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Characterization at the Molecular Level using Robust Biochemical Approaches of a New Kinase Protein Béatrice Vallée, Michel Doudeau, Fabienne Godin, Hélène Bénédetti To cite this version: Béatrice Vallée, Michel Doudeau, Fabienne Godin, Hélène Bénédetti. Characterization at the Molec- ular Level using Robust Biochemical Approaches of a New Kinase Protein. Journal of visualized experiments : JoVE, JoVE, 2019, pp.e59820. 10.3791/59820. hal-02282630 HAL Id: hal-02282630 https://hal.archives-ouvertes.fr/hal-02282630 Submitted on 17 Jun 2020 HAL is a multi-disciplinary open access L’archive ouverte pluridisciplinaire HAL, est archive for the deposit and dissemination of sci- destinée au dépôt et à la diffusion de documents entific research documents, whether they are pub- scientifiques de niveau recherche, publiés ou non, lished or not. The documents may come from émanant des établissements d’enseignement et de teaching and research institutions in France or recherche français ou étrangers, des laboratoires abroad, or from public or private research centers. publics ou privés. Characterization at the Molecular Level using Robust Biochemical Approaches of a New Kinase Protein AUTHORS 1 1 1 1 Béatrice Vallée M Michel Doudeau F Fabienne Godin Hélène Bénédetti AFFILIATIONS 1 Centre de Biophysique Moléculaire, CNRS, UPR 4301, University of Orléans and INSERM CORRESPONDING AUTHOR Béatrice Vallée [email protected] Abstract actosidase-based-screening-assay-for-identification Extensive whole genome sequencing has identified many engineered to its sequence or via an antibody specifically Open Reading Frames (ORFs) providing many potential targeting it. These protein complexes may then be separated proteins. These proteins may have important roles for the by SDS-PAGE (Sodium Dodecyl Sulfate PolyAcrylamide Gel) cell and may unravel new cellular processes. Among and identified using mass spectrometry. Immunoprecipitated proteins, kinases are major actors as they belong to cell LIMK2-1 was also used to test its kinase activity in vitro by 32 signaling pathways and have the ability to switch on or off γ[ P] ATP labeling. This well-established assay may use many many processes crucial to the fate of the cell, such as cell different substrates, and mutated versions of the bait may be growth, division, differentiation, motility, and death. used to assess the role of specific residues. The effects of phar- macological agents may also be evaluated since this technique In this study, we focused on a new potential kinase protein, is both highly sensitive and quantitative. Nonetheless, radioac- LIMK2-1. We demonstrated its existence by Western Blot tivity handling requires particular caution. Kinase activity may using a specific antibody. We evaluated its interaction with also be assessed with specific antibodies targeting the phospho an upstream regulating protein using coimmunoprecipita- group of the modified amino acid. These kinds of antibodies tion experiments. Coimmunoprecipitation is a very powerful are not commercially available for all the phospho modified technique able to detect the interaction between two target residues. proteins. It may also be used to detect new partners of a bait protein. The bait protein may be purified either via a tag 1 Introduction determine the thermodynamic constants of the binding, for For many decades, numerous signaling pathways have been which physical techniques such as Surface Plasmon Reso- elucidated and their involvement in crucial cellular processes nance, Isothermal Titration Calorimetry or Microscale 13,14 such as cell division, differentiation, motility, programmed cell Thermophoresis are required . death, immunity and neurobiology, has been shown. Kinases Kinase activity may be assessed using multiple techniques. play a significant role in these signaling pathways as they Herein, we focused on phospho-specific antibodies and in vitro often finely regulate their activation or inactivation and are γ[32P] ATP (Adenosine TriPhosphate) labeling. Phospho-spe- part of transient versatile complexes that respond to external cific antibodies target the phosphate modification of a particu- stimuli1,2,3. Mutation and dysregulation of kinases often lead lar residue within a protein. They may be used in Western Blot to diseases in humans, and they have therefore become one of or ELISA (Enzyme-Linked ImmunoSorbent Assay) after cell the most important drug targets over the past forty years4. lysis, for immunohistochemistry, and also on intact cells using In this context, it is important to be able to detect kinase inter- flow cytometry or immunofluorescence. Their drawbacks may action with their upstream regulators or downstream include their lack of specificity, which can be evaluated using substrates and to identify new partners. Affinity purification a mutated version of the target protein, and their not being 32 and immunoprecipitation are very powerful techniques for the commercially available for all proteins. In vitro γ[ P] ATP isolation of protein complexes5. The bait protein or kinase may labeling is a very robust, well-established and highly sensitive 15 be tagged with a specific peptide sequence allowing the use of method . Immunoprecipitated or recombinant proteins may commercial beads covalently coupled with antibodies target- be used, and different substrates may be tested. The effects of ing the peptide. This material permits a high reproducibility in drugs may also be assessed as this method is quantitative. Its experiments6,7,8. Endogenous proteins may also be immuno- major drawback is that the radioactivity associated with the precipitated using antibodies targeting directly the bait approach requires handling with caution. Alternative methods protein. The antibodies may be cross-linked to Protein A or are also possible based on the measurement of fluorescent or Protein G agarose beads or simply incubated with these beads luminescent peptide substrates and taking advantage of prior to adding lysate. Lysis buffers must be optimized to allow altered fluorescent/luminescent properties upon phosphoryl- protein solubilization without losing interaction and to avoid ation. Such methods also allow high throughput, which is protein degradation. A major drawback of this approach is required, for example, in the screening of molecules that may that the interaction is detected upon cell lysis; therefore, be potential inhibitors of the target kinase. Indeed, kinases transient or weak interactions, together with those requiring represent one of the largest classes of drug targets pursued by 16 subcellular context may be missed. Other techniques may be pharmaceutical companies . used to work directly in the cell such as Proximity Ligation In this study, we focused on the LIMK2-1 protein (LIMK2-1 Assay (PLA)9, in vivo cross-linking-assisted affinity purifica- stands for Lin11, Isle1, Mec3 Kinase isoform 2-1). The LIMK2 tion (XAP)10, Bioluminescence Resonance Energy Transfer kinase protein was first described in 199517. Three isoforms of (BRET) or Förster Resonance Energy Transfer (FRET)11,12. LIMK2 are produced by alternative splicing: LIMK2a, LIMK2b Furthermore, immunoprecipitation is not appropriate to and LIMK2-1. At present, LIMK2-1 has only been described at 2 the mRNA level in a single study18. Herein, we characterize 4. Add 8 mL of DMEM supplemented with 10% fetal this potential new kinase protein at the molecular level using calf serum. Keep the prepared plates within the robust biochemical approaches. Firstly, we demonstrate that biosafety cabinet. LIMK2-1 is indeed synthesized. Similar to its two counter- 3. Take HEK-293 plates from step 1.1 within the biosafety parts, LIMK2a and LIMK2b, it interacts with the upstream cabinet. Remove the medium from the plates and discard kinase ROCK (Rho-associated protein kinase). We show it in a dedicated bleach trash. Add 2 mL of supplemented LIMK2-1 has a kinase activity on Myelin Basic Protein (MBP), DMEM, and flush the cells to detach them using the flow but not on cofilin, the canonical substrate of LIM kinases. of a 1 mL micropipette, taking care to avoid making foam. Protocol 4. Collect the cells in a 15 mL tube and add 4 mL of supple- 1. Cell preparation for transfection mented DMEM. Homogenize with a 10 mL pipette, by CAUTION: All the steps of the cell culture must be pipetting up and down 3 times. performed in a dedicated laboratory, and cells are manipu- 5. Take 2 mL of this cell solution and add them to colla- lated within a Class 2 microbiological cabinet. gen-treated plates containing supplemented DMEM from 1. Seed HEK-293 (Human Embryonic Kidney) cells in Ø 10 step 1.2.4. cm plates in 10 mL of DMEM (Dulbecco's Modified 6. Grow for 24 hours in the incubator at 5% CO and 37 °C. Eagles Medium) supplemented with 10% fetal calf 2 Cells should be 50-80% confluent at the time of transfec- serum. Culture for 3 to 5 days under 5% CO , at 37 °C, 2 tion. until the cells reach ~90% confluence. 2. Treat Ø 10 cm plates with Collagen R to increase cell 2. Transient transfections adhesion to the plastic plates. 1. Remove the medium from the plates, discard it in a 1. Add 5 mL of a solution of Collagen R, 200-fold dilut- dedicated bleach trash, and add 10 mL of fresh supple- ed in Phosphate Saline Buffer (PBS) in a Ø 10 cm mented DMEM. Put back the plates into the incubator at plate. Overlay the liquid over the whole surface of 37 °C while preparing the transfection mixture. the plate. 2. In a 15 mL tube, add 450 µL of a 10 mM Tris-HCl pH 2. Incubate at room temperature for at least 1 h within 7.5/1 mM EDTA (Tris stands for Tris the biosafety cabinet. (hydroxymethyl)aminomethane, and EDTA for ethylenedinitrilotetraacetic acid) solution and 50 µL of a 3. Remove collagen solution, and discard it. Add 5 mL 2.5 M CaCl solution. Mix by inversion. 2 of PBS, spread it all over the surface of the plate, remove it and discard it. Repeat this wash once. 3. Add 10 µg of plasmidic DNA (Deoxyribonucleic acid) prepared from a midi preparation on a liquid culture of 3 bacteria transformed with the dedicated plasmid.

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