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>

<ol><li>Chong, H., Vikis, H. G., Guan, K. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Mechanisms+of+regulating+the+Raf+kinase+family%5BTitle%5D)+AND+%22Cellular+Signalling%22%5BJournal%5D)">Mechanisms of regulating the Raf kinase family.</a> Cellular Signalling. 15 (5), 463-469 (2003).</li>
<li>Wellbrock, C., Karasarides, M., Marais, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+RAF+proteins+take+center+stage%5BTitle%5D)+AND+%22Nature+Reviews+Molecular+Cell+Biology%22%5BJournal%5D)">The RAF proteins take center stage.</a> Nature Reviews Molecular Cell Biology. 5 (11), 875-885 (2004).</li>
<li>Baccarini, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Second+nature%3A+biological+functions+of+the+Raf-1+%22kinase%22%5BTitle%5D)+AND+%22FEBS+Letter%22%5BJournal%5D)">Second nature: biological functions of the Raf-1 "kinase".</a> FEBS Letter. 579 (15), 3271-3277 (2005).</li>
<li>Lavioe, H., Therrien, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Regulation+of+RAF+protein+kinases+in+ERK+signaling%5BTitle%5D)+AND+%22Nature+Reviews+Molecular+Cell+Biology%22%5BJournal%5D)">Regulation of RAF protein kinases in ERK signaling.</a> Nature Reviews Molecular Cell Biology. 16 (5), 281-298 (2015).</li>
<li>Schreck, R., Rapp, U. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Raf+kinases%3A+oncogenesis+and+drug+discovery%5BTitle%5D)+AND+%22International+Journal+of+Cancer%22%5BJournal%5D)">Raf kinases: oncogenesis and drug discovery.</a> International Journal of Cancer. 119 (10), 2261-2271 (2006).</li>
<li>Roberts, P. J., Der, C. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Targeting+the+Raf-MEK-ERK+mitogen-activated+protein+kinase+cascade+for+the+treatment+of+cancer%5BTitle%5D)+AND+%22Oncogene%22%5BJournal%5D)">Targeting the Raf-MEK-ERK mitogen-activated protein kinase cascade for the treatment of cancer.</a> Oncogene. 26 (22), 3291-3310 (2007).</li>
<li>McCubrey, J. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Roles+of+the+Raf%2FMEK%2FERK+pathway+in+cell+growth%2C+malignant+transformation+and+drug+resistance%5BTitle%5D)+AND+%22Biochemistry+Biophysics+Acta%22%5BJournal%5D)">Roles of the Raf/MEK/ERK pathway in cell growth, malignant transformation and drug resistance.</a> Biochemistry Biophysics Acta. 1773 (8), 1263-1284 (2007).</li>
<li>Schubbert, S., Shannon, K., Bollag, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Hyperactive+Ras+in+developmental+disorders+and+cancer%5BTitle%5D)+AND+%22Nature+Reviews+Cancer%22%5BJournal%5D)">Hyperactive Ras in developmental disorders and cancer.</a> Nature Reviews Cancer. 7 (4), 295-308 (2007).</li>
<li>Davies, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Mutations+of+the+BRAF+gene+in+human+cancer%5BTitle%5D)+AND+%22Nature%22%5BJournal%5D)">Mutations of the BRAF gene in human cancer.</a> Nature. 417 (6892), 949-954 (2002).</li>
<li>Garnett, M. J., Marais, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Guilty+as+charged%3A+B-RAF+is+a+human+oncogene%5BTitle%5D)+AND+%22Cancer+Cell%22%5BJournal%5D)">Guilty as charged: B-RAF is a human oncogene.</a> Cancer Cell. 6 (4), 313-319 (2004).</li>
<li>Pandit, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Gain-of-function+RAF1+mutations+cause+Noonan+and+LEOPARD+syndromes+with+hypertrophic+cardiomyopathy%5BTitle%5D)+AND+%22Nature+Genetics%22%5BJournal%5D)">Gain-of-function RAF1 mutations cause Noonan and LEOPARD syndromes with hypertrophic cardiomyopathy.</a> Nature Genetics. 39 (8), 1007-1012 (2007).</li>
<li>Mason, C. S., Springer, C. J., Cooper, R. G., Superti-Furga, G., Marshall, C. J., Marais, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Serine+and+tyrosine+phosphorylations+cooperate+in+Raf-1%2C+but+not+B-Raf+activation%5BTitle%5D)+AND+%22EMBO+Journal%22%5BJournal%5D)">Serine and tyrosine phosphorylations cooperate in Raf-1, but not B-Raf activation.</a> EMBO Journal. 18 (8), 2137-2148 (1999).</li>
<li>Hu, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Allosteric+activation+of+functionally+asymmetric+RAF+kinase+dimers%5BTitle%5D)+AND+%22Cell%22%5BJournal%5D)">Allosteric activation of functionally asymmetric RAF kinase dimers.</a> Cell. 154 (5), 1036-1046 (2013).</li>
<li>Desideri, E., Cavallo, A. L., Baccarini, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Alike+but+different%3A+RAF+paralogs+and+their+signaling+outputs%5BTitle%5D)+AND+%22Cell%22%5BJournal%5D)">Alike but different: RAF paralogs and their signaling outputs.</a> Cell. 161 (5), 967-970 (2015).</li>
<li>Yuan, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+dimer-dependent+catalytic+activity+of+RAF+family+kinases+is+revealed+through+characterizing+their+oncogenic+mutants%5BTitle%5D)+AND+%22Oncogene%22%5BJournal%5D)">The dimer-dependent catalytic activity of RAF family kinases is revealed through characterizing their oncogenic mutants.</a> Oncogene. 37 (43), 5719-5734 (2018).</li>
<li>Wan, P. T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Mechanism+of+activation+of+the+RAF-ERK+signaling+pathway+by+oncogenic+mutations+of+B-RAF%5BTitle%5D)+AND+%22Cell%22%5BJournal%5D)">Mechanism of activation of the RAF-ERK signaling pathway by oncogenic mutations of B-RAF.</a> Cell. 116 (6), 855-867 (2004).</li>
<li>Shaw, A. S., Kornev, A. P., Hu, J., Ahuja, L. G., Taylor, S. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Kinases+and+pseudokinases%3A+lessons+from+RAF%5BTitle%5D)+AND+%22Molecular+and+Cellular+Biology%22%5BJournal%5D)">Kinases and pseudokinases: lessons from RAF.</a> Molecular and Cellular Biology. 34 (9), 1538-1546 (2014).</li>
<li>Taylor, S. S., Shaw, A. S., Hu, J., Meharena, H. S., Kornev, A. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Pseudokinases+from+a+structural+perspective%5BTitle%5D)+AND+%22Biochemistry+Society+Transactions%22%5BJournal%5D)">Pseudokinases from a structural perspective.</a> Biochemistry Society Transactions. 41 (4), 981-986 (2013).</li>
<li>Rajakulendran, T., Sahmi, M., Lefrançois, M., Sicheri, F., Therrien, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+dimerization-dependent+mechanism+drives+RAF+catalytic+activation%5BTitle%5D)+AND+%22Nature%22%5BJournal%5D)">A dimerization-dependent mechanism drives RAF catalytic activation.</a> Nature. 461 (7263), 542-545 (2009).</li>
<li>Heidorn, S. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Kinase-dead+BRAF+and+oncogenic+RAS+cooperate+to+drive+tumor+progression+through+CRAF%5BTitle%5D)+AND+%22Cell%22%5BJournal%5D)">Kinase-dead BRAF and oncogenic RAS cooperate to drive tumor progression through CRAF.</a> Cell. 140 (2), 209-221 (2010).</li>
<li>Yuan, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Activating+mutations+in+MEK1+enhance+homodimerization+and+promote+tumorigenesis%5BTitle%5D)+AND+%22Science+Signaling%22%5BJournal%5D)">Activating mutations in MEK1 enhance homodimerization and promote tumorigenesis.</a> Science Signaling. 11 (554), 6795(2018).</li>
<li>Yuan, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+AMPK+inhibitor+overcomes+the+paradoxical+effect+of+RAF+inhibitors+through+blocking+phospho-Ser-621+in+the+C+terminus+of+CRAF%5BTitle%5D)+AND+%22Journal+of+Biological+Chemistry%22%5BJournal%5D)">The AMPK inhibitor overcomes the paradoxical effect of RAF inhibitors through blocking phospho-Ser-621 in the C terminus of CRAF.</a> Journal of Biological Chemistry. 293 (37), 14276-14284 (2018).</li>
<li>Hu, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Kinase+regulation+by+hydrophobic+spine+assembly+in+cancer%5BTitle%5D)+AND+%22Molecular+and+Cellular+Biology%22%5BJournal%5D)">Kinase regulation by hydrophobic spine assembly in cancer.</a> Molecular and Cellular Biology. 35 (1), 264-276 (2015).</li>
<li>Poulikakos, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((RAF+inhibitor+resistance+is+mediated+by+dimerization+of+aberrantly+spliced+BRAF%28V600E%29%5BTitle%5D)+AND+%22Nature%22%5BJournal%5D)">RAF inhibitor resistance is mediated by dimerization of aberrantly spliced BRAF(V600E).</a> Nature. 480 (7377), 387-390 (2011).</li>
<li>Hu, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Mutation+that+blocks+ATP+binding+creates+a+pseudokinase+stabilizing+the+scaffolding+function+of+kinase+suppressor+of+Ras%2C+CRAF+and+BRAF%5BTitle%5D)+AND+%22Proceedings+of+the+National+Academy+of+Sciences+of+the+United+States+of+America%22%5BJournal%5D)">Mutation that blocks ATP binding creates a pseudokinase stabilizing the scaffolding function of kinase suppressor of Ras, CRAF and BRAF.</a> Proceedings of the National Academy of Sciences of the United States of America. 108 (15), 6067-6072 (2011).</li>
<li>Taylor, S. S., Kornev, A. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Protein+kinases%3A+evolution+of+dynamic+regulatory+proteins%5BTitle%5D)+AND+%22Trends+Biochemistry+Sciences%22%5BJournal%5D)">Protein kinases: evolution of dynamic regulatory proteins.</a> Trends Biochemistry Sciences. 36 (2), 65-77 (2011).</li>
<li>Farrar, M. A., Alberol-Ila, J., Perlmutter, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Activation+of+the+Raf-1+kinase+cascade+by+coumermycin-induced+dimerization%5BTitle%5D)+AND+%22Nature%22%5BJournal%5D)">Activation of the Raf-1 kinase cascade by coumermycin-induced dimerization.</a> Nature. 383 (6596), 178-181 (1996).</li>
<li>Luo, Z., Tzivion, G., Belshaw, P. J., Vavvas, D., Marshall, M., Avruch, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Oligomerization+activates+c-Raf-1+through+a+Ras-dependent+mechanism%5BTitle%5D)+AND+%22Nature%22%5BJournal%5D)">Oligomerization activates c-Raf-1 through a Ras-dependent mechanism.</a> Nature. 383 (6596), 181-185 (1996).</li>
<li>Weber, C. K., Slupsky, J. R., Kalmes, H. A., Rapp, U. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Active+Ras+induces+heterodimerization+of+cRaf+and+BRaf%5BTitle%5D)+AND+%22Cancer+Research%22%5BJournal%5D)">Active Ras induces heterodimerization of cRaf and BRaf.</a> Cancer Research. 61 (9), 3595-3598 (2001).</li>
<li>Garnett, M. J., Rana, S., Paterson, H., Barford, D., Marais, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Wild-type+and+mutant+B-RAF+activate+C-RAF+through+distinct+mechanisms+involving+heterodimerization%5BTitle%5D)+AND+%22Molecular+Cell%22%5BJournal%5D)">Wild-type and mutant B-RAF activate C-RAF through distinct mechanisms involving heterodimerization.</a> Molecular Cell. 20 (6), 963-969 (2005).</li>
<li>Hatzivassiliou, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((RAF+inhibitors+prime+wild-type+RAF+to+activate+the+MAPK+pathway+and+enhance+growth%5BTitle%5D)+AND+%22Nature%22%5BJournal%5D)">RAF inhibitors prime wild-type RAF to activate the MAPK pathway and enhance growth.</a> Nature. 464 (7287), 431-435 (2010).</li>
<li>Poulikakos, P. I., Zhang, C., Bollag, G., Shokat, K. M., Rosen, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((RAF+inhibitors+transactivate+RAF+dimers+and+ERK+signalling+in+cells+with+wild-type+BRAF%5BTitle%5D)+AND+%22Nature%22%5BJournal%5D)">RAF inhibitors transactivate RAF dimers and ERK signalling in cells with wild-type BRAF.</a> Nature. 464 (7287), 427-430 (2010).</li>
<li>Kolch, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Meaningful+relationships%3A+the+regulation+of+the+Ras%2FRAF%2FMEK%2FERK+pathway+by+protein+interactions%5BTitle%5D)+AND+%22Biochemistry+Journal%22%5BJournal%5D)">Meaningful relationships: the regulation of the Ras/RAF/MEK/ERK pathway by protein interactions.</a> Biochemistry Journal. 351, Pt 2 289-305 (2000).</li>
<li>Cseh, B., Doma, E., Baccarini, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((%22RAF%22+neighborhood%3A+protein-protein+interaction+in+the+Raf%2FMek%2FErk+pathway%5BTitle%5D)+AND+%22FEBS+Letters%22%5BJournal%5D)">"RAF" neighborhood: protein-protein interaction in the Raf/Mek/Erk pathway.</a> FEBS Letters. 588 (15), 2398-2406 (2014).</li>
<li>García-Gómez, R., Bustelo, X. R., Crespo, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Protein-Protein+Interactions%3A+Emerging+Oncotargets+in+the+RAS-ERK+Pathway%5BTitle%5D)+AND+%22Trends+Cancer%22%5BJournal%5D)">Protein-Protein Interactions: Emerging Oncotargets in the RAS-ERK Pathway.</a> Trends Cancer. 4 (9), 616-633 (2018).</li>
<li>Luker, K. E., Smith, M. C., Luker, G. D., Gammon, S. T., Piwnica-Worms, H., Piwnica-Worms, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Kinetics+of+regulated+protein%E2%80%93protein+interactions+revealed+with+firefly+luciferase+complementation+imaging+in+cells+and+living+animals%5BTitle%5D)+AND+%22Proceedings+of+the+National+Academy+of+Sciences+of+the+United+States+of+America%22%5BJournal%5D)">Kinetics of regulated protein–protein interactions revealed with firefly luciferase complementation imaging in cells and living animals.</a> Proceedings of the National Academy of Sciences of the United States of America. 101 (33), 12288-12293 (2004).</li>
<li>Chen, S. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Oncogenic+BRAF+deletions+that+function+as+homodimers+and+are+sensitive+to+inhibition+by+RAF+dimer+inhibitor+LY3009120%5BTitle%5D)+AND+%22Cancer+Discovery%22%5BJournal%5D)">Oncogenic BRAF deletions that function as homodimers and are sensitive to inhibition by RAF dimer inhibitor LY3009120.</a> Cancer Discovery. 6 (3), 300-315 (2016).</li>
<li>Foster, S. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Activation+mechanism+of+oncogenic+deletion+mutations+in+BRAF%2C+EGFR%2C+and+HER2%5BTitle%5D)+AND+%22Cancer+Cell%22%5BJournal%5D)">Activation mechanism of oncogenic deletion mutations in BRAF, EGFR, and HER2.</a> Cancer Cell. 29 (4), 477-493 (2016).</li>
</ol>

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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Research

JoVE Journal

Cancer Research

6.0K 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

In Vivo Functional Study of Disease-associated Rare Human Variants Using Drosophila

Advances in sequencing technology have made whole-genome and whole-exome datasets more accessible for both clinical diagnosis and cutting-edge human genetics research. Although a number of in silico algorithms have been developed to predict the pathogenicity of variants identified in these datasets, functional studies are critical to determining how specific genomic variants affect protein function, especially for missense variants. In the Undiagnosed Diseases Network (UDN) and other rare disease research consortia, model organisms (MO) including Drosophila, C. elegans, zebrafish, and mice are actively used to assess the function of putative human disease-causing variants. This protocol describes a method for the functional assessment of rare human variants used in the Model Organisms Screening Center Drosophila Core of the UDN. The workflow begins with gathering human and MO information from multiple public databases, using the MARRVEL web resource to assess whether the variant is likely to contribute to a patient&#39;s condition as well as design effective experiments based on available knowledge and resources. Next, genetic tools (e.g., T2A-GAL4 and UAS-human cDNA lines) are generated to assess the functions of variants of interest in Drosophila. Upon development of these reagents, two-pronged functional assays based on rescue and overexpression experiments can be performed to assess variant function. In the rescue branch, the endogenous fly genes are &#34;humanized&#34;&#160;by replacing the orthologous Drosophila gene with reference or variant human transgenes. In the overexpression branch, the reference and variant human proteins are exogenously driven in a variety of tissues. In both cases, any scorable phenotype (e.g., lethality, eye morphology, electrophysiology) can be used as a read-out, irrespective of the disease of interest. Differences observed between reference and variant alleles suggest a variant-specific effect, and thus likely pathogenicity. This protocol allows rapid, in vivo assessments of putative human disease-causing variants of genes with known and unknown functions.

In Vivo Functional Study of Disease-associated Rare Human Variants Using Drosophila

The goal of this protocol is to outline the design and performance of in vivo experiments in Drosophila melanogaster to assess the functional consequences of rare gene variants associated with human diseases.

Advances in sequencing technology have made whole-genome and whole-exome datasets more accessible for both clinical diagnosis and cutting-edge human ...

Genetics

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 ...

Temporal Regulation of Phosphatase Activity with the Rapamycin-Regulated System

Development and Application of Rapamycin-regulated Tyrosine Phosphatases

Tyrosine phosphatases are an important family of enzymes that regulate critical physiological functions. They are often dysregulated in human diseases, making them key targets of biological studies. Tools that enable the regulation of phosphatase activity are instrumental in the dissection of their function. Traditional approaches, such as overexpression of constitutively active or dominant negative mutants, or downregulation using siRNA, lack temporal control. Phosphatase inhibitors often have poor specificity, and they only allow researchers to determine what processes are affected by the inhibition of the phosphatase. 
We developed a chemogenetic approach, the Rapamycin-regulated (RapR) system, which allows for allosteric regulation of a phosphatase catalytic domain that enables tight temporal control of phosphatase activation. The RapR system consists of an iFKBP domain inserted into an allosteric site in the phosphatase. The intrinsic structural dynamics of the RapR domain disrupt the catalytic domain, leading to the inactivation of the enzyme. The addition of rapamycin mediates the formation of a complex between iFKBP and a co-expressed FRB protein, which stabilizes iFKBP and restores activity to the phosphatase's catalytic domain. 
This system provides high specificity and tight temporal control of phosphatase activation in living cells. The unique capabilities of this system enable the identification of transient events and interrogation of individual signaling pathways downstream of a phosphatase. This protocol describes guidelines for the development of a RapR-phosphatase, its biochemical characterization, and the analysis of its effects on downstream signaling and regulation of cell morphodynamics. It also provides a detailed description of a protein engineering strategy, in vitro assays analyzing phosphatase activity, and live cell imaging experiments identifying changes in cell morphology.

Author Spotlight: Developing Tools to Tune the Activity of Tyrosine Phosphatases

This protocol describes the design, creation, and application of rapamycin-regulated phosphatases. This method provides high specificity and tight temporal control of phosphatase activation in living cells.

Tyrosine phosphatases are an important family of enzymes that regulate critical physiological functions. They are often dysregulated in human diseases, ...

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

Summary

Explore More Videos

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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

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