate an EGFR phosphatase via exclusive interaction with RhoB-GTP

Nishiyama, Kanako; Maekawa, Mamoru; Nakagita, Tomoya; Nakayama, Jun; Kiyoi, Takeshi; Chosei, Mami; Murakami, Akari; Kamei, Yasuhiro; Takeda, Hiroyuki; Takada, Yasutsugu; Higashiyama, Shigeki

doi:10.26508/lsa.202101095

Cited by 14 publications

(10 citation statements)

References 70 publications

(99 reference statements)

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“…3A ). CNKSR1 has previously been reported in different cell-based contexts to regulate other intracellular signal transduction processes, including NF-κB, c-JUN, RhoGTPase signaling, or AKT signaling ( 24, 30–33 ). To identify leads for intracellular signal transduction cues associated with CNKSR1 function in pancreas cancer, we compared the transcriptome of pancreas cancer cell lines with high CNKSR1 expression levels to cell lines with no or minimal CNKSR1 expression next ( Fig.…”

Section: Resultsmentioning

confidence: 99%

Scaffolding Protein Connector Enhancer of Kinase Suppressor of Ras 1 (CNKSR1) Regulates MAPK Inhibition Responsiveness in Pancreas Cancer via Crosstalk with AKT Signaling

Miermont

Sable

et al. 2023

Molecular Cancer Research

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Combinatorial molecular therapy in pancreatic ductal adenocarcinoma (PDAC) has yielded largely disappointing results in clinical testing to-date as a multitude of adaptive resistance mechanisms is making selection of patients via molecular markers which capture essential, intersecting signaling routes challenging. Here, we report the scaffolding protein connector enhancer of kinase suppressor of Ras 1 (CNKSR1) as mediator of resistance to mitogen-activated protein kinase (MEK) inhibition. MEK inhibition in CNKSR1high cancer cells induces translocation of CNKSR1 to the plasma membrane where the scaffolding protein interacts with and stabilizes the phosphorylated form of AKT. CNKSR1-mediated AKT activation following MEK inhibition was associated with increased cellular p-PRAS40 levels and reduced nuclear translocation and cellular levels of FoxO1, a negative regulator of AKT signaling. In clinical PDAC specimens, high cytoplasmatic CNKSR1 levels correlated with increased cellular phospho-AKT and mTOR levels. Pharmacological co-blockade of AKT and MEK ranked top in induced synergies with MEK inhibition in CNKSR1high pancreas cancer cells among other inhibitor combinations targeting known CNKSR1 signaling. In vivo, CNKSR1high pancreatic tumors treated with AKT and MEK inhibitors showed improved outcome in the combination arm compared to single agent treatment, an effect not observed in CNKSR1low models. Our results identify CNKSR1 as regulator of adaptive resistance to MEK inhibition by promoting crosstalk to AKT signaling via a scaffolding function for the phosphorylated form of AKT. CNSKR1 expression might be a possible molecular marker to enrich patients for future AKT-MEK inhibitor precision medicine studies. Implications: The CNKSR1 scaffold, identified within a RNAi screen as a novel mediator of resistance to MEK inhibition in pancreas cancer, connects MAPK pathway and AKT signaling and may be adopted as a biomarker to select patients for combined MEK AKT blockade.

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Section: Resultsmentioning

confidence: 99%

Scaffolding Protein Connector Enhancer of Kinase Suppressor of Ras 1 (CNKSR1) Regulates MAPK Inhibition Responsiveness in Pancreas Cancer via Crosstalk with AKT Signaling

Miermont

Sable

et al. 2023

Molecular Cancer Research

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“…The set of 250 recombinant human channels is a relatively compact protein array; however, it may be an important tool for drug discovery because channels are important drug target proteins. Protein arrays are profoundly useful in comprehensive analysis, such as the discovery of enzymes or substrates, the identification of binding partners, antibody specificity analysis, and the target discovery of small-molecule drugs [ 41 , 46 , 47 , 48 ]. A promising application for the channel array is the discovery of drug seeds by affinity selection.…”

Section: Discussionmentioning

confidence: 99%

Evaluation of Cell-Free Synthesized Human Channel Proteins for In Vitro Channel Research

et al. 2022

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Despite channel proteins being important drug targets, studies on channel proteins remain limited, as the proteins are difficult to express and require correct complex formation within membranes. Although several in vitro synthesized recombinant channels have been reported, considering the vast diversity of the structures and functions of channel proteins, it remains unclear which classes of channels cell-free synthesis can be applied to. In this study, we synthesized 250 clones of human channels, including ion channel pore-forming subunits, gap junction proteins, porins, and regulatory subunits, using a wheat cell-free membrane protein production system, and evaluated their synthetic efficiency and function. Western blotting confirmed that 95% of the channels were successfully synthesized, including very large channels with molecular weights of over 200 kDa. A subset of 47 voltage-gated potassium ion channels was further analyzed using a planar lipid bilayer assay, out of which 80% displayed a voltage-dependent opening in the assay. We co-synthesized KCNB1 and KCNS3, a known heteromeric complex pair, and demonstrated that these channels interact on a liposome. These results indicate that cell-free protein synthesis provides a promising solution for channel studies to overcome the bottleneck of in vitro protein production.

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“…CUL3/KCTD10 ubiquitinated RhoB for K63-linked ubiquitin degradation and promote HER2-positive breast cancer cell proliferation (137,138). Two downstream proteins of RhoB, RAC1 (Rho GTPase) and CNKSR1 (connector enhancer of kinase suppressor of Ras1), were found to be significantly correlated with the prognosis of HER2-positive breast cancer patients (138,139). Beyond small-molecule inhibitors, proteolysis-targeting chimeras (PROTACs), which can induce the recruitment of E3 to target protein, have recently emerged as significant future therapeutic opportunities (140,141).…”

Section: Discussionmentioning

confidence: 99%

E3 Ubiquitin Ligases in Breast Cancer Metastasis: A Systematic Review of Pathogenic Functions and Clinical Implications

et al. 2021

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Female breast cancer has become the most commonly occurring cancer worldwide. Although it has a good prognosis under early diagnosis and appropriate treatment, breast cancer metastasis drastically causes mortality. The process of metastasis, which includes cell epithelial–mesenchymal transition, invasion, migration, and colonization, is a multistep cascade of molecular events directed by gene mutations and altered protein expressions. Ubiquitin modification of proteins plays a common role in most of the biological processes. E3 ubiquitin ligase, the key regulator of protein ubiquitination, determines the fate of ubiquitinated proteins. E3 ubiquitin ligases target a broad spectrum of substrates. The aberrant functions of many E3 ubiquitin ligases can affect the biological behavior of cancer cells, including breast cancer metastasis. In this review, we provide an overview of these ligases, summarize the metastatic processes in which E3s are involved, and comprehensively describe the roles of E3 ubiquitin ligases. Furthermore, we classified E3 ubiquitin ligases based on their structure and analyzed them with the survival of breast cancer patients. Finally, we consider how our knowledge can be used for E3s’ potency in the therapeutic intervention or prognostic assessment of metastatic breast cancer.

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CNKSR1 serves as a scaffold to activate an EGFR phosphatase via exclusive interaction with RhoB-GTP

Cited by 14 publications

References 70 publications

Scaffolding Protein Connector Enhancer of Kinase Suppressor of Ras 1 (CNKSR1) Regulates MAPK Inhibition Responsiveness in Pancreas Cancer via Crosstalk with AKT Signaling

Scaffolding Protein Connector Enhancer of Kinase Suppressor of Ras 1 (CNKSR1) Regulates MAPK Inhibition Responsiveness in Pancreas Cancer via Crosstalk with AKT Signaling

Evaluation of Cell-Free Synthesized Human Channel Proteins for In Vitro Channel Research

E3 Ubiquitin Ligases in Breast Cancer Metastasis: A Systematic Review of Pathogenic Functions and Clinical Implications

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