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W tym Artykule

  • Podsumowanie
  • Streszczenie
  • Wprowadzenie
  • Protokół
  • Wyniki
  • Dyskusje
  • Ujawnienia
  • Podziękowania
  • Materiały
  • Odniesienia
  • Przedruki i uprawnienia

Podsumowanie

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.

Streszczenie

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.

Wprowadzenie

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>CRAF>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' 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.

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Protokół

1. In Vitro Kinase Assay for Measuring the Catalytic Activity of RAF Mutants

  1. Construct vectors encoding RAF mutants (Figure 1A) with FLAG(DYKDDDDK) tag at C-terminus by using Gibson Assembly or traditional molecular cloning methods.
    1. 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’s protocols. Use the following conditions for PCR reactions: (1) 95 °C, 2 min; (2) 95 °C, 30 s; (3) 59 °C, 30 s; (4) 68 °C, 3 min; (5) 20 cycles of (2 to 4); (6) 4 °C hold.
      NOTE: The PCR primers for cloning: 5- AAATTAATACGACTCACTATAGGGAGACCC-3 and 5-CAGCGGGTTTAAACGGGCCCTCTA-3.
    2. 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.
    3. Validate all vectors by DNA sequencings before transfection.
  2. 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’s protocol of transfection reagents (Table of Materials).
  3. Replace the culture medium 24 h after transfection.
  4. Aspirate the culture medium 48 h after transfection and add 400 µL/well of lysis buffer (25 mM Tris·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.
  5. Transfer the cell lysates to a 1.5 mL tube, and spin down by 12,000 x g for 10 min at 4 °C to deplete cell debris.
  6. Transfer 300 µL per sample of clean whole cell lysates to 1.5 mL tubes, add 20 µL per sample of anti-FLAG affinity beads, and rotate in a cold room (4 °C) for 1 h. Also take 40 µL 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.
  7. 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 µL of kinase reaction mixture (2 μg of MEK1(K97A) and 100 µM ATP in 20 µL 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 °C in a cold room.
  8. Incubate the kinase reactions at room temperature (25 °C) for 10 min, and flip the tubes containing kinase reactions with fingers every other minute during incubation.
  9. Add 5 µL of 5x SDS sample buffer (375 mM Tris·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 °C for 5 min.
  10. 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 γ32P-ATP incorporation. Briefly, 10 µM γ32P-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.

2. RAF Co-activation Assay for Evaluating the Allosteric Activity of Kinase-dead RAF Mutants

  1. 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.
  2. 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.
  3. 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.
  4. Mix the clean whole cell lysate with 5x SDS sample buffer quickly at room temperature (25 °C) and then boil at 90 °C for 5 min.
  5. 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.

3. Complimentary Split Luciferase Assay for Measuring the Relative Dimer Affinity/Stability of RAF Mutants

  1. 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.
  2. Transfect 293T cells with a pair of vectors encoding different Nluc-RAF mutants and Cluc-RAF mutants as described in step 1.2.
  3. 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).
  4. 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).
  5. 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.
  6. 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.
  7. 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.

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Wyniki

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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Dyskusje

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,

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Ujawnienia

The authors declare that they have no competing financial interests.

Podziękowania

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).

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Materiały

NameCompanyCatalog NumberComments
anti-phosphoERK1/2Cell Signaling Technologies4370
anti-phosphoMEK1/2Cell Signaling Technologies9154
anti-ERK1/2AB clonalA0229
anti-MEK1/2Cell Signaling Technologies9124
anti-FLAG(mouse)Sigma-AldrichF3165
anti-HANovus BiologicalsMAB6875
anti-FLAG(Rabbit)Cell Signaling Technologies14793
anti-β-actinSigma-AldrichA2228
anti-FLAG beads(M2)Sigma-AldrichA4596
HRP-conjugated anti-mouse IgGJackson Laboratories115-035-003
HRP-conjugated anti-Rabbit IgGJackson Laboratories111-035-144
pcDNA3.1(+)In vitrogenV79020
Gibson Assembly Cloning  KitNew England BiolabsE5510
T4 DNA ligaseNew England BiolabsM0202
Lipofectamine 2000Invitrogen11668019
Fugene 6Roche11 814 443 001
DMEM w/o phenol redInvitrogen21063-029
D-luciferin GoldBioLUCK-100
6xhis-tagged MEK1 (K97A) prepared in our previous studiesN.A.Reference 15.
GloMax-Multi Detection System.PromegaE7041

Odniesienia

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

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RAF Family KinasesDisease related MutantsCharacterization MethodsCatalytic ActivityFlag Tech Mutations293T Cell CulturesCell LysatesLysis BufferAnti FLAG Affinity BeadsKinase Reaction MixtureElectrophoresis GelPhospho MAPK DetectionImmunoblot Analysis

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