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

Podsumowanie

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.

Streszczenie

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 γ[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.

Wprowadzenie

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 stimuli^1,2,3. 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 experiments^6,7,8. 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 Förster 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 required^13,14.

Kinase activity may be assessed using multiple techniques. Herein, we focused on phospho-specific antibodies and in vitro γ[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 γ[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.

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Protokół

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.

1. Seed HEK-293 (Human Embryonic Kidney) cells in Ø 10 cm plates in 10 mL of DMEM (Dulbecco's Modified Eagles Medium) supplemented with 10% fetal calf serum. Culture for 3 to 5 days under 5% CO₂, at 37 °C, until the cells reach ~90% confluence.
2. Treat Ø 10 cm plates with Collagen R to increase cell adhesion to the plastic plates.
   1. Add 5 mL of a solution of Collagen R, 200-fold diluted in Phosphate Saline Buffer (PBS) in a Ø 10 cm plate. Overlay the liquid over the whole surface of the plate.
   2. Incubate at room temperature for at least 1 h within the biosafety cabinet.
   3. 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.
   4. Add 8 mL of DMEM supplemented with 10% fetal calf serum. Keep the prepared plates within the biosafety cabinet.
3. 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.
4. 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.
5. Take 2 mL of this cell solution and add them to collagen-treated plates containing supplemented DMEM from step 1.2.4.
6. Grow for 24 hours in the incubator at 5% CO₂ and 37 °C. Cells should be 50-80% confluent at the time of transfection.

2. Transient transfections

1. 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 °C while preparing the transfection mixture.
2. In a 15 mL tube, add 450 μL 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 μL of a 2.5 M CaCl₂ solution. Mix by inversion.
3. Add 10 μg of plasmidic DNA (Deoxyribonucleic acid) prepared from a midi preparation on a liquid culture of bacteria transformed with the dedicated plasmid. Mix by inversion.
4. Under smooth agitation on a vortex, add 500 μL 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.
5. 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.
6. 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.
7. Incubate for 24 to 72 hours in the incubator at 37 °C. Usually maximum protein expression is reached within 48 hours.

3. Lysis

NOTE: Work on ice, and with cold buffers to prevent protein degradation.

1. 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 β-glycerophosphate, 10 μg/mL aprotinin, 0.05 μg/mL okaidic acid, 1 μg/mL leupeptin, and 1 mM PMSF (phenylmethylsulfonyl fluoride). Around 4 mL of lysis buffer are required for each transfected plate.
2. Remove plates with transfected cells from the incubator. Put them on ice.
   NOTE: From this step, it is possible to work on a “normal” bench.
3. Remove the media, and discard it in a dedicated bleach trash.
4. 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.
5. Add 500 μL of cold lysis buffer on the transfected washed cells. Spread it all over the surface of the plate.
6. Incubate for 10 min on ice. From time to time (at least twice), spread again the buffer all over the surface of the plate.
7. Scrap the cells and collect them in a microcentrifuge tube.
8. Centrifuge for 10 min at 10,000 x g at 4 °C.
9. 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.
10. Collect an aliquot of this fraction to a new microcentrifuge tube (around 50 μL). This fraction corresponds to the “TOTAL fraction” or “Cell Lysate” or “Whole Cell extract” 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 be added to the sample, which is then heated at 95 °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 °C.

4. Immunoprecipitation

1. Gently resuspend agarose beads coupled with the appropriate antibody: HA (Human influenza hemagglutinin), Flag, or GFP (Green Fluorescent Protein) by smooth inversion.
2. Cut the end of a 200 μL 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.
3. Take 40 μL of beads in a microcentrifuge tube.
4. Add 500 μL 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 °C. Remove carefully supernatant and discard it. Add 500 μL of TENET. Incubate for at least 1 hour at 4 °C on a rotating wheel.
   NOTE: This pre-incubation step in TENET allows to reduce non-specific interactions and so to decrease background signal.
5. Wash the beads twice with 500 μL of lysis buffer.
   1. Centrifuge 2 min at 1,000 x g at 4°C.
   2. Remove carefully supernatant buffer, and discard it.
      NOTE: Take care not to aspirate beads during these washing steps.
   3. Add 500 μL of lysis buffer. Homogenize by inverting the tube.
   4. Repeat steps 4.5.1- 4.5.3.
6. Centrifuge for 2 min at 1,000 x g at 4°C. Carefully remove supernatant buffer, and discard it.
7. Incubate beads with the lysate from step 3.9 for 2 to 4 hours at 4 °C on a rotating wheel.
8. 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 °C. Alternatively, beads may be washed twice with the lysis buffer and then three times with the kinase buffer to perform γ[³²P] ATP labeling.

5. Coimmunoprecipitation analyses

1. Wash immunoprecipitated beads from step 4.7 with the lysis buffer.
   1. Centrifuge for 2 min at 1,000 x g in a refrigerated centrifuge at 4 °C.
   2. Carefully remove the supernatant, and discard it.
   3. Add 500 μL of lysis buffer. Homogenize by inverting the tube.
   4. Repeat steps 5.1.1-5.1.3 four times.
2. Elution
   1. Centrifuge for 2 min at 1,000 x g in a refrigerated centrifuge at 4 °C.
   2. 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.
   3. Add 40 μL of 4x Laemmli buffer (200 mM Tris/HCl pH 6.8, 4% SDS, 40% glycerol, 0.5 M β-mercaptoethanol, 0.02% bromophenol blue). Homogenize by gently tapping the tube.
   4. Incubate for 5 min at room temperature.
   5. Centrifuge for 5 min at 10,000 x g at room temperature.
   6. With a Hamilton syringe, remove the supernatant and collect it in a new microcentrifuge tube. This fraction corresponds to the “Eluate”, which may be stored at -80 °C, or directly analyzed by Western Blot.

6. Kinase assay

1. 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 β-glycerophosphate, 1 μg/mL leupeptin, and 1 mM PMSF. Around 2 mL of the kinase buffer is required for each immunoprecipitation condition.
2. Wash immunoprecipitated beads from step 4.7 twice with 500 μL of lysis buffer.
   1. Centrifuge for 2 min at 1,000 x g in a refrigerated centrifuge at 4 °C.
   2. Carefully remove supernatant, and discard it. Add 500 μL of lysis buffer. Homogenize by inversion.
   3. Repeat steps 6.2.1 and 6.2.2.
3. Wash immunoprecipitated beads three times with 500 μL of kinase buffer.
   1. Centrifuge for 2 min at 1,000 x g in a refrigerated centrifuge at 4 °C.
   2. Carefully remove supernatant, and discard it. Add 500 μL of kinase buffer. Homogenize by inversion.
   3. Repeat steps 6.3.1 and 6.3.2 twice.
4. Centrifuge for 2 min at 1,000 x g in a refrigerated centrifuge at 4 °C.
5. 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.
6. Add 40 μL of kinase buffer to the immunoprecipitated beads. Resuspend the beads by gently tapping the tube.
7. Prepare the mix for the γ[³²P] ATP labeling (final volume is 22.5 μL, complete with kinase buffer) in a safe lock-tube.
   1. Add the required volume of kinase buffer to reach a final volume of 22.5 μL.
   2. Cut the end of a 20 μL tip of a micropipette. Resuspend the immunoprecipitated beads from step 6.6 by pipetting up and down several times. Collect 10 μL of these beads into the safe-lock tube.
   3. Add ATP from a 10 μM 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 μL, to be sure the volume is correct.
   4. Add 2.5 μg of substrate (cofilin or Myelin Basic Protein, MBP in the present study case).
8. Add 5 μCi of γ[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).
9. Incubate for 20 min at 30 °C.
10. Stop the reaction with 6 μL of 5x Laemmli buffer.
11. Heat at 95° C for 5 min.
12. Centrifuge at 10,000 x g for 5 min at room temperature.
13. Load on a SDS-PAGE. Proceed to migration.
    NOTE: Take care that the front line of free γ[³²P] ATP does not exit the gel to avoid contamination of the migration tank.
14. Stain the gel at room temperature.
    1. Remove the gel from the glass plates.
    2. Proceed with three baths in water at room temperature.
    3. Stain the gel overnight with Coomassie Blue at room temperature.
    4. Destain the gel with several wash baths with water at room temperature.
15. Wrap the gel with plastic wrap.
16. Expose for one night or more on a phosphorimager screen.
17. Read the screen on a phosphorimager to detect labeled bands.

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Wyniki

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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Dyskusje

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...

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Materiały

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