Name: characterization at the molecular level using robust biochemical approaches of a new kinase protein
Uploaded: 2019-06-30T14:00:00
Description: Watch this Scientific Journal Video about Characterization at the Molecular Level using Robust Biochemical Approaches of a New Kinase Protein at JoVE.com

This work was supported by La Ligue contre le Cancer, l&#8217;Association Neurofibromatoses et Recklinghausen, and la R&#233;gion Centre Val de Loire. Many thanks to Aur&#233;lie Cosson and D&#233;borah Casas for flow cytometry data, and to Keyron Hickman-Lewis for thorough proofreading of the manuscript.

1. Cell preparation for transfection
CAUTION: All the steps of the cell culture must be performed in a dedicated laboratory, and cells are manipulated within a Class 2 microbiological cabinet.
<ol>
	<li>Seed HEK-293 (Human Embryonic Kidney) cells in &#216; 10 cm plates in 10 mL of DMEM (Dulbecco&#39;s Modified Eagles Medium) supplemented with 10% fetal calf serum. Culture for 3 to 5 days under 5% CO2, at 37 &#176;C, until the cells reach ~90% confluence.</li>
	<li>Treat &#216; 10 ...

<ol>
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Herein, we have used robust biochemical tools to characterize at the molecular level a new protein, LIMK2-1, believed to be a kinase based on its sequence and on its homologs, LIMK2a and LIMK2b20.
Firstly, we demonstrated the existence of LIMK2-1 at the protein level using Western Blot analysis with a specific antibody. Following this, we evaluated its interaction with the upstream kinase ROCK1, which is known to regulate LIMK2a and LIMK2b, the homologs of LIMK2-1. Fina...

For many decades, numerous signaling pathways have been elucidated and their involvement in crucial cellular processes such as cell division, differentiation, motility, programmed cell death, immunity and neurobiology, has been shown. Kinases play a significant role in these signaling pathways as they often finely regulate their activation or inactivation and are part of transient versatile complexes that respond to external stimuli1,2,3. Mutation and dysregulation of kinases often lead to diseases in humans, and they have therefore become one of the most important drug targets over the past forty years4.
In this context, it is important to be able to detect kinase interaction with their upstream regulators or downstream substrates and to identify new partners. Affinity purification and immunoprecipitation are very powerful techniques for the isolation of protein complexes5. The bait protein or kinase may be tagged with a specific peptide sequence allowing the use of commercial beads covalently coupled with antibodies targeting the peptide. This material permits a high reproducibility in experiments6,7,8. Endogenous proteins may also be immunoprecipitated using antibodies targeting directly the bait protein. The antibodies may be cross-linked to Protein A or Protein G agarose beads or simply incubated with these beads prior to adding lysate. Lysis buffers must be optimized to allow protein solubilization without losing interaction and to avoid protein degradation. A major drawback of this approach is that the interaction is detected upon cell lysis; therefore, transient or weak interactions, together with those requiring subcellular context may be missed. Other techniques may be used to work directly in the cell such as Proximity Ligation Assay (PLA)9, in vivo cross-linking-assisted affinity purification (XAP)10, Bioluminescence Resonance Energy Transfer (BRET) or F&#246;rster Resonance Energy Transfer (FRET)11,12. Furthermore, immunoprecipitation is not appropriate to determine the thermodynamic constants of the binding, for which physical techniques such as Surface Plasmon Resonance, Isothermal Titration Calorimetry or Microscale Thermophoresis are required13,14.
Kinase activity may be assessed using multiple techniques. Herein, we focused on phospho-specific antibodies and in vitro &#947;[32P] ATP (Adenosine TriPhosphate) labeling. Phospho-specific antibodies target the phosphate modification of a particular residue within a protein. They may be used in Western Blot or ELISA (Enzyme-Linked ImmunoSorbent Assay) after cell lysis, for immunohistochemistry, and also on intact cells using flow cytometry or immunofluorescence. Their drawbacks may include their lack of specificity, which can be evaluated using a mutated version of the target protein, and their not being commercially available for all proteins. In vitro &#947;[32P] ATP labeling is a very robust, well-established and highly sensitive method15. Immunoprecipitated or recombinant proteins may be used, and different substrates may be tested. The effects of drugs may also be assessed as this method is quantitative. Its major drawback is that the radioactivity associated with the approach requires handling with caution. Alternative methods are also possible based on the measurement of fluorescent or luminescent peptide substrates and taking advantage of altered fluorescent/luminescent properties upon phosphorylation. Such methods also allow high throughput, which is required, for example, in the screening of molecules that may be potential inhibitors of the target kinase. Indeed, kinases represent one of the largest classes of drug targets pursued by pharmaceutical companies16.
In this study, we focused on the LIMK2-1 protein (LIMK2-1 stands for Lin11, Isle1, Mec3 Kinase isoform 2-1). The LIMK2 kinase protein was first described in 199517. Three isoforms of LIMK2 are produced by alternative splicing: LIMK2a, LIMK2b and LIMK2-1. At present, LIMK2-1 has only been described at the mRNA level in a single study18. Herein, we characterize this potential new kinase protein at the molecular level using robust biochemical approaches. Firstly, we demonstrate that LIMK2-1 is indeed synthesized. Similar to its two counterparts, LIMK2a and LIMK2b, it interacts with the upstream kinase ROCK (Rho-associated protein kinase). We show LIMK2-1 has a kinase activity on Myelin Basic Protein (MBP), but not on cofilin, the canonical substrate of LIM kinases.

<table>
	<tbody>
		<tr>
			<td>Antibody anti-actin</td>
			<td>Sigma-Aldrich</td>
			<td>A1978</td>
			<td>for Western Blot</td>
		</tr>
		<tr>
			<td>Antibody anti-c-Myc</td>
			<td>Invitrogen</td>
			<td>MA1-21316</td>
			<td>for Western Blot</td>
		</tr>
		<tr>
			<td>Antibody anti-cofilin</td>
			<td>Cell signaling Technology</td>
			<td>3312/5175</td>
			<td>for Western Blot</td>
		</tr>
		<tr>
			<td>Antibody anti-GFP</td>
			<td>Santa Cruz</td>
			<td>sc-9996</td>
			<td>for Western Blot</td>
		</tr>
		<tr>
			<td>Antibody anti-HA</td>
			<td>Roche Applied Science</td>
			<td>11687423001</td>
			<td>for Western Blot</td>
		</tr>
		<tr>
			<td>Antibody anti-phospho-cofilin</td>
			<td>Cell signaling Technology</td>
			<td>3313</td>
			<td>for Western Blot</td>
		</tr>
		<tr>
			<td>Antibody Anti-PP1i</td>
			<td>Eurogentec</td>
			<td>designed for this study</td>
			<td>for Western Blot</td>
		</tr>
		<tr>
			<td>Aprotinin</td>
			<td>Euromedex</td>
			<td>A-162B</td>
			<td>for lysis buffer</td>
		</tr>
		<tr>
			<td>ATP</td>
			<td>Invitrogen</td>
			<td>PV3227</td>
			<td>for g[32P] labeling</td>
		</tr>
		<tr>
			<td>g[32P] ATP</td>
			<td>Perkin Elmer</td>
			<td>NEG502A</td>
			<td>for g[32P] labeling</td>
		</tr>
		<tr>
			<td>BES buffered saline</td>
			<td>Sigma-Aldrich</td>
			<td>14280</td>
			<td>for transfection</td>
		</tr>
		<tr>
			<td>b-glycerophosphate</td>
			<td>Sigma-Aldrich</td>
			<td>G9422</td>
			<td>for lysis and kinase buffer</td>
		</tr>
		<tr>
			<td>b-mercaptoethanol</td>
			<td>Sigma-Aldrich</td>
			<td>M3148</td>
			<td>for Laemmli</td>
		</tr>
		<tr>
			<td>BSA</td>
			<td>Sigma-Aldrich</td>
			<td>A3059</td>
			<td>for blocking buffer</td>
		</tr>
		<tr>
			<td>Bromophenol Blue</td>
			<td>Sigma-Aldrich</td>
			<td>B0126</td>
			<td>for Laemmli</td>
		</tr>
		<tr>
			<td>CaCl2</td>
			<td>Sigma-Aldrich</td>
			<td>C3881</td>
			<td>for transfection</td>
		</tr>
		<tr>
			<td>Centrifuge</td>
			<td>Sigma</td>
			<td>111-541</td>
			<td></td>
		</tr>
		<tr>
			<td>Collagen R</td>
			<td>Pan Biotech</td>
			<td>P06-20166</td>
			<td>for transfection</td>
		</tr>
		<tr>
			<td>Control siRNA</td>
			<td>Ambion</td>
			<td>AM4611</td>
			<td>for PP1i antibody specificity</td>
		</tr>
		<tr>
			<td>Coomassie PageBlue Protein Staining Solution</td>
			<td>Thermo-Fisher</td>
			<td>24620</td>
			<td>for gel staining</td>
		</tr>
		<tr>
			<td>EDTA</td>
			<td>Sigma-Aldrich</td>
			<td>3690</td>
			<td>for lysis buffer</td>
		</tr>
		<tr>
			<td>Electrophoresis Unit</td>
			<td>Biorad</td>
			<td>Mini-Protean</td>
			<td>for Western Blot</td>
		</tr>
		<tr>
			<td>EZview Red anti-HA affinity gel</td>
			<td>Sigma-Aldrich</td>
			<td>E6779</td>
			<td>for immunoprecipitation</td>
		</tr>
		<tr>
			<td>GeneSys software</td>
			<td>Ozyme</td>
			<td></td>
			<td>for Western Blot acquisition</td>
		</tr>
		<tr>
			<td>GeneTolls software</td>
			<td>Ozyme</td>
			<td></td>
			<td>for Western Blot quantification</td>
		</tr>
		<tr>
			<td>GFP-trap beads</td>
			<td>Chromtek</td>
			<td></td>
			<td>for immunoprecipitation</td>
		</tr>
		<tr>
			<td>Glycine</td>
			<td>Euromedex</td>
			<td>26-128-6405</td>
			<td>for transfer buffer</td>
		</tr>
		<tr>
			<td>GST-cofilin</td>
			<td>Upstate Cell signaling</td>
			<td>12-556</td>
			<td>for g[32P] labeling</td>
		</tr>
		<tr>
			<td>Hamilton syringe 100 mL</td>
			<td>Hamilton</td>
			<td>710</td>
			<td>to remove carefully supernatant from beads without aspirating them</td>
		</tr>
		<tr>
			<td>HEPES</td>
			<td>Sigma-Aldrich</td>
			<td>H3375</td>
			<td>for kinase buffer</td>
		</tr>
		<tr>
			<td>ImageQuant TL software</td>
			<td>GE Healthcare</td>
			<td></td>
			<td>for radioactivity acquisition and quantification</td>
		</tr>
		<tr>
			<td>LIMK2 siRNA</td>
			<td>Ambion</td>
			<td>s8191</td>
			<td>for PP1i antibody specificity</td>
		</tr>
		<tr>
			<td>Leupeptin</td>
			<td>Sigma-Aldrich</td>
			<td>SP-04-2217</td>
			<td>for lysis and kinase buffer</td>
		</tr>
		<tr>
			<td>MBP</td>
			<td>Upstate Cell signaling</td>
			<td>13-173</td>
			<td>for g[32P] labeling</td>
		</tr>
		<tr>
			<td>MgCl2</td>
			<td>Sigma-Aldrich</td>
			<td>M8266</td>
			<td>for kinase buffer</td>
		</tr>
		<tr>
			<td>MnCl2</td>
			<td>Sigma-Aldrich</td>
			<td>244589</td>
			<td>for kinase buffer</td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>Euromedex</td>
			<td>1112</td>
			<td>for lysis and kinase buffer</td>
		</tr>
		<tr>
			<td>NaF</td>
			<td>Sigma-Aldrich</td>
			<td>S-1504</td>
			<td>for lysis and kinase buffer</td>
		</tr>
		<tr>
			<td>Okaidic acid</td>
			<td>Euromedex</td>
			<td>0-2220</td>
			<td>for lysis buffer</td>
		</tr>
		<tr>
			<td>PMSF</td>
			<td>Sigma-Aldrich</td>
			<td>78830</td>
			<td>for lysis and kinase buffer</td>
		</tr>
		<tr>
			<td>p-nitrophenylphosphate</td>
			<td>Euromedex</td>
			<td>1026</td>
			<td>for lysis buffer</td>
		</tr>
		<tr>
			<td>PVDF membrane Immobillon-P</td>
			<td>Merck-Millipore</td>
			<td>IPVH00010 pore size 0,45 mm</td>
			<td>for Western Blot</td>
		</tr>
		<tr>
			<td>Rotating wheel</td>
			<td>Labinco</td>
			<td></td>
			<td>for bead incubation</td>
		</tr>
		<tr>
			<td>Safe lock eppendorf</td>
			<td>Eppendorf</td>
			<td>0030120.086</td>
			<td>for kinase assay</td>
		</tr>
		<tr>
			<td>SDS</td>
			<td>Sigma-Aldrich</td>
			<td>5030</td>
			<td>for Laemmli and migration buffer</td>
		</tr>
		<tr>
			<td>Sodium orthovanadate</td>
			<td>LC Laboratories</td>
			<td>S8507</td>
			<td>for lysis and kinase buffer</td>
		</tr>
		<tr>
			<td>Sodium pyrophosphate</td>
			<td>Fluka</td>
			<td>71501</td>
			<td>for lysis buffer</td>
		</tr>
		<tr>
			<td>Super Signal West Dura</td>
			<td>Protein Biology</td>
			<td>34075</td>
			<td>for Western Blot</td>
		</tr>
		<tr>
			<td>Syngene Pxi</td>
			<td>Ozyme</td>
			<td></td>
			<td>for Western Blot</td>
		</tr>
		<tr>
			<td>Tissue extracts 
			 
			&#160;</td>
			<td>Biochain 
			 
			&#160;</td>
			<td>P1234035 Brain 
			P12345152 Lung 
			P1234149 Liver 
			P1234188 Pancreas 
			P1234260 Testis</td>
			<td>for Western Blot analysis 
			 
			&#160;</td>
		</tr>
		<tr>
			<td>Transfer Unit</td>
			<td>Biorad</td>
			<td>Mini-Trans-Blot</td>
			<td>for Western Blot</td>
		</tr>
		<tr>
			<td>Tris</td>
			<td>Euromedex</td>
			<td>26-128-3094 B</td>
			<td>for lysis buffer</td>
		</tr>
		<tr>
			<td>Tween-20</td>
			<td>Sigma-Aldrich</td>
			<td>P7949</td>
			<td>for blocking buffer</td>
		</tr>
		<tr>
			<td>Typhoon FLA9500</td>
			<td>GE Healthcare</td>
			<td></td>
			<td>to read autoradiography</td>
		</tr>
		<tr>
			<td>Typhoon Trio</td>
			<td>Amersham Bioscience</td>
			<td></td>
			<td>to read autoradiography</td>
		</tr>
		<tr>
			<td>Whatman paper</td>
			<td>GE Healthcare</td>
			<td>3030-672</td>
			<td>for Western Blot</td>
		</tr>
	</tbody>
</table>

characterization at the molecular level using robust biochemical approaches of a new kinase protein

Extensive whole genome sequencing has identified many Open Reading Frames (ORFs) providing many potential proteins. These proteins may have important roles for the cell and may unravel new cellular processes. Among proteins, kinases are major actors as they belong to cell signaling pathways and have the ability to switch on or off many processes crucial to the fate of the cell, such as cell growth, division, differentiation, motility, and death.
In this study, we focused on a new potential kinase protein, LIMK2-1. We demonstrated its existence by Western Blot using a specific antibody. We evaluated its interaction with an upstream regulating protein using coimmunoprecipitation experiments. Coimmunoprecipitation is a very powerful technique able to detect the interaction between two target proteins. It may also be used to detect new partners of a bait protein. The bait protein may be purified either via a tag engineered to its sequence or via an antibody specifically targeting it. These protein complexes may then be separated by SDS-PAGE (Sodium Dodecyl Sulfate PolyAcrylamide Gel) and identified using mass spectrometry. Immunoprecipitated LIMK2-1 was also used to test its kinase activity in vitro by &#947;[32P] ATP labeling. This well-established assay may use many different substrates, and mutated versions of the bait may be used to assess the role of specific residues. The effects of pharmacological agents may also be evaluated since this technique is both highly sensitive and quantitative. Nonetheless, radioactivity handling requires particular caution. Kinase activity may also be assessed with specific antibodies targeting the phospho group of the modified amino acid. These kinds of antibodies are not commercially available for all the phospho modified residues.

LIMK2-1 protein is synthesized 
LIMK2-1 is mentioned in databanks, but thus far only one paper has shown the existence of its mRNA18. Compared to its two homologs, LIMK2a and LIMK2b, LIMK2-1 has an extra C-terminal domain identified as a Protein Phosphatase 1 Inhibitory domain (PP1i). We designed an antibody that targets a peptide of this domain, amino acids 671-684 (Figure 1A). 
BLAST research against human ...

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Characterization at the Molecular Level using Robust Biochemical Approaches of a New Kinase Protein

We characterized a new kinase protein using robust biochemical approaches: Western Blot analysis with a dedicated specific antibody on different cell lines and tissues, interactions by coimmunoprecipitation experiments, kinase activity detected by Western Blot using a phospho-specific antibody and by &#947;[32P] ATP labeling.

Extensive whole genome sequencing has identified many Open Reading Frames (ORFs) providing many potential proteins. These proteins may have important ...

Immunoprecipitation is a very powerful technique to isolate and purify a target protein. In smooth conditions, proteins, regulators, or substrates may be co-immunoprecipitated. A new interaction network may then be discovered.The activity of the immunoprecipitated protein may also be assessed. In particular, activity of of a kinase may be tested on different substrates by P32-ATP labeling. Nevertheless, activity of inhibitors targeting a kinase involved in a disease may be evaluated.They may constitute lead compounds to develop new drugs. This method can be extended from mammals to yeast or bacteria. This technique is not that difficult.To key points:ensure to have a negative control to be sure the detected interaction is specific, and carefully choose lysis conditions to keep interactions and activity. Small details and gestures are important to ensure the success of this technique, and they are never described in materials and methods of articles. Demonstrating the procedure will be Fabienne Godin, a technician from our laboratory.After cells are prepared in the cell culture room within the biosafety cabinet, use a 10 milliliter pipette to remove the media from the plates and discard it in a dedicated waste bottle with bleach. Then, add 10 milliliters of fresh supplemented DMEM, and put the plates back into an incubator at 37 degrees Celsius. To prepare the transfection mixture, in a 15 milliliter tube, add 450 microliters of 10 millimolar Tris hydrochloride solution at pH 7.5, supplemented with one millimolar EDTA and 50 microliters of 2.5 molar calcium chloride solution.Mix by inversion. Into the tube, add a volume corresponding to 10 micrograms of plasma DNA, amplified by a DNA midiprep kit, and diluted with the elution buffer of this kit, and whose concentration has been determined. Invert the tube to mix.Then, with smooth agitation on a vortex mixer, slowly add 500 microliters of BES buffered saline 2X concentrate, drop by drop into the tube. Move it very carefully, not to disturb the complex formation between DNA and calcium phosphate. Incubate for at least 15 minutes, or up to 45 minutes, at room temperature.Now, transfer the plate to transfect from the incubator to the safety cabinet. Add the prepared DNA complexes drop by drop onto the cells all over the surface of the plate. Incubate for 24 to 72 hours in the incubator at 37 degrees Celsius.To prevent protein degradation, prepare lysis buffer on ice, taking into consideration four milliliters of lysis buffer for each transfected plate. Take the plate with transfected cells from the incubator. Remove the media and discard it in a dedicated bleach waste bottle.To wash the cells, add three milliliters of cold PBS on the side of the plate drop by drop, to avoid detaching transfected cells. And gently swirl the plate to spread the buffer all over the surface. Tilt the plate to remove the PBS and discard it into the waste bottle.Repeat the washing once more. Place the plate on ice. At the end, carefully remove the rest of the PBS.Next, add 500 microliters of cold lysis buffer to the transfected washed cells. Spread it all over the surface of the plate. Incubate for 10 minutes on ice.From time to time, again spread the buffer all over the surface of the plate. After that, use a cell spatula to scrape the cells off the surface and collect them in a microcentrifuge tube. Centrifuge for 10 minutes at 10, 000 times G and four degrees Celsius.Collect the supernatant lysate into a new microcentrifuge tube and collect a 50 microliter aliquot of this fraction to another new microcentrifuge tube. This aliquot will be used to check if transfected proteins are well expressed. Discard the pellet in the trash.First, gently resuspend the stock solution of agarose beads coupled with the appropriate antibody. Cut the end of a 200 microliter tip to make a one to two millimeter opening. Attach the tip to a micropipette and draw up 40 microliters of the solution, allowing beads to enter the tip.Pipette the beads up and down several times to saturate the tip to ensure the correct volume of beads and transfer the beads to a microcentrifuge tube. Add 500 microliters of TNET buffer, mix by inversion, and centrifuge for two minutes at 1, 000 times G and four degrees Celsius. Carefully remove the supernatant, and add 500 microliters of TNET buffer.Incubate on a rotating wheel for at least one hour at four degrees Celsius. Then, centrifuge the tube for two minutes at 1, 000 times G and four degrees Celsius and wash the beads twice with 500 microliters of lysis buffer by inverting and centrifuging. After washing, transfer the previously collected total fraction lysate into the tube and incubate on a rotating wheel at four degrees Celsius for two to four hours.Now, centrifuge the tube containing the immunoprecipitated beads for two minutes at 1, 000 times G and four degrees Celsius. Wash the beads five times with 500 microliters of lysis buffer by inverting and centrifuging. When washing the beads, take care not to go too close from the beads while removing supernatant.Otherwise, beads will be aspirated and at the end of the experiment, no more beads will be left. For elution, first centrifuge for two minutes at 1, 000 times G and four degrees Celsius. Use a one milliliter micropipette to carefully remove the supernatant.Then, with a Hamilton syringe equipped with a cemented needle, remove the last drops of the supernatant to avoid aspiration of the beads. Next, add 40 microliters of 4X Laemmli buffer. Homogenize by gently tapping the tube.Incubate for five minutes at room temperature. Then, centrifuge for five minutes at 10, 000 times G and room temperature. With a Hamilton syringe, transfer the supernatant eluate into a new microcentrifuge tube.Store at minus 80 degrees Celsius. HEK-293 cells were transfected with untagged LIMK 2-1 or one of the HA tagged isoforms of LIMK 2. LIMK 2-1 is well expressed in the different conditions.Anti LIMK 2-1 antibody does not recognize LIMK 2a and LIMK 2b isoforms, and it is specific, as its signal decreases when cells are transfected with LIMK2 small interfering RNA, compared to control small interfering RNA. In various human cell lines, LIMK 2-1 appeared to be expressed in HEK-293 and HeLa, but not in C6.Coimmunoprecipitation experiments were used to assess the interaction of LIMK2-1 with upstream kinase ROCK. Each of the different proteins encoded by the transfected vectors were well expressed.The three isoforms of LIMK2, as well as larp6, were efficiently immunoprecipitated. In the eluates, ROCK was detected and thus coimmunoprecipitated with the three isoforms of LIMK2, but not with larp6. The detected interaction is then specific.Coimmunoprecipitated LIMK 2-1 was tested for its kinase activity via gamma P32 ATP labeling. LIMK 2-1 did not phosphorylate cofilin, whereas it did phosphorylate myelin basic protein, or MBP. LIMK 2-1 is efficiently immunoprecipitated in this test.It is critical to have negative control while performing immunoprecipitation to be sure the interaction is specific. The composition of a lysis buffer must be carefully set up to preserve interaction and activity. Upon immunoprecipitation, kinase activity may be assessed to count different substrates.Mutation may be easily introduced and may unravel the role of specific residues. Functional studies may also be performed to understand the physiological role of new highlighted interactions. Cells must be cultured in a dedicated culture room within a biosafety cabinet.P32 ATP has to be handled with specific caution. Shield to protect from radiation, Geiger counter, specific waste collect, personal breast and finger badges to detect radioactive exposure and filter tips.

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