Name: characterize disease-related mutants of raf family kinases by using a set of practical and feasible methods
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Description: Watch this Scientific Journal Video about Characterize Disease-related Mutants of RAF Family Kinases by Using a Set of Practical and Feasible Methods at JoVE.com

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 protocol...

<ol>
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P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pseudokinases+from+a+structural+perspective.">Pseudokinases from a structural perspective.</a> Biochemistry Society Transactions. 41 (4), 981-986 (2013).</li><li>Rajakulendran, T., Sahmi, M., Lefran&#231;ois, M., Sicheri, F., Therrien, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+dimerization-dependent+mechanism+drives+RAF+catalytic+activation.">A dimerization-dependent mechanism drives RAF catalytic activation.</a> Nature. 461 (7263), 542-545 (2009).</li><li>Heidorn, S. 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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 border="0" cellpadding="0" cellspacing="0" style="width:660px;" width="660">
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	</colgroup>
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:195px;">anti-phosphoERK1/2</td>
			<td style="width:197px;">Cell Signaling Technologies</td>
			<td style="width:104px;">4370</td>
			<td style="width:164px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:195px;">anti-phosphoMEK1/2</td>
			<td style="width:197px;">Cell Signaling Technologies</td>
			<td style="width:104px;">9154</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:195px;">anti-ERK1/2</td>
			<td style="width:197px;">AB clonal</td>
			<td style="width:104px;">A0229</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:195px;">anti-MEK1/2</td>
			<td style="width:197px;">Cell Signaling Technologies</td>
			<td style="width:104px;">9124</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:195px;">anti-FLAG(mouse)</td>
			<td style="width:197px;">Sigma-Aldrich</td>
			<td style="width:104px;">F3165</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:195px;">anti-HA</td>
			<td style="width:197px;">Novus Biologicals</td>
			<td style="width:104px;">MAB6875</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:195px;">anti-FLAG(Rabbit)</td>
			<td style="width:197px;">Cell Signaling Technologies</td>
			<td style="width:104px;">14793</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:195px;">anti-&#946;-actin</td>
			<td style="width:197px;">Sigma-Aldrich</td>
			<td style="width:104px;">A2228</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:195px;">anti-FLAG beads(M2)</td>
			<td style="width:197px;">Sigma-Aldrich</td>
			<td style="width:104px;">A4596</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:195px;">HRP-conjugated anti-mouse IgG</td>
			<td style="width:197px;">Jackson Laboratories</td>
			<td style="width:104px;">115-035-003</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:195px;">HRP-conjugated anti-Rabbit IgG</td>
			<td style="width:197px;">Jackson Laboratories</td>
			<td style="width:104px;">111-035-144</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:195px;">pcDNA3.1(+)</td>
			<td style="width:197px;">In vitrogen</td>
			<td style="width:104px;">V79020</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:195px;">Gibson Assembly Cloning&#160; Kit</td>
			<td style="width:197px;">New England Biolabs</td>
			<td style="width:104px;">E5510</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:195px;">T4 DNA ligase</td>
			<td style="width:197px;">New England Biolabs</td>
			<td style="width:104px;">M0202</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:195px;">lipofectamine 2000</td>
			<td style="width:197px;">Invitrogen</td>
			<td align="right" style="width:104px;">11668019</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:195px;">Fugene 6</td>
			<td style="width:197px;">Roche</td>
			<td style="width:104px;">11 814 443 001</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:195px;">DMEM w/o phenol red</td>
			<td style="width:197px;">Invitrogen</td>
			<td style="width:104px;">21063-029</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">D-luciferin&#160;</td>
			<td style="width:197px;">GoldBio</td>
			<td style="width:104px;">LUCK-100</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:195px;">6xhis-tagged MEK1 (K97A)</td>
			<td style="width:197px;">&#160;prepared in our previous studies</td>
			<td style="width:104px;">N.A.</td>
			<td>Reference 15.</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:195px;">GloMax-Multi Detection System.</td>
			<td style="width:197px;">Promega</td>
			<td style="width:104px;">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 ...

This method facilitates the effective characterization of disease related RAF kinase methods. This technique is highly sensitive and reproducible, cost effective, user friendly, and does not require any advanced equipment. To measure the catalytic activity of RAF mutants, introduce flag tech mutations into the RAF coding sequence by PCR, before inserting whole sequences into the appropriate vector according to standard protocols.Insert the glutathione as transferase coding sequence upstream of the RAF mutant coding sequences, to generate vectors encoding glutathione as transferase fused RAF mutants, in the same manner. Transvect 80%to 90%confluent 293T cell cultures with the vectors encoding the flag tagged RAF kinases or their mutants, according to standards protocols. After 24 hours of culture, replace the medium in each culture.After 48 hours of culture, add 400 microliters of lysis buffer supplemented with protease and phosphatase inhibitors, to each well on ice. After five to 10 minutes transfer the cell lysates to a 1.5 milliliter tube for centrifugation to sediment the cell debris. Transfer 300 microliters of each sample into individual 1.5 milliliter micro centrifuge tubes.Set aside 40 microliters per sample for downstream wholesale lysate fossil extra cellular signal regulated kinase one and two expression and activity amino blot analysis. Add 20 microliters of anti-FLAG affinity beads to each tube, for a one hour incubation with rotation in a four degrees Celsius cold room. At the end of the incubation quickly and gently wash the anti flag beads one time with lysis buffer in the cold room and one time with kinase reaction buffer before adding 20 microliters of kinase reaction mixture.Graph Mutants with a low Dima affinity may lose their catalytic activity in vitro because of Dima dissociation. Therefore, the quick and gentle wash is critical. After 10 minutes at room temperature with flipping every other minute, stop the kinase reactions with five microliters of five XSDS sample buffer, per sample and boil the samples at 90 degrees Celsius for five minutes.At the end of the incubation, run the samples on a nine to 12%polyacrylamide electrophoresis gel with 0.1%SDS, then transfer the proteins to a nitrocellulose membrane to allow detection of the levels of phospho mitogen-activated protein kinase and RAF mutants in each sample, by immuno blotting. To evaluate the Allosteric activity of kinase dead RAF mutants, construct vectors encoding the RAF receiver or the kinase dead RAF activators. Transvect 293 T cells with two vectors encoding both the RAF receiver and the kinase dead RAF activator, or a single vector encoding one of the proteins as demonstrated.Harvest the whole cell lysates after 48 hours of culture as demonstrated, and quickly mix the clean whole cell lysate with five XSDS sample buffer at room temperature, Before boiling the lysates for five minutes at 90 degrees Celsius. Then run the boiled whole cell lysate samples on a nine to 12%polyacrylamide electrophoresis gel that's 0.1%SDS and transfer the proteins to a nitrocellulose membrane for detecting the levels of phospho extra cellular signal regulated kinase one and two and control proteins by immune blotting as demonstrated. To measure the relative dimer affinity and or stability of the RAF mutants construct vectors encoding flag tech draft mutants fused to the terminus of Firefly luciferase or the C Terminus a firefly luciferase.Transvect 293 T cells with a pair of vectors encoding different and Terminus of firefly luciferase RAF mutants and C Terminus a firefly luciferase RAF mutants as demonstrated. 24 hours after the transvection replate two times 10 to the fifth 293 T cell transactions per well into crystal black image plates in color free medium. 48 hours after the transvestion add the luciferin to 293 T cell transvectents and incubate for 30 minutes.Before measuring the luciferase signals on a multi detection system. At the end of the measurement aspirate the supernatents, and blice the transacted 293 T cells with lysas buffer as demonstrated. Run the whole cell lysate samples on a nine to 12%polyacrylamide electrophoresis gel with 0.1%SDS.Detect the expression levels of the end terminus of Firefly luciferase RAF mutants, or the C terminus of Firefly luciferase RAF mutants by anti flag amino blot as demonstrated. Then normalize the luciferase signals of the 293 T cell transvectants according to the expression levels of the end terminus of Firefly luciferase and C terminus of Firefly luciferase RAF mutants. The in vitro kinase asa can be used to effectively probe the catalytic activity of some constitutively active R-spine mutants, but not that of kinase dead mutants with a fused C-spine.The catalytic activity of constitutively active RAF mutants with a weak dimer affinity or stability however, might not be probed by using this ASA as their dimers are broken during the purification process. To avoid the loss of catalytic activity of RAF mutants with weak dimer affinity or stability, these mutants can be fused with glutothionas transferase to rescue the catalytic activity of the mutants. The RAF co-activation ASA can be used to evaluate the allosteric activity of kinase dead RAF Mutants.As illustrated kinase dead mutants with an acidic end terminal motif and C spine fusion, function as allosteric activators to trigger the catalytic activity of mutants with a non phosphor relatable acidic end terminal motif, when co expressed in 293T cells. Further a complimentary split luciferase SA can be used for measuring the relative dimer affinity or stability of RAF family kinases and their mutants. Using a lysas buffer with a low concentration of detergent, and a quick gentle wash procedure during the sample purification is essential for a successful in vitro kinase SA.This technique will help researchers determine precisely how disease relay the RAF mutants, activate the downstream pathway, facilitating the selection of appropriate therapeutic strategies for disease treatment.

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