The authors would like to acknowledge the Hairy Cell Leukemia Fellowship for support of Yuan Jimin. This work was supported by Asia Fund Cancer Research (AFCR2017/2019-JH), Duke-NUS Khoo Bridge Funding Award (Duke-NUS-KBrFA/2018/0014), NCCRF bridging grant (NCCRF-YR2018-JUL-BG4), NCCRF pilot grant (NCCRF-YR2017-JUL-PG3), and SHF Academic Medicine Research Grant (AM/TP011/2018).

1. In Vitro Kinase Assay for Measuring the Catalytic Activity of RAF Mutants
<ol>
	<li>Construct vectors encoding RAF mutants (Figure 1A) with FLAG(DYKDDDDK) tag at C-terminus by using Gibson Assembly or traditional molecular cloning methods.
	<ol>
		<li>Introduce the FLAG tag and mutations into the RAF coding sequences by PCRs, and then insert whole sequences into pCDNA3.1(+) vector by using Gibson assembly or T4 DNA ligation and following the manufacture&#8217;s protocols. Use the following conditions for PCR reactions: (1) 95 &#176;C, 2 min; (2) 95 &#176;C, 30 s; (3) 59 &#176;C, 30 s; (4) 68 &#176;C, 3 min; (5) 20 cycles of (2 to 4); (6) 4 &#176;C hold. 
		NOTE: The PCR primers for cloning: 5- AAATTAATACGACTCACTATAGGGAGACCC-3 and 5-CAGCGGGTTTAAACGGGCCCTCTA-3.</li>
		<li>Insert the GST coding sequence upstream of RAF mutant coding sequences to generate vectors encoding GST-fused RAF mutants by using same methods as described in step 1.1.1.</li>
		<li>Validate all vectors by DNA sequencings before transfection.</li>
	</ol>
	</li>
	<li>Plate 293T cells in 6-well plates at a density of 5 x 105 cells/well one day before transfection. When the cell density reaches 80~90% confluence on the second day, transfect with vectors encoding FLAG-tagged RAF kinases or their mutants from step 1.1 into cells by following the manufacture&#8217;s protocol of transfection reagents (Table of Materials).</li>
	<li>Replace the culture medium 24 h after transfection.</li>
	<li>Aspirate the culture medium 48 h after transfection and add 400 &#181;L/well of lysis buffer (25 mM Tris&#183;HCl, 150 mM NaCl, 1 mM ethylenediaminetetraacetic acid (EDTA), 0.25% NP-40, pH 7.2) supplemented with protease and phosphatase inhibitors to lyse cells on ice. 
	NOTE: The concentration of NP-40 in lysis buffer is critical for detecting the catalytic activity of RAF mutants with moderate dimer affinity/stability in vitro. A high concentration of detergent or a strong detergent in lysis buffer may break RAF dimers and thereby kill the catalytic activity of RAF kinases or their mutants.</li>
	<li>Transfer the cell lysates to a 1.5 mL tube, and spin down by 12,000 x g for 10 min at 4 &#176;C to deplete cell debris.</li>
	<li>Transfer 300 &#181;L per sample of clean whole cell lysates to 1.5 mL tubes, add 20 &#181;L per sample of anti-FLAG affinity beads, and rotate in a cold room (4 &#176;C) for 1 h. Also take 40 &#181;L per sample of clean whole cell lysate aside for detecting the expression and activity (phospho-ERK1/2) of RAF mutants by immunoblots as described below.</li>
	<li>Wash the anti-FLAG beads once with lysis buffer, then once with kinase reaction buffer (20 mM HEPES, 10 mM MgCl2, 0.5 mM Na3VO4, 0.5 mM DTT, pH 7.2), and add 20 &#181;L of kinase reaction mixture (2 &#956;g of MEK1(K97A) and 100 &#181;M ATP in 20 &#181;L of kinase reaction buffer) per sample. 
	NOTE: The bead washing should be completed gently and quickly, the residual buffer should be aspirated completely before adding kinase reaction mixture, and all operations at this step should be carried out at 4 &#176;C in a cold room.</li>
	<li>Incubate the kinase reactions at room temperature (25 &#176;C) for 10 min, and flip the tubes containing kinase reactions with fingers every other minute during incubation.</li>
	<li>Add 5 &#181;L of 5x SDS sample buffer (375 mM Tris&#183;HCl, 9% sodium dodecyl sulfate (SDS), 50% Glycerol, 0.03% Bromophenol Blue) per sample to stop kinase reactions, and then heat the samples at 90 &#176;C for 5 min.</li>
	<li>Run the samples in 9~12% polyacrylamide gel electrophoresis (PAGE) with 0.1% SDS, transfer the proteins to nitrocellulose membrane, and detect the levels of phospho-MEK and RAF mutants in samples by immunoblots. 
	NOTE: The phospho-MEK can also be quantified by using &#947;32P-ATP incorporation. Briefly, 10 &#181;M &#947;32P-ATP is added to the kinase reaction buffer, and the amount of phosphorylated MEK is then quantified after PAGE separation by using standard autoradiography, phosphorimaging, or liquid scintillation counting techniques as appropriate.</li>
</ol>
2. RAF Co-activation Assay for Evaluating the Allosteric Activity of Kinase-dead RAF Mutants
<ol>
	<li>Construct vectors encoding the RAF receiver (CRAF kinase domain with unphosphorylatable NtA motif, AAFF) or the kinase-dead RAF activators (RAF kinase domain with phosphorylation-mimicked NtA motif, SSDD, DDEE or DGEE) (Figure 1A) as described in step 1.1.</li>
	<li>Transfect 293T cells with two vectors encoding both the RAF receiver and the kinase-dead RAF activator or a single vector encoding one of proteins as described in steps 1.2 and 1.3.</li>
	<li>Replace the culture medium at 24 h after transfection, and harvest 293T transfectants at 48 h to prepare the whole cell lysates as described in steps 1.4 and 1.5.</li>
	<li>Mix the clean whole cell lysate with 5x SDS sample buffer quickly at room temperature (25 &#176;C) and then boil at 90 &#176;C for 5 min.</li>
	<li>Run the boiled whole cell lysate samples in 9~12% PAGE with 0.1% SDS, transfer the proteins to nitrocellulose membrane, and detect the levels of phospho-ERK1/2 and control proteins by immunoblots.</li>
</ol>
3. Complimentary Split Luciferase Assay for Measuring the Relative Dimer Affinity/Stability of RAF Mutants
<ol>
	<li>Construct vectors encoding FLAG-tagged RAF mutants fused to the N-terminus of Nluc (N-terminus of firefly luciferase, aa2-416) or the C-terminus of Cluc (C-terminus of firefly luciferase, aa398-550) as described in step 1.1.</li>
	<li>Transfect 293T cells with a pair of vectors encoding different Nluc-RAF mutants and Cluc-RAF mutants as described in step 1.2.</li>
	<li>At 24 h after transfection, replate 293T cell transfectants into Krystal black image plates at the cell density of 2x105 per well with color-free medium (i.e., DMEM without phenol red).</li>
	<li>24 h later, add D-luciferin (0.2 mg/mL) to 293T cell transfectants, incubate for 30 min, and measure the luciferase signals by using a multi detection system (Table of Materials).</li>
	<li>After measuring the luciferase signals, aspirate the medium and lyse 293T transfectants with lysis buffer to prepare the whole cell lysates as described in steps 1.4 and 1.5.</li>
	<li>Run the whole cell lysate samples in 9~12% PAGE with 0.1% SDS and detect the expression levels of Nluc-RAF mutants and Cluc-RAF mutants by anti-FLAG immunoblot as described in step 2.5. The relative expression levels of both Nluc-RAF mutant and Cluc-RAF mutant in 293T transfectants are quantified by using image J from their immunoblots.</li>
	<li>Normalize the luciferase signals of 293T cell transfectants according to the expression levels of Nluc-RAF mutants and Cluc-RAF mutants. Briefly, this is achieved by dividing the raw luciferase signal by the relative expression levels of Nluc-RAF mutants and Cluc-RAF mutants from step 3.6.</li>
</ol>

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The authors declare that they have no competing financial interests.

In this article, we presented three methods for characterizing disease-related RAF mutants, which include in vitro kinase assay, RAF co-activation assay, and complimentary split luciferase assay. Since RAF kinases have both catalytic activity and allosteric activity, various RAF mutants can activate the downstream signaling through two distinct mechanisms13,14,16,17,<sup class="xref"...

The RAF family kinases are a key component of RAS/RAF/MEK/ERK signaling cascade, which transmit a signal from RAS to activate mitogen-activated protein kinase (MEK)1,2,3,4. This family of kinases plays a crucial role in cell growth, survival and differentiation, and their alterations induce many diseases, notably cancer5,6,7,8. Recently, genomic sequencings have identified many disease-related RAF mutants that exhibit different properties in the signal transmission of RAS/RAF/MEK/ERK cascade9,10,11. A careful characterization of RAF mutants will help us understand the molecular mechanisms of how RAF mutants alter the signal output of RAS/RAF/MEK/ERK cascade, eventually select appropriate approaches for treating various RAF mutant-driven diseases.
The RAF family kinases include three members, CRAF, BRAF, and ARAF, which have similar molecular structures but different abilities to activate downstream signaling1,2,3,4. Among these paralogs, BRAF has the highest activity by virtue of its constitutively phosphorylated NtA (N-terminal acidic) motif12,13,14, while ARAF has the lowest activity arising from its non-canonical APE motif15. This may explain the different mutation frequencies of RAF paralogs in diseases: BRAF&#62;CRAF&#62;ARAF. Moreover, within the same RAF paralog, mutations in different sites may trigger downstream signaling in distinct manners, which adds another layer of complexity to the regulation of RAF family kinases. Recent studies have demonstrated that all RAF kinases have both catalytic and allosteric activities13,14,16,17,18. Constitutively active RAF mutants turn on the downstream signaling directly by phosphorylating MEK, whereas kinase-dead RAF mutants can transactivate their wild-type counterparts through side-to-side dimerization and activate MEK-ERK signaling16,19,20. The dimer affinity/stability is a key parameter that not only determines the allosteric activity of kinase-dead RAF mutants but also affects the catalytic activity of constitutively active RAF mutants15,21,22. The kinase-dead RAF mutants with high dimer affinity/stability can transactivate the endogenous wild-type RAFs directly15, while those with intermediate dimer affinity/stability requires a coordination of active Ras or an elevated level of wild-type RAF molecules to function13,15,20,21,23. Similarly, constitutively active RAF mutants phosphorylate MEK in a dimer-dependent manner, and those with low dimer affinity/stability lose their catalytic activity in vitro upon immunoprecipitation that breaks the weak RAF dimers15,21,22. The dimer affinity/stability also determines the sensitivity of RAF mutants to their inhibitors, and positively correlates to the resistance of RAF inhibitors24. Therefore, to characterize disease-related RAF mutants, it is necessary to measure their catalytic and allosteric activities, and dimer affinity/stability.
In recent years, our laboratory and others have developed various methods to characterize RAF family kinases and their mutants. According to our laboratory and others&#39;&#160;experience, we think that the following three assays have advantages in defining disease-related RAF mutants: (1) the in vitro kinase assay that can be carried out with ease to detect the catalytic activity of constitutively active RAF mutants15; (2) the RAF co-activation assay that is a reliable and convenient method to measure the allosteric activity of kinase-dead RAF mutants13,15,21,22,23,25; (3) the complimentary split luciferase assay that has much higher sensitivity in measuring the relative dimer affinity/stability of RAF mutants in contrast to the traditional co-immunoprecipitation assay, and is able to carry out without advanced equipment in contrast to the quantitative analytic methods such as SPR (Surface Plasmon Resonance) analysis15,22. Combining these three assays, we can understand easily how a disease-related RAF mutant alters the downstream signaling and thereby utilize an appropriate therapeutic strategy to treat the disease caused by this RAF mutation.

<table><tbody><tr><td>anti-phosphoERK1/2</td><td>Cell Signaling Technologies</td><td>4370</td><td></td></tr><tr><td>anti-phosphoMEK1/2</td><td>Cell Signaling Technologies</td><td>9154</td><td></td></tr><tr><td>anti-ERK1/2</td><td>AB clonal</td><td>A0229</td><td></td></tr><tr><td>anti-MEK1/2</td><td>Cell Signaling Technologies</td><td>9124</td><td></td></tr><tr><td>anti-FLAG(mouse)</td><td>Sigma-Aldrich</td><td>F3165</td><td></td></tr><tr><td>anti-HA</td><td>Novus Biologicals</td><td>MAB6875</td><td></td></tr><tr><td>anti-FLAG(Rabbit)</td><td>Cell Signaling Technologies</td><td>14793</td><td></td></tr><tr><td>anti-&amp;beta;-actin</td><td>Sigma-Aldrich</td><td>A2228</td><td></td></tr><tr><td>anti-FLAG beads(M2)</td><td>Sigma-Aldrich</td><td>A4596</td><td></td></tr><tr><td>HRP-conjugated anti-mouse IgG</td><td>Jackson Laboratories</td><td>115-035-003</td><td></td></tr><tr><td>HRP-conjugated anti-Rabbit IgG</td><td>Jackson Laboratories</td><td>111-035-144</td><td></td></tr><tr><td>pcDNA3.1(+)</td><td>In vitrogen</td><td>V79020</td><td></td></tr><tr><td>Gibson Assembly Cloning&amp;nbsp; Kit</td><td>New England Biolabs</td><td>E5510</td><td></td></tr><tr><td>T4 DNA ligase</td><td>New England Biolabs</td><td>M0202</td><td></td></tr><tr><td>Lipofectamine 2000</td><td>Invitrogen</td><td>11668019</td><td></td></tr><tr><td>Fugene 6</td><td>Roche</td><td>11 814 443 001</td><td></td></tr><tr><td>DMEM w/o phenol red</td><td>Invitrogen</td><td>21063-029</td><td></td></tr><tr><td>D-luciferin&amp;nbsp;</td><td>GoldBio</td><td>LUCK-100</td><td></td></tr><tr><td>6xhis-tagged MEK1 (K97A)</td><td>&amp;nbsp;prepared in our previous studies</td><td>N.A.</td><td>Reference 15.</td></tr><tr><td>GloMax-Multi Detection System.</td><td>Promega</td><td>E7041</td><td></td></tr></tbody></table>

characterize disease-related mutants of raf family kinases by using a set of practical and feasible methods

The rapidly accelerated fibrosarcoma (RAF) family kinases play a central role in cell biology and their dysfunction leads to cancers and developmental disorders. A characterization of disease-related RAF mutants will help us select appropriate therapeutic strategies for treating these diseases. Recent studies have shown that RAF family kinases have both catalytic and allosteric activities, which are tightly regulated by dimerization. Here, we constructed a set of practical and feasible methods to determine the catalytic and allosteric activities and the relative dimer affinity/stability of RAF family kinases and their mutants. Firstly, we amended the classical in vitro kinase assay by reducing the detergent concentration in buffers, utilizing a gentle quick wash procedure, and employing a glutathione S-transferase (GST) fusion to prevent RAF dimers from dissociating during purification. This enables us to measure the catalytic activity of constitutively active RAF mutants appropriately. Secondly, we developed a novel RAF co-activation assay to evaluate the allosteric activity of kinase-dead RAF mutants by using N-terminal truncated RAF proteins, eliminating the requirement of active Ras in current protocols and thereby achieving a higher sensitivity. Lastly, we generated a unique complementary split luciferase assay to quantitatively measure the relative dimer affinity/stability of various RAF mutants, which is more reliable and sensitive compared to the traditional co-immunoprecipitation assay. In summary, these methods have the following advantages: (1) user-friendly; (2) able to carry out effectively without advanced equipment; (3) cost-effective; (4) highly sensitive and reproducible.

The RAF family kinases have both catalytic and allosteric activities, which enable their disease-related mutants to turn on the downstream signaling through different mechanisms13,14,16,17,18. The constitutively active RAF mutants directly phosphorylate their substrates, while the kinase-dead RAF mutants fulfill their function through transactivating wild-type...

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Characterize Disease-related Mutants of RAF Family Kinases by Using a Set of Practical and Feasible Methods

In this article, we presented a set of practical and feasible methods for characterizing disease-related mutants of RAF family kinases, which include in vitro kinase assay, RAF co-activation assay, and complementary split luciferase assay.

The rapidly accelerated fibrosarcoma (RAF) family kinases play a central role in cell biology and their dysfunction leads to cancers and developmental ...

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Universidad San Sebastian

Research

JoVE Journal

Cancer Research

5.9K Views.  National Cancer Centre Singapore. In this article, we presented a set of practical and feasible methods for characterizing disease-related mutants of RAF family kinases, which include in vitro kinase assay, RAF co-activation assay, and complementary split luciferase assay.

Video: Characterize Disease-related Mutants of RAF Family Kinases by Using a Set of Practical and Feasible Methods

Assaying Protein Kinase Activity with Radiolabeled ATP

Protein kinases are able to govern large-scale cellular changes in response to complex arrays of stimuli, and much effort has been directed at uncovering allosteric details of their regulation. Kinases comprise signaling networks whose defects are often hallmarks of multiple forms of cancer and related diseases, making an assay platform amenable to manipulation of upstream regulatory factors and validation of reaction requirements critical in the search for improved therapeutics. Here, we describe a basic kinase assay that can be easily adapted to suit specific experimental questions including but not limited to testing the effects of biochemical and pharmacological agents, genetic manipulations such as mutation and deletion, as well as cell culture conditions and treatments to probe cell signaling mechanisms. This assay utilizes radiolabeled [&#947;-32P] ATP, which allows for quantitative comparisons and clear visualization of results, and can be modified for use with immunoprecipitated or recombinant kinase, specific or typified substrates, all over a wide range of reaction conditions.

Protein kinases are highly evolved signaling enzymes and scaffolds that are critical for inter- and intracellular signal transduction. We present a protocol for measuring kinase activity through the use of radiolabeled adenosine triphosphate ([&#947;-32P] ATP), a reliable method to aid in elucidation of cellular signaling regulation.

Protein kinases are able to govern large-scale cellular changes in response to complex arrays of stimuli, and much effort has been directed at uncovering ...

Biochemistry

Generation of High-Throughput Three-Dimensional Tumor Spheroids for Drug Screening

Cancer cells have routinely been cultured in two dimensions (2D) on a plastic surface. This technique, however, lacks the true environment a tumor mass is exposed to in vivo. Solid tumors grow not as a sheet attached to plastic, but instead as a collection of clonal cells in a three-dimensional (3D) space interacting with their neighbors, and with distinct spatial properties such as the disruption of normal cellular polarity. These interactions cause 3D-cultured cells to acquire morphological and cellular characteristics which are more relevant to in vivo tumors. Additionally, a tumor mass is in direct contact with other cell types such as stromal and immune cells, as well as the extracellular matrix from all other cell types. The matrix deposited is comprised of macromolecules such as collagen and fibronectin.
In an attempt to increase the translation of research findings in oncology from bench to bedside, many groups have started to investigate the use of 3D model systems in their drug development strategies. These systems are thought to be more physiologically relevant because they attempt to recapitulate the complex and heterogeneous environment of a tumor. These systems, however, can be quite complex, and, although amenable to growth in 96-well formats, and some now even in 384, they offer few choices for large-scale growth and screening. This observed gap has led to the development of the methods described here in detail to culture tumor spheroids in a high-throughput capacity in 1536-well plates. These methods represent a compromise to the highly complex matrix-based systems, which are difficult to screen, and conventional 2D assays. A variety of cancer cell lines harboring different genetic mutations are successfully screened, examining compound efficacy by using a curated library of compounds targeting the Mitogen-Activated Protein Kinase or MAPK pathway. The spheroid culture responses are then compared to the response of cells grown in 2D, and differential activities are reported. These methods provide a unique protocol for testing compound activity in a high-throughput 3D setting.

Across a wide variety of disease indications, more physiologically relevant models are being developed and implemented into drug discovery programs. The new model system described here demonstrates how three-dimensional tumor spheroids can be cultured and screened in a high-throughput 1536-well plate-based system to search for new oncology drugs.

Cancer cells have routinely been cultured in two dimensions (2D) on a plastic surface. This technique, however, lacks the true environment a tumor mass is ...

Light-mediated Reversible Modulation of the Mitogen-activated Protein Kinase Pathway during Cell Differentiation and Xenopus Embryonic Development

Kinase activity is crucial for a plethora of cellular functions, including cell proliferation, differentiation, migration, and apoptosis. During early embryonic development, kinase activity is highly dynamic and widespread across the embryo. Pharmacological and genetic approaches are commonly used to probe kinase activities. Unfortunately, it is challenging to achieve superior spatial and temporal resolution using these strategies. Furthermore, it is not feasible to control the kinase activity in a reversible fashion in live cells and multicellular organisms. Such a limitation remains a bottleneck for achieving a quantitative understanding of kinase activity during development and differentiation. This work presents an optogenetic strategy that takes advantage of a bicistronic system containing photoactivatable proteins Arabidopsis thaliana cryptochrome 2 (CRY2) and the N-terminal domain of cryptochrome-interacting basic-helix-loop-helix (CIBN). Reversible activation of the mitogen-activated protein kinase (MAPK) signaling pathway is achieved through light-mediated protein translocation in live cells. This approach can be applied to mammalian cell cultures and live vertebrate embryos. This bicistronic system can be generalized to control the activity of other kinases with similar activation mechanisms and can be applied to other model systems.

Light-mediated Reversible Modulation of the Mitogen-activated Protein Kinase Pathway during Cell Differentiation and Xenopus Embryonic Development

This protocol describes an optogenetic strategy to modulate mitogen-activated protein kinase (MAPK) activity during cell differentiation and Xenopus embryonic development. This method allows for the reversible activation of the MAPK signaling pathway in mammalian cell culture and in multicellular live organisms, like Xenopus embryos, with high spatial and temporal resolution.

Kinase activity is crucial for a plethora of cellular functions, including cell proliferation, differentiation, migration, and apoptosis. During early ...

Developmental Biology

Kinase Inhibitor Screening In Self-assembled Human Protein Microarrays

The screening of kinase inhibitors is crucial for better understanding properties of a drug and for the identification of potentially new targets with clinical implications. Several methodologies have been reported to accomplish such screening. However, each has its own limitations (e.g., the screening of only ATP analogues, restriction to using purified kinase domains, significant costs associated with testing more than a few kinases at a time, and lack of flexibility in screening protein kinases with novel mutations). Here, a new protocol that overcomes some of these limitations and can be used for the unbiased screening of kinase inhibitors is presented. A strength of this method is its ability to compare the activity of kinase inhibitors across multiple proteins, either between different kinases or different variants of the same kinase. Self-assembled protein microarrays generated through the expression of protein kinases by a human-based in vitro transcription and translation system (IVTT) are employed. The proteins displayed on the microarray are active, allowing for measurement of the effects of kinase inhibitors. The following procedure describes the protocol steps in detail, from the microarray generation and screening to the data analysis.

A detailed protocol for the generation of self-assembled human protein microarrays for the screening of kinase inhibitors is presented.

The screening of kinase inhibitors is crucial for better understanding properties of a drug and for the identification of potentially new targets with ...

Identification of EGFR and RAS Inhibitors using Caenorhabditis elegans

The changes in the plasma membrane localization of the epidermal growth factor receptor (EGFR) and its downstream effector RAS have been implicated in several diseases including cancer. The free-living nematode C. elegans possesses an evolutionary and functionally conserved EGFR-RAS-ERK MAP signal cascade which is central for the development of the vulva. Gain of function mutations in RAS homolog LET-60 and EGFR homolog LET-23 induce the generation of visible nonfunctional ectopic pseudovulva along the ventral body wall of these worms. Previously, the multivulval (Muv) phenotype in these worms has been shown to be inhibited by small chemical molecules. Here we describe a protocol for using the worm in a liquid-based assay to identify inhibitors that abolish the activities of EGFR and RAS proteins. Using this assay, we show R-fendiline, an indirect inhibitor of K-RAS, suppresses the Muv phenotype expressed in the let-60(n1046) and let-23(sa62) mutant worms. The assay is simple, inexpensive, is not time consuming to setup, and can be used as an initial platform for the discovery of anticancer therapeutics.

Identification of EGFR and RAS Inhibitors using Caenorhabditis elegans

The genetically tractable nematode Caenorhabditis elegans can be used as a simple and inexpensive model for drug discovery. Described here is a protocol to identify anticancer therapeutics that inhibit the downstream signaling of RAS and EGFR proteins.

The changes in the plasma membrane localization of the epidermal growth factor receptor (EGFR) and its downstream effector RAS have been implicated in ...

CRAF-14-3-3 Interaction Analysis in Live Cells Using a BRET-Based Approach

Bioluminescence Resonance Energy Transfer (BRET)-Based Assay for Measuring Interactions of CRAF with 14-3-3 Proteins in Live Cells

CRAF is a primary effector of RAS GTPases and plays a critical role in the tumorigenesis of several KRAS-driven cancers. In addition, CRAF is a hotspot for germline mutations, which are shown to cause the developmental RASopathy, Noonan syndrome. All RAF kinases contain multiple phosphorylation-dependent binding sites for 14-3-3 regulatory proteins. The differential binding of 14-3-3 to these sites plays essential roles in the formation of active RAF dimers at the plasma membrane under signaling conditions and in maintaining RAF autoinhibition under quiescent conditions. Understanding how these interactions are regulated and how they can be modulated is critical for identifying new therapeutic approaches that target RAF function. Here, I describe a bioluminescence resonance energy transfer (BRET)-based assay for measuring the interactions of CRAF with 14-3-3 proteins in live cells. Specifically, this assay measures the interactions of CRAF fused to a Nano luciferase donor and 14-3-3 fused to a Halo tag acceptor, where the interaction of RAF and 14-3-3 results in donor-to-acceptor energy transfer and the generation of the BRET signal. The protocol further shows that this signal can be disrupted by mutations shown to prevent 14-3-3 binding to each of its high-affinity RAF docking sites. This protocol describes the procedures for seeding, transfecting, and replating the cells, along with detailed instructions for reading BRET emissions, performing data analysis, and confirming protein expression levels. In addition, example assay results, along with optimization and troubleshooting steps, are provided.

Author Spotlight: Integrating BRET-Based Assays and Rare Mutation Analysis to Decipher RAF Kinase Regulation in Live Cells

This protocol describes a BRET-based assay for measuring the interactions of the CRAF kinase with 14-3-3 proteins in live cells. The protocol outlines steps for preparing the cells, reading BRET emissions, and data analysis. An example result with identification of appropriate controls and troubleshooting for assay optimization is also presented.

CRAF is a primary effector of RAS GTPases and plays a critical role in the tumorigenesis of several KRAS-driven cancers. In addition, CRAF is a hotspot ...

An Efficient Protocol to Assess ERK Activity Modulation in Early Zebrafish Noonan Syndrome Models via Live FRET Microscopy and Immunofluorescence

RASopathies are genetic syndromes caused by ERK hyperactivation and resulting in multisystemic diseases that can also lead to cancer predisposition. Despite a broad genetic heterogeneity, germline gain-of-function mutations in key regulators of the RAS-MAPK pathway underlie the majority of the cases, and, thanks to advanced sequencing techniques, potentially pathogenic variants affecting the RAS-MAPK pathway continue to be identified. Functional validation of the pathogenicity of these variants, essential for accurate diagnosis, requires fast and reliable protocols, preferably in vivo. Given the scarcity of effective treatments in early childhood, such protocols, especially if scalable in cost-effective animal models, can be instrumental in offering a preclinical ground for drug repositioning/repurposing. 
Here we describe step-by-step the protocol for rapid generation of transient RASopathy models in zebrafish embryos and direct inspection of live disease-associated ERK activity changes occurring already during gastrulation through real-time multispectral F&#246;rster resonance energy transfer (FRET) imaging. The protocol uses a transgenic ERK reporter recently established and integrated with the hardware of commercial microscopes. We provide an example application for Noonan syndrome (NS) zebrafish models obtained by expression of the Shp2D61G. We describe a straightforward method that enables registration of ERK signal change in the NS fish model before and after pharmacological signal modulation by available low-dose MEK inhibitors. We detail how to generate, retrieve, and assess ratiometric FRET signals from multispectral acquisitions before and after treatment and how to cross-validate the results via classical immunofluorescence on whole embryos at early stages. We then describe how, via examining standard morphometric parameters, to query late changes in embryo shape, indicative of a resulting impairment of gastrulation, in the same embryos whose ERK activity is assessed by live FRET at 6 h post fertilization.

RASopathies are multisystem genetic syndromes caused by RAS-MAPK pathway hyperactivation. Potentially pathogenic variants awaiting validation emerge continuously while poor preclinical evidence limits therapy. Here, we describe our in vivo protocol to test and cross-validate RASopathy-associated ERK activation levels and its pharmacological modulation during embryogenesis by live FRET imaging in Teen-reporter zebrafish.

RASopathies are genetic syndromes caused by ERK hyperactivation and resulting in multisystemic diseases that can also lead to cancer predisposition. ...

In Vivo Modeling of the Morbid Human Genome using Danio rerio

Here, we present methods for the development of assays to query potentially clinically significant nonsynonymous changes using in vivo complementation in zebrafish. Zebrafish (Danio rerio) are a useful animal system due to their experimental tractability; embryos are transparent to enable facile viewing, undergo rapid development ex vivo, and can be genetically manipulated.1 These aspects have allowed for significant advances in the analysis of embryogenesis, molecular processes, and morphogenetic signaling. Taken together, the advantages of this vertebrate model make zebrafish highly amenable to modeling the developmental defects in pediatric disease, and in some cases, adult-onset disorders. Because the zebrafish genome is highly conserved with that of humans (~70% orthologous), it is possible to recapitulate human disease states in zebrafish. This is accomplished either through the injection of mutant human mRNA to induce dominant negative or gain of function alleles, or utilization of morpholino (MO) antisense oligonucleotides to suppress genes to mimic loss of function variants. Through complementation of MO-induced phenotypes with capped human mRNA, our approach enables the interpretation of the deleterious effect of mutations on human protein sequence based on the ability of mutant mRNA to rescue a measurable, physiologically relevant phenotype. Modeling of the human disease alleles occurs through microinjection of zebrafish embryos with MO and/or human mRNA at the 1-4 cell stage, and phenotyping up to seven days post fertilization (dpf). This general strategy can be extended to a wide range of disease phenotypes, as demonstrated in the following protocol. We present our established models for morphogenetic signaling, craniofacial, cardiac, vascular integrity, renal function, and skeletal muscle disorder phenotypes, as well as others.

In Vivo Modeling of the Morbid Human Genome using Danio rerio

Here, we present a systematic approach for developing physiologically relevant, sensitive and specific in vivo assays for interpreting variation in human pathology. Transient genetic manipulation via microinjection of WT and mutant human mRNA and morpholino (MO) antisense oligonucleotides harness the tractability of the developing zebrafish embryo to rapidly assay pathogenic mutations, especially, but not exclusively, in the context of human developmental disorders.

Here,  we present methods for the development of assays to query potentially clinically  significant nonsynonymous changes using in  vivo complementation ...

Biology

A Method for Screening and Validation of Resistant Mutations Against Kinase Inhibitors

The discovery of BCR/ABL as a driver oncogene in chronic myeloid leukemia (CML) resulted in the development of Imatinib, which, in fact, demonstrated the potential of targeting the kinase in cancers by effectively treating the CML patients. This observation revolutionized drug development to target the oncogenic kinases implicated in various other malignancies, such as, EGFR, B-RAF, KIT and PDGFRs. However, one major drawback of anti-kinase therapies is the emergence of drug resistance mutations rendering the target to have reduced or lost affinity for the drug. Understanding the mechanisms employed by resistant variants not only helps in developing the next generation inhibitors but also gives impetus to clinical management using personalized medicine. We reported a retroviral vector based screening strategy to identify the spectrum of resistance conferring mutations in BCR/ABL, which has helped in developing the next generation BCR/ABL inhibitors. Using Ruxolitinib and JAK2 as a drug target pair, here we describe in vitro screening methods that utilizes the mouse BAF3 cells expressing the random mutation library of JAK2 kinase.

Emergence of genetic resistance against kinase inhibitor therapy poses significant challenge for effective cancer therapy. Identification and characterization of resistant mutations against a newly developed drug helps in better clinical management and next generation drug design. Here, we describe our protocol for in vitro screening and validation of resistant mutations.

The discovery of BCR/ABL as a driver oncogene in chronic myeloid leukemia (CML) resulted in the development of Imatinib, which, in fact, demonstrated the ...

Employing Digital Droplet PCR to Detect BRAF V600E Mutations in Formalin-fixed Paraffin-embedded Reference Standard Cell Lines

ddPCR is a highly sensitive PCR method that utilizes a water-oil emulsion system. Using a droplet generator, an extracted nucleic acid sample is partitioned into ~20,000 nano-sized, water-in-oil droplets, and PCR amplification occurs in individual droplets. The ddPCR approach is in identifying sequence mutations, copy number alterations, and select structural rearrangements involving targeted genes. Here, we demonstrate the use of ddPCR as a powerful technique for precisely quantitating rare BRAF V600E mutations in FFPE reference standard cell lines, which is helpful in identifying individuals with cancer. In conclusion, ddPCR technique offers the potential to precisely profile the specific rare mutations in different genes in various types of FFPE samples.

The goal of this video is to demonstrate how to perform&#160;automated DNA extraction from formalin-fixed paraffin-embedded (FFPE) reference standard cell lines and digital droplet PCR&#160;(ddPCR) analysis to detect rare mutations in a clinical setting. Detecting mutations in FFPE samples demonstrates the clinical utility of ddPCR in FFPE samples.

Characterize Disease-related Mutants of RAF Family Kinases by Using a Set of Practical and Feasible Methods

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Assaying Protein Kinase Activity with Radiolabeled ATP

Generation of High-Throughput Three-Dimensional Tumor Spheroids for Drug Screening

Light-mediated Reversible Modulation of the Mitogen-activated Protein Kinase Pathway during Cell Differentiation and Xenopus Embryonic Development

Kinase Inhibitor Screening In Self-assembled Human Protein Microarrays

Identification of EGFR and RAS Inhibitors using Caenorhabditis elegans

Bioluminescence Resonance Energy Transfer (BRET)-Based Assay for Measuring Interactions of CRAF with 14-3-3 Proteins in Live Cells

An Efficient Protocol to Assess ERK Activity Modulation in Early Zebrafish Noonan Syndrome Models via Live FRET Microscopy and Immunofluorescence

In Vivo Modeling of the Morbid Human Genome using Danio rerio

A Method for Screening and Validation of Resistant Mutations Against Kinase Inhibitors

Employing Digital Droplet PCR to Detect BRAF V600E Mutations in Formalin-fixed Paraffin-embedded Reference Standard Cell Lines