All reagents were of ultrapure, metals-free grade where possible. Buffer 1 is 50 mM Tris-HCl (pH = 7.5 at 25 °C), 10 mM MgCl2, 1 mM EGTA, 2 mM DTT and 0.01% Brij-35-P. Buffer 2 is 50 mM Tris (pH = 7.5 at 25 °C), 150 mM NaCl, 50 mM β-glycerophosphate, 10 mM sodium pyrophosphate, 30 mM NaF, 1% Triton X-100, 2 mM EGTA, 100 μM Na3VO4, 1 mM DTT, protease inhibitor cocktail III (10 μL/mL, Calbiochem, 539134), and phosphatase inhibitor cocktail 1 (10 μL/mL, Sigma, P2825).
Synthesis of kinase activity probes
Sensors were synthesized and characterized as described previously (Lukovic et al., 2008
; Lukovic et al., 2009
; Stains et al., 2011
). All peptide-based sensors were acetyl-capped at the N-terminus and included a C-terminal amide. Concentrations were determined based on the Sox chromophore by measuring the absorbance at 355 nm in 0.1 M NaOH containing 1 mM Na2
EDTA (extinction coefficient = 8427 M-1
) (Lukovic et al., 2008
Cell culture and lysate preparation
HepG2 human hepatocarcinoma cells (ATCC, HB-8065) and HT-29 colorectal adenocarcinoma cells (ATCC, HTB-38) were propagated on tissue culture plastic in either Eagle's minimum essential medium (EMEM, ATCC, HepG2) or McCoy's 5a modified medium (ATCC, HT-29) supplemented with 10% FBS (Hyclone, Thermo Scientific) and 1% penicillin/streptomycin (Invitrogen) in a humidified incubator at 37 °C and 5% CO2. Prior to stimulation to activate specific kinases, HepG2 cells were plated at 1×105 cells/cm2 on collagen-I coated 6-well plates (BD Biocoat, Becton Dickinson) and HT-29 cells were plated at 1×105 cells/cm2 on tissue culture treated plastic 6-well plates or 10 cm dishes for inhibitor studies and immmunodepletions, respectively. Both cell types were grown for 24 hours in their respective full medium. Cells were then serum-starved in serum-free medium for 18 hours and dosed with either NaCl (250 mM) for 30 minutes, insulin (Sigma, I9278, 500 ng/mL) for 5 minutes, or forskolin (Cell Signaling, 3828, 30 μM) for 15 minutes. Pretreatment with the upstream kinase inhibitors SB202190 (Calbiochem, 559388), LY294002 (Calbiochem, 440204), or wortmannin (Calbiochem, 681675) was accomplished by adding the indicated molar concentrations to serum-free media 1 hr prior to stimulation. Inhibitor dilutions were prepared such that cells experienced a constant DMSO concentration of 0.1% for all dosing conditions. At the indicated time points, cells were washed with ice cold PBS and lysed on ice in Buffer 2. Lysates were incubated on ice for 15 minutes and clarified by centrifugation. Supernatants were flash frozen in liquid nitrogen and stored at -80 °C. These lysates were used to validate second generation CSox-based activity probes for MK2, Akt, and PKA. These experiments demonstrated that less sensor and cell lysate could be used to obtain robust signal-to-noise with the second generation of probes and that inhibitors could be removed from assays for MK2.
HeLa cells were propagated in DMEM (Invitrogen, 11995) supplemented with 10% heat-inactivated FBS, and 1% penicillin/streptomycin in a humidified incubator at 37 °C and 5% CO2. NIH-3T3 cells were propagated in DMEM supplemented with 10% FBS, and 1% penicillin/streptomycin in a humidified incubator at 37 °C and 5% CO2. Prior to stimulation cells were starved for 18 hours in DMEM supplemented with 2 mM L-Gln, and 1% penicillin/streptomycin. Sorbitol was then added to a final concentration of 300 mM and lysates were prepared after 1 hr as indicated above using Buffer 2. C2C12 myoblasts (ATCC, CRL-1772) were propagated in DMEM supplemented with 20% FBS and grown in a humidified incubator with a 10% CO2 atmosphere. Prior to mitogen withdrawal cells were seeded onto 150 mm collagen-I coated dishes (BD Biosciences, 354551) at ˜40% confluency. At 95% confluency (time 0) cells were switched to DMEM supplemented with 2% horse serum. This medium was replenished daily during differentiation. Lysates were prepared from two separate passages of cells at the indicated time points using the following procedure. Cells were washed with ice cold PBS and lysed on ice in Buffer 2. Lysates were clarified by centrifugation and supernatants were flash frozen in liquid nitrogen and stored at -80 °C. Total protein concentrations for all cell lysates were determined using the BioRad protein assay (500-0006) with BSA as a standard. Lysates were prepared from two separate passages of cells.
Images and videos were acquired using an Olympus IX50 inverted microscope equipped with a 40× phase contrast objective and a QImaging Retiga 2000R camera.
Hierarchical clustering analysis
Fold changes in kinase activity were determined relative to time 0 and hierarchical clustering analysis was performed in MATLAB using the built-in Bioinformatics Toolbox with the Euclidean distance metric.
HT-29 cells were propagated, plated, starved, stimulated with insulin and lysed as described above. The flash frozen lysates were thawed on ice and aliquoted in replicates of 500 μg total protein for depleted and control conditions and brought to 100 μL final volume in buffer 2. Lysates were then incubated with either 4 μg of anti-Akt/PKB PH domain 1 antibody (Millipore, 05-591) for Akt immunodepletion or 4 μg of normal mouse IgG (Santa Cruz, sc-2025) as a naïve antibody control for 2 hours at 4°C on a rotator. Samples were then incubated with 40 μL of Protein G sepharose beads (GE Healthcare, 17-0618-01) for 1 hour at 4°C on a rotator. Beads were then pelleted and supernatants were collected and subjected to two additional rounds of immunodepletion. An input control lysate was aliquoted and kept on ice during all rounds of immunodepletion to account for sample and kinase activity loss during incubation and liquid transfer steps. All lysates were then assayed for Akt kinase activity as described below.
Tissue lysate preparation
Tissues were obtained from surgical discards through the National Disease Research Interchange (NDRI) in accordance with an IRB approved protocol from the MIT Committee On the Use of Humans as Experimental Subjects. Tissue samples were flash frozen in liquid nitrogen as soon as possible (˜1 hr) after surgery. The guidelines for procurement of snap frozen tissue may vary slightly as tissues are procured from various surgical sites. Frozen tissues were dissected into ˜100 mg sections, placed in a 50 mL conical tube and washed thoroughly with ice cold PBS. Lysates were prepared by the addition of 3 volumes (in μL) of Buffer 2 per mg of tissue and subsequent homogenization on ice with an Omni tissue homogenizer equipped with a plastic homogenizing probe for hard tissues. Samples were then incubated on ice for 1 hr, followed by clarification by centrifugation. The supernatants were collected by piercing the lipid layer and were flash frozen and stored at -80 °C. Total protein concentrations were determined using the BioRad protein assay (500-0006) with BSA as the standard.
Cell and tissue lysate assays
Assays were conducted as previously described (Lukovic et al., 2009
; Stains et al., 2011
) using the following concentrations of substrates and amounts of lysate for each of the indicated kinases: p38α (1 μM substrate and 10 μg lysate), MK2 (2.5-5 μM substrate and 10-20 μg lysate), ERK1/2 (5 μM substrate and 40 μg lysate), Akt (2.5-5 μM substrate and 10-20 μg lysate), and PKA (10 μM substrate and 20 μg lysate). Inhibitors of off-target kinases were included in assays for p38α (Stains et al., 2011
) (1 μM staurosporine), Akt (Shults et al., 2005
) (4 μM PKC inhibitor peptide, 4 μM calmidazolium, and 5 μM GF109203X; control experiments demonstrated that addition of PKItide did not influence the rate of phosphorylation of the Akt sensor), and PKA (Shults et al., 2005
) (4 μM PKC inhibitor peptide, 4 μM calmidazolium, and 5 μM GF109203X). The activity of p38α was determined by background subtraction in each lysate using 1 μM SB203580 (Stains et al., 2011
). Reactions were prepared in Buffer 1 and contained 1 mM ATP, final reaction volumes were 120 μLs. Assays were performed in half-area 96-well plates (Corning, 3992), and fluorescence was monitored at 485 nm by exciting at 360 nm using a 455 nm cutoff on a Spectramax Gemini XS plate reader (Molecular Devices) at 30 °C. Slopes were determined using linear fits from Excel during the time in which fluorescence increases were linear with respect to time (typically 15 – 60 min); fits are corrected for lag times in the reaction. Assays with the direct MK2 inhibitor, MK2 Inhibitor III (Calbiochem, 475864), were conducted using NaCl-stimulated HepG2 lysates in presence of the indicated concentration of inhibitor, DMSO concentrations were 1% in the assay. Assays with the direct PKA inhibitors, H89 (Cell Signaling, 9844) and PKItide (Shults et al., 2005
), were conducted using forskolin-stimulated HepG2 lysates in presence of the indicated concentration of inhibitor, in the case of H89 DMSO concentrations were 3% in the assay. The average fold changes for biological replicates of C2C12 lysate and tissues preparations were determined from each individual assay and errors were propagated accordingly.
384-well plate assays
Assays for MK2 activity in 384-well plates (MatriCal, MP101-1-PS) were conducted in a total volume of 30 μL using 5 μM MK2 substrate and 5 μg lysate; reactions were overlaid with 20 μL of white, light mineral oil prior to recording fluorescence.
Western blot analysis
Lysates (20-100 μg total protein) were separated using 12% SDS-PAGE gels, except for myosin heavy chain which was resolved on a 4-20% gradient gel (BioRad, 161-1159). Proteins were transferred to a nitrocellulose membrane. Blots were probed with primary antibodies for myogenin (Santa Cruz Biotechnology, sc-12732), myosin heavy chain (R&D Systems, MAB4470), tubulin (Abcam, ab59680), phospho-p38 (Cell Signaling, 9215), total p38 (Cell Signaling, 9212), p38α (Cell Signaling, 9218), p38γ (Cell Signaling, 2307), phospho-ERK1/2 (Cell Signaling, 4377), total ERK1/2 (Millipore, 06-182), phospho-Akt (Cell Signaling, 9271), total Akt (Cell Signaling, 4685), or β-actin (Abcam, ab8227) which were detected using an HRP conjugated goat anti-rabbit (Pierce, 32460) or goat anti-mouse (Pierce, 32430) secondary antibody where appropriate. Blots were visualized by enhanced chemiluminescence (Pierce, 34075).