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 cm plates with Collagen R to increase cell adhesion to the plastic plates.
	<ol>
		<li>Add 5 mL of a solution of Collagen R, 200-fold diluted in Phosphate Saline Buffer (PBS) in a &#216; 10 cm plate. Overlay the liquid over the whole surface of the plate.</li>
		<li>Incubate at room temperature for at least 1 h within the biosafety cabinet.</li>
		<li>Remove collagen solution, and discard it. Add 5 mL of PBS, spread it all over the surface of the plate, remove it and discard it. Repeat this wash once.</li>
		<li>Add 8 mL of DMEM supplemented with 10% fetal calf serum. Keep the prepared plates within the biosafety cabinet.</li>
	</ol>
	</li>
	<li>Take HEK-293 plates from step 1.1 within the biosafety cabinet. Remove the medium from the plates and discard it in a dedicated bleach trash. Add 2 mL of supplemented DMEM, and flush the cells to detach them using the flow of a 1 mL micropipette, taking care to avoid making foam.</li>
	<li>Collect the cells in a 15 mL tube and add 4 mL of supplemented DMEM. Homogenize with a 10 mL pipette, by pipetting up and down 3 times.</li>
	<li>Take 2 mL of this cell solution and add them to collagen-treated plates containing supplemented DMEM from step 1.2.4.</li>
	<li>Grow for 24 hours in the incubator at 5% CO2 and 37 &#176;C. Cells should be 50-80% confluent at the time of transfection.</li>
</ol>
2. Transient transfections
<ol>
	<li>Remove the medium from the plates, discard it in a dedicated bleach trash, and add 10 mL of fresh supplemented DMEM. Put back the plates into the incubator at 37 &#176;C while preparing the transfection mixture.</li>
	<li>In a 15 mL tube, add 450 &#956;L of a 10 mM Tris-HCl pH 7.5/1 mM EDTA (Tris stands for Tris(hydroxymethyl)aminomethane, and EDTA for ethylenedinitrilotetraacetic acid) solution and 50 &#956;L of a 2.5 M CaCl2 solution. Mix by inversion.</li>
	<li>Add 10 &#956;g of plasmidic DNA (Deoxyribonucleic acid) prepared from a midi preparation on a liquid culture of bacteria transformed with the dedicated plasmid. Mix by inversion.</li>
	<li>Under smooth agitation on a vortex, add 500 &#956;L of BES buffered saline 2x concentrate (composition: BES, 10.7 g/L, NaCl, 16.0 g/L, Na2HPO4, 0.27 g/L; BES stands for N,N-Bis(2-hydroxyethyl)-2-aminoethanesulfonic acid, N,N-Bis(2-hydroxyethyl)taurine) slowly drop by drop.</li>
	<li>Incubate for at least 15 min (up to 45 min) at room temperature within the biosafety cabinet. Do not vortex, do not mix! Move the tubes very carefully not to disturb the complex formation between DNA and calcium phosphate.</li>
	<li>Take the plates from step 2.1 to the safety cabinet. Add the DNA complexes very carefully, drop by drop, onto the cells all over the surface of the plate.</li>
	<li>Incubate for 24 to 72 hours in the incubator at 37 &#176;C. Usually maximum protein expression is reached within 48 hours.</li>
</ol>
3. Lysis
NOTE: Work on ice, and with cold buffers to prevent protein degradation.
<ol>
	<li>Prepare lysis buffer: 50 mM Tris-HCl, pH 7.5, 100 mM NaCl, 5 mM EDTA, 0.1% Triton X-100, 50 mM NaF, 10 mM sodium pyrophosphate, 1 mM Na3VO4, 20 mM p-nitrophenyl phosphate, 20 mM &#946;-glycerophosphate, 10 &#956;g/mL aprotinin, 0.05 &#956;g/mL okaidic acid, 1 &#956;g/mL leupeptin, and 1 mM PMSF (phenylmethylsulfonyl fluoride). Around 4 mL of lysis buffer are required for each transfected plate.</li>
	<li>Remove plates with transfected cells from the incubator. Put them on ice. 
	NOTE: From this step, it is possible to work on a &#8220;normal&#8221; bench.</li>
	<li>Remove the media, and discard it in a dedicated bleach trash.</li>
	<li>Wash twice with 3 mL of cold PBS: add 3 mL of PBS on the side of the plate drop by drop to avoid detaching transfected cells, spread all over the surface of the plate, remove PBS and discard it; repeat this step once more. At the end, remove carefully the rest of PBS by tilting the plate to prevent lysis buffer dilution for next step.</li>
	<li>Add 500 &#956;L of cold lysis buffer on the transfected washed cells. Spread it all over the surface of the plate.</li>
	<li>Incubate for 10 min on ice. From time to time (at least twice), spread again the buffer all over the surface of the plate.</li>
	<li>Scrap the cells and collect them in a microcentrifuge tube.</li>
	<li>Centrifuge for 10 min at 10,000 x g at 4 &#176;C.</li>
	<li>Collect supernatant in a new microcentrifuge tube. This fraction corresponds to the lysate (whole cell extract). Discard the pellet which corresponds to cell membrane debris.</li>
	<li>Collect an aliquot of this fraction to a new microcentrifuge tube (around 50 &#956;L). This fraction corresponds to the &#8220;TOTAL fraction&#8221; or &#8220;Cell Lysate&#8221; or &#8220;Whole Cell extract&#8221; allowing analysis of whether transfected proteins are expressed by Western Blot. 
	NOTE: At this point, samples can be directly used for Western Blot analyses. Laemmli buffer must&#160;be added to the sample, which is then heated at 95 &#176;C for 5 min, centrifuged at 10,000 x g for 5 min, and loaded on appropriate SDS-PAGE. Samples may also be stored at -80 &#176;C.</li>
</ol>
4. Immunoprecipitation
<ol>
	<li>Gently resuspend agarose beads coupled with the appropriate antibody: HA (Human influenza hemagglutinin), Flag, or GFP (Green Fluorescent Protein) by smooth inversion.</li>
	<li>Cut the end of a 200 &#956;L tip from a micropipette to allow beads to enter the tip. Pipette the beads up and down several times to saturate the tip to ensure to take the correct volume of beads.</li>
	<li>Take 40 &#956;L of beads in a microcentrifuge tube.</li>
	<li>Add 500 &#956;L of TENET (30 mM Tris-HCl pH 7.5, 120 mM NaCl, 5 mM EDTA, 1% Triton X-100) buffer. Mix by inversion. Centrifuge for 2 min at 1,000 x g at 4 &#176;C. Remove carefully supernatant and discard it. Add 500 &#956;L of TENET. Incubate for at least 1 hour at 4 &#176;C on a rotating wheel. 
	NOTE: This pre-incubation step in TENET allows to reduce non-specific interactions and so to decrease background signal.</li>
	<li>Wash the beads twice with 500 &#956;L of lysis buffer.
	<ol>
		<li>Centrifuge 2 min at 1,000 x g at 4&#176;C.</li>
		<li>Remove carefully supernatant buffer, and discard it. 
		NOTE: Take care not to aspirate beads during these washing steps.</li>
		<li>Add 500 &#956;L of lysis buffer. Homogenize by inverting the tube.</li>
		<li>Repeat steps 4.5.1- 4.5.3.</li>
	</ol>
	</li>
	<li>Centrifuge for 2 min at 1,000 x g at 4&#176;C. Carefully remove supernatant buffer, and discard it.</li>
	<li>Incubate beads with the lysate from step 3.9 for 2 to 4 hours at 4 &#176;C on a rotating wheel.</li>
	<li>Wash immunoprecipitated beads. 
	NOTE: At this point, the immunoprecipitated beads may be washed five times with the lysis buffer, and then eluted with Laemmli buffer. The eluate may be used for Western Blot analyses or stored at -80 &#176;C. Alternatively, beads may be washed twice with the lysis buffer and then three times with the kinase buffer to perform &#947;[32P] ATP labeling.</li>
</ol>
5. Coimmunoprecipitation analyses
<ol>
	<li>Wash immunoprecipitated beads from step 4.7 with the lysis buffer.
	<ol>
		<li>Centrifuge for 2 min at 1,000 x g in a refrigerated centrifuge at 4 &#176;C.</li>
		<li>Carefully remove the supernatant, and discard it.</li>
		<li>Add 500 &#956;L of lysis buffer. Homogenize by inverting the tube.</li>
		<li>Repeat steps 5.1.1-5.1.3 four times.</li>
	</ol>
	</li>
	<li>Elution
	<ol>
		<li>Centrifuge for 2 min at 1,000 x g in a refrigerated centrifuge at 4 &#176;C.</li>
		<li>Carefully remove the supernatant, and discard it. Remove the last drops of supernatant with a Hamilton syringe in order to avoid aspiration of the beads, and discard the supernatant.</li>
		<li>Add 40 &#956;L of 4x Laemmli buffer (200 mM Tris/HCl pH 6.8, 4% SDS, 40% glycerol, 0.5 M &#946;-mercaptoethanol, 0.02% bromophenol blue). Homogenize by gently tapping the tube.</li>
		<li>Incubate for 5 min at room temperature.</li>
		<li>Centrifuge for 5 min at 10,000 x g at room temperature.</li>
		<li>With a Hamilton syringe, remove the supernatant and collect it in a new microcentrifuge tube. This fraction corresponds to the &#8220;Eluate&#8221;, which may be stored at -80 &#176;C, or directly analyzed by Western Blot.</li>
	</ol>
	</li>
</ol>
6. Kinase assay
<ol>
	<li>Prepare the kinase buffer: 50 mM HEPES-NaOH pH 7.5 (HEPES stands for 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid), 150 mM NaCl, 5 mM MgCl2, 5 mM MnCl2, 50 mM NaF, 1 mM Na3VO4, 20 mM &#946;-glycerophosphate, 1 &#956;g/mL leupeptin, and 1 mM PMSF. Around 2 mL of the kinase buffer is required for each immunoprecipitation condition.</li>
	<li>Wash immunoprecipitated beads from step 4.7 twice with 500 &#956;L of lysis buffer.
	<ol>
		<li>Centrifuge for 2 min at 1,000 x g in a refrigerated centrifuge at 4 &#176;C.</li>
		<li>Carefully remove supernatant, and discard it. Add 500 &#956;L of lysis buffer. Homogenize by inversion.</li>
		<li>Repeat steps 6.2.1 and 6.2.2.</li>
	</ol>
	</li>
	<li>Wash immunoprecipitated beads three times with 500 &#956;L of kinase buffer.
	<ol>
		<li>Centrifuge for 2 min at 1,000 x g in a refrigerated centrifuge at 4 &#176;C.</li>
		<li>Carefully remove supernatant, and discard it. Add 500 &#956;L of kinase buffer. Homogenize by inversion.</li>
		<li>Repeat steps 6.3.1 and 6.3.2 twice.</li>
	</ol>
	</li>
	<li>Centrifuge for 2 min at 1,000 x g in a refrigerated centrifuge at 4 &#176;C.</li>
	<li>Carefully remove supernatant, and discard it. Remove the last drops of supernatant with a Hamilton syringe in order to avoid aspiration of the beads, and discard the supernatant.</li>
	<li>Add 40 &#956;L of kinase buffer to the immunoprecipitated beads. Resuspend the beads by gently tapping the tube.</li>
	<li>Prepare the mix for the &#947;[32P] ATP labeling (final volume is 22.5 &#956;L, complete with kinase buffer) in a safe lock-tube.
	<ol>
		<li>Add the required volume of kinase buffer to reach a final volume of 22.5 &#956;L.</li>
		<li>Cut the end of a 20 &#956;L tip of a micropipette. Resuspend the immunoprecipitated beads from step 6.6 by pipetting up and down several times. Collect 10 &#956;L of these beads into the safe-lock tube.</li>
		<li>Add ATP from a 10 &#956;M stock solution to reach 50 mM of finale concentration. According to the number of sample treated, a dilution of the stock solution of ATP in the kinase buffer is suggested to allow to pipette 1 to 2 &#956;L, to be sure the volume is correct.</li>
		<li>Add 2.5 &#956;g of substrate (cofilin or Myelin Basic Protein, MBP in the present study case).</li>
	</ol>
	</li>
	<li>Add 5 &#956;Ci of &#947;[32P] ATP (3,000 Ci/mmol) to initiate the reaction. Mix by pipetting up and down slowly. 
	CAUTION: From this point, work must be done in a safety place dedicated for radioactivity manipulations with cautious precaution, dedicated protections and appropriate controls (radioactive shields, Geiger counter, specific waste collect, personal breast and finger badges to detect radioactive exposure, filter tips).</li>
	<li>Incubate for 20 min at 30 &#176;C.</li>
	<li>Stop the reaction with 6 &#956;L of 5x Laemmli buffer.</li>
	<li>Heat at 95&#176; C for 5 min.</li>
	<li>Centrifuge at 10,000 x g for 5 min at room temperature.</li>
	<li>Load on a SDS-PAGE. Proceed to migration. 
	NOTE: Take care that the front line of free &#947;[32P] ATP does not exit the gel to avoid contamination of the migration tank.</li>
	<li>Stain the gel at room temperature.
	<ol>
		<li>Remove the gel from the glass plates.</li>
		<li>Proceed with three baths in water at room temperature.</li>
		<li>Stain the gel overnight with Coomassie Blue at room temperature.</li>
		<li>Destain the gel with several wash baths with water at room temperature.</li>
	</ol>
	</li>
	<li>Wrap the gel with plastic wrap.</li>
	<li>Expose for one night or more on a phosphorimager screen.</li>
	<li>Read the screen on a phosphorimager to detect labeled bands.</li>
</ol>

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The authors have nothing to disclose.

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 &amp;#947;[&lt;sup&gt;32&lt;/sup&gt;P] labeling</td></tr><tr><td>&amp;#947;[&lt;sup&gt;32&lt;/sup&gt;P] ATP</td><td>Perkin Elmer</td><td>NEG502A</td><td>for &amp;#947;[&lt;sup&gt;32&lt;/sup&gt;P] labeling</td></tr><tr><td>BES buffered saline</td><td>Sigma-Aldrich</td><td>14280</td><td>for transfection</td></tr><tr><td>&amp;#946;-glycerophosphate</td><td>Sigma-Aldrich</td><td>G9422</td><td>for lysis and kinase buffer</td></tr><tr><td>&amp;#946;-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>CaCl&lt;sub&gt;2&lt;/sub&gt;</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 &amp;#947;[&lt;sup&gt;32&lt;/sup&gt;P] 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 &amp;#947;[&lt;sup&gt;32&lt;/sup&gt;P] labeling</td></tr><tr><td>MgCl&lt;sub&gt;2&lt;/sub&gt;</td><td>Sigma-Aldrich</td><td>M8266</td><td>for kinase buffer</td></tr><tr><td>MnCl&lt;sub&gt;2&lt;/sub&gt;</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&lt;br/&gt; &lt;br/&gt; &amp;nbsp;</td><td>Biochain&lt;br/&gt; &lt;br/&gt; &amp;nbsp;</td><td>P1234035 Brain&lt;br/&gt; P12345152 Lung&lt;br/&gt; P1234149 Liver&lt;br/&gt; P1234188 Pancreas&lt;br/&gt; P1234260 Testis</td><td>for Western Blot analysis&lt;br/&gt; &lt;br/&gt; &amp;nbsp;</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 ...

characterization-at-molecular-level-using-robust-biochemical

Universidad San Sebastian

Research

JoVE Journal

Biochemistry

6.1K Views.  University of Orléans and INSERM. 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 γ[32P] ATP labeling.

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

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 ...

Oligopeptide Competition Assay for Phosphorylation Site Determination

Protein phosphorylation at specific sites determines its conformation and interaction with other molecules. Thus, protein phosphorylation affects biological functions and characteristics of the cell. Currently, the most common method for discovering phosphorylation sites is by liquid chromatography/mass spectrometry (LC/MS) analysis, a rapid and sensitive method. However, relatively labile phosphate moieties are often released from phosphopeptides during the fragmentation step, which often yields false-negative signals. In such cases, a traditional in vitro kinase assay using site-directed mutants would be more accurate, but this method is laborious and time-consuming. Therefore, an alternative method using peptide competition may be advantageous. The consensus recognition motif of 5&#39; adenosine monophosphate-activated protein kinase (AMPK) has been established1 and was validated using a positional scanning peptide library assay2. Thus, AMPK phosphorylation sites for a novel substrate could be predicted and confirmed by the peptide competition assays. In this report, we describe the detailed steps and procedures for the in vitro oligopeptide-competing kinase assay by illustrating AMPK-mediated nuclear factor erythroid 2-related factor 2 (Nrf2) phosphorylation. To authenticate the phosphorylation site, we carried out a sequential in vitro kinase assay using a site-specific mutant. Overall, the peptide competition assay provides a method to screen multiple potential phosphorylation sites and to identify sites for validation by the phosphorylation site mutants.

Peptide competition assays are widely used in a variety of molecular and immunological experiments. This paper describes a detailed method for an in vitro oligopeptide-competing kinase assay and the associated validation procedures, which may be useful to find specific phosphorylation sites.

Protein phosphorylation at specific sites determines its conformation and interaction with other molecules. Thus, protein phosphorylation affects ...

Identification of Cyclin-dependent Kinase 1 Specific Phosphorylation Sites by an In Vitro Kinase Assay

Cyclin-dependent kinase 1 (Cdk1) is a master controller for the cell cycle in all eukaryotes and phosphorylates an estimated 8 - 13% of the proteome; however, the number of identified targets for Cdk1, particularly in human cells is still low. The identification of Cdk1-specific phosphorylation sites is important, as they provide mechanistic insights into how Cdk1 controls the cell cycle. Cell cycle regulation is critical for faithful chromosome segregation, and defects in this complicated process lead to chromosomal aberrations and cancer.
Here, we describe an in vitro kinase assay that is used to identify Cdk1-specific phosphorylation sites. In this assay, a purified protein is phosphorylated in vitro by commercially available human Cdk1/cyclin B. Successful phosphorylation is confirmed by SDS-PAGE, and phosphorylation sites are subsequently identified by mass spectrometry. We also describe purification protocols that yield highly pure and homogeneous protein preparations suitable for the kinase assay, and a binding assay for the functional verification of the identified phosphorylation sites, which probes the interaction between a classical nuclear localization signal (cNLS) and its nuclear transport receptor karyopherin &#945;. To aid with experimental design, we review approaches for the prediction of Cdk1-specific phosphorylation sites from protein sequences. Together these protocols present a very powerful approach that yields Cdk1-specific phosphorylation sites and enables mechanistic studies into how Cdk1 controls the cell cycle. Since this method relies on purified proteins, it can be applied to any model organism and yields reliable results, especially when combined with cell functional studies.

Identification of Cyclin-dependent Kinase 1 Specific Phosphorylation Sites by an In Vitro Kinase Assay

Cyclin-dependent kinase 1 (Cdk1) is activated in the G2 phase of the cell cycle and regulates many cellular pathways. Here, we present a protocol for an in vitro kinase assay with Cdk1, which allows the identification of Cdk1-specific phosphorylation sites for establishing cellular targets of this important kinase.

Cyclin-dependent kinase 1 (Cdk1) is a master controller for the cell cycle in all eukaryotes and phosphorylates an estimated 8 - 13% of the proteome; ...

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.

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 ...

Cancer Research

Identification of Novel CK2 Kinase Substrates Using a Versatile Biochemical Approach

The study of kinase-substrate relationships is essential to gain a complete understanding of the functions of these enzymes and their downstream targets in both physiological and pathological states. CK2 is an evolutionarily conserved serine/threonine kinase with a growing list of hundreds of substrates involved in multiple cellular processes. Due to its pleiotropic properties, identifying and characterizing a comprehensive set of CK2 substrates has been particularly challenging and remains a hurdle in the study of this important enzyme. To address this challenge, we have devised a versatile experimental strategy that enables the targeted enrichment and identification of putative CK2 substrates. This protocol takes advantage of the unique dual co-substrate specificity of CK2 allowing for specific thiophosphorylation of its substrates in a cell or tissue lysate. These substrate proteins are subsequently alkylated, immunoprecipitated, and identified by liquid chromatography/tandem mass spectrometry (LC-MS/MS). We have previously used this approach to successfully identify CK2 substrates from Drosophila ovaries and here we extend the application of this protocol to human glioblastoma cells, illustrating the adaptability of this method to investigate the biological roles of this kinase in various model organisms and experimental systems.

The objective of this protocol is to label, enrich, and identify substrates of protein kinase CK2 from a complex biological sample such as a cell lysate or tissue homogenate. This method leverages unique aspects of CK2 biology for this purpose.

The study of kinase-substrate relationships is essential to gain a complete understanding of the functions of these enzymes and their downstream targets ...

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 ...

A Mass Spectrometry-Based Approach to Identify Phosphoprotein Phosphatases and their Interactors

Most cellular processes are regulated by dynamic protein phosphorylation. More than three-quarters of proteins are phosphorylated, and phosphoprotein phosphatases (PPPs) coordinate over 90% of all cellular serine/threonine dephosphorylation. Deregulation of protein phosphorylation has been implicated in the pathophysiology of various diseases, including cancer and neurodegeneration. Despite their widespread activity, the molecular mechanisms controlling PPPs and those controlled by PPPs are poorly characterized. Here, a proteomic approach termed phosphatase inhibitor beads and mass spectrometry (PIB-MS) is described to identify and quantify PPPs, their posttranslational modifications, and their interactors in as little as 12 h using any cell line or tissue. PIB-MS utilizes a non-selective PPP inhibitor, microcystin-LR (MCLR), immobilized on sepharose beads to capture and enrich endogenous PPPs and their associated proteins (termed the PPPome). This method does not require the exogenous expression of tagged versions of PPPs or the use of specific antibodies. PIB-MS offers an innovative way to study the evolutionarily conserved PPPs and expand our current understanding of dephosphorylation signaling.

Here, we present a protocol for the enrichment of endogenous phosphoprotein phosphatases and their interacting proteins from cells and tissues and their identification and quantification by mass spectrometry-based proteomics.

Most cellular processes are regulated by dynamic protein phosphorylation. More than three-quarters of proteins are phosphorylated, and phosphoprotein ...

Assaying the Kinase Activity of LRRK2 in vitro

Leucine Rich Repeat Kinase 2 (LRRK2) is a 2527 amino acid member of the ROCO family of proteins, possessing a complex, multidomain structure including a GTPase domain (termed ROC, for Ras of Complex proteins) and a kinase domain1. The discovery in 2004 of mutations in LRRK2 that cause Parkinson's disease (PD) resulted in LRRK2 being the focus of a huge volume of research into its normal function and how the protein goes awry in the disease state2,3. Initial investigations into the function of LRRK2 focused on its enzymatic activities4-6. Although a clear picture has yet to emerge of a consistent alteration in these due to mutations, data from a number of groups has highlighted the importance of the kinase activity of LRRK2 in cell death linked to mutations7,8. Recent publications have reported inhibitors targeting the kinase activity of LRRK2, providing a key experimental tool9-11. In light of these data, it is likely that the enzymatic properties of LRRK2 afford us an important window into the biology of this protein, although whether they are potential drug targets for Parkinson's is open to debate.
A number of different approaches have been used to assay the kinase activity of LRRK2. Initially, assays were carried out using epitope tagged protein overexpressed in mammalian cell lines and immunoprecipitated, with the assays carried out using this protein immobilised on agarose beads4,5,7. Subsequently, purified recombinant fragments of LRRK2 in solution have also been used, for example a GST tagged fragment purified from insect cells containing residues 970 to 2527 of LRRK212. Recently, Dani&euml;ls et al. reported the isolation of full length LRRK2 in solution from human embryonic kidney cells, however this protein is not widely available13. In contrast, the GST fusion truncated form of LRRK2 is commercially available (from Invitrogen, see table 1 for details), and provides a convenient tool for demonstrating an assay for LRRK2 kinase activity. Several different outputs for LRRK2 kinase activity have been reported. Autophosphorylation of LRRK2 itself, phosphorylation of Myelin Basic Protein (MBP) as a generic kinase substrate and phosphorylation of an artificial substrate - dubbed LRRKtide, based upon phosphorylation of threonine 558 in Moesin - have all been used, as have a series of putative physiological substrates including &alpha;-synuclein, Moesin and 4-EBP14-17. The status of these proteins as substrates for LRRK2 remains unclear, and as such the protocol described below will focus on using MBP as a generic substrate, noting the utility of this system to assay LRRK2 kinase activity directed against a range of potential substrates.

Assaying the Kinase Activity of LRRK2 in vitro

Leucine Rich Repeat Kinase 2 is a large multidomain kinase, mutations in which are the most common genetic cause of Parkinson's disease. Analysis of the kinase activity of this protein has proven to be a crucial tool in understanding the biology and dysfunction of this protein. In this paper, in vitro assaying of the kinase activity of LRRK2 and a selection of its mutants is described, providing an experimental system to examine phosphorylation of putative substrates and potential dysfunction of LRRK2 in disease.

Leucine Rich Repeat Kinase 2 (LRRK2) is a 2527 amino acid member of the ROCO family of proteins, possessing a complex, multidomain structure including a ...

Biology

Identification of Post-translational Modifications of Plant Protein Complexes

Plants adapt quickly to changing environments due to elaborate perception and signaling systems. During pathogen attack, plants rapidly respond to infection via the recruitment and activation of immune complexes. Activation of immune complexes is associated with post-translational modifications (PTMs) of proteins, such as phosphorylation, glycosylation, or ubiquitination. Understanding how these PTMs are choreographed will lead to a better understanding of how resistance is achieved.
Here we describe a protein purification method for nucleotide-binding leucine-rich repeat (NB-LRR)-interacting proteins and the subsequent identification of their post-translational modifications (PTMs). With small modifications, the protocol can be applied for the purification of other plant protein complexes. The method is based on the expression of an epitope-tagged version of the protein of interest, which is subsequently partially purified by immunoprecipitation and subjected to mass spectrometry for identification of interacting proteins and PTMs.
This protocol demonstrates that: i). Dynamic changes in PTMs such as phosphorylation can be detected by mass spectrometry; ii). It is important to have sufficient quantities of the protein of interest, and this can compensate for the lack of purity of the immunoprecipitate; iii). In order to detect PTMs of a protein of interest, this protein has to be immunoprecipitated to get a sufficient quantity of protein.

We describe here a protocol for the purification and characterization of plant protein complexes. We demonstrate that by immunoprecipitating a single protein within a complex, so we can identify its post-translational modifications and its interacting partners.

Plants adapt quickly to changing environments due to elaborate perception and signaling systems. During pathogen attack, plants rapidly respond to ...

Probing High-density Functional Protein Microarrays to Detect Protein-protein Interactions

High-density functional protein microarrays containing ~4,200 recombinant yeast proteins are examined for kinase protein-protein interactions using an affinity purified yeast kinase fusion protein containing a V5-epitope tag for read-out. Purified kinase is obtained through culture of a yeast strain optimized for high copy protein production harboring a plasmid containing a Kinase-V5 fusion construct under a GAL inducible promoter. The yeast is grown in restrictive media with a neutral carbon source for 6 hr followed by induction with 2% galactose. Next, the culture is harvested and kinase is purified using standard affinity chromatographic techniques to obtain a highly purified protein kinase for use in the assay. The purified kinase is diluted with kinase buffer to an appropriate range for the assay and the protein microarrays are blocked prior to hybridization with the protein microarray. After the hybridization, the arrays are probed with monoclonal V5 antibody to identify proteins bound by the kinase-V5 protein. Finally, the arrays are scanned using a standard microarray scanner, and data is extracted for downstream informatics analysis1,2 to determine a high confidence set of protein interactions for downstream validation in vivo.

Using protein microarrays containing nearly the entire S. cerevisiae proteome is probed for rapid unbiased interrogation of thousands of protein-protein interactions in parallel. This method can be utilized for protein-small molecule, posttranslational modification, and other assays in high-throughput.

High-density functional protein microarrays containing ~4,200 recombinant yeast proteins are examined for kinase protein-protein interactions using an ...

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

Summary

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Chapters in this video

Assaying Protein Kinase Activity with Radiolabeled ATP

Oligopeptide Competition Assay for Phosphorylation Site Determination

Identification of Cyclin-dependent Kinase 1 Specific Phosphorylation Sites by an In Vitro Kinase Assay

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

Identification of Novel CK2 Kinase Substrates Using a Versatile Biochemical Approach

Kinase Inhibitor Screening In Self-assembled Human Protein Microarrays

A Mass Spectrometry-Based Approach to Identify Phosphoprotein Phosphatases and their Interactors

Assaying the Kinase Activity of LRRK2 in vitro

Identification of Post-translational Modifications of Plant Protein Complexes

Probing High-density Functional Protein Microarrays to Detect Protein-protein Interactions