Screening of protein kinase candidates in silico and in vitro

Screening of protein kinase candidates in silico and in vitro 

I. In silico screen:

The recognition sequence for phosphorylation varies from kinase to kinase and contains a variable number of amino acids. These can be located both before and after the phosphorylated amino acid. The type of amino acids required for recognition also varies greatly. The in silico analysis was used to generate an overview of kinases predicted to phosphorylate Su(H) at S269. To this end we applied the GPS 3.0 software programme, which has an extensive database containing a large number of human kinases and recognition motifs (Xue et al., 2011; PMID: 21062758). Please note that there are more recent databases like GPS 6.0 software (Chen et al., 2023; PMID: 37158278) or the atlas of substrate specificities for the human Ser/Thr kinome (Johnson et al., 2023; PMID: 36631611), which should be used preferentially nowadays.

Simplified, this simulation results in a ‘score’ and a ‘cut-off’ value, which have been specified for each human kinase. The ‘score’ indicates the likelihood that the kinase in question will phosphorylate the site. The ‘cut-off’ value indicates the threshold below which phosphorylation of the site under investigation is unlikely. The higher the ‘score’ and the greater the difference between the ‘score’ and the ‘cut-off’ value, the more likely it is that the analysed kinase can phosphorylate the substrate in vitro. Although the prediction is always based on an experimentally confirmed phosphorylation, it does not automatically equate to a phosphorylation in vivo. Furthermore, an excluded site can serve as a substrate for a given kinase in vivo. In addition, both the ‘cut-off’ value and the calculated ‘score’ for the site under investigation are always specific to a particular kinase. For this reason, a comparison of the ‘scores’ of different kinases is not allowed. 

We used the GPS3.0 software at the lowest threshold levels, concentrating subsequently on the 40 kinases with the highest difference between score and cut-off value. The corresponding 30 Drosophila kinases were determined with the help of flybase according to (Morrison et al., 2000; PMID: 10908587).

II. In vitro screen:

We aimed at screening a set of human Ser/Thr kinases provided by ProQinase GmbH (Freiburg, Germany) using theSu(H) beta-trefoil domain BTD as substrate. To this end, we expressed and purified the BTD polypeptide to be send out for the ‘KinaseFinder assay service’.

(A) Cloning of a GST-BTD expression construct

A 525 bp cDNA fragment encoding the Su(H) beta-trefoil domain (corresponding to codons 247-414) was PCR-amplified using PCR primers Su(H) BTD_UP (5’AAG GGA TCC TCG CTA AAG AAT GCC GAT CTG TG 3’) and Su(H) BTD_LP (5’CAT GAA TTC TCA GAA CTG GTA CTC AGC CTT GTC GG 3’), respectively, at 52.5°C annealing temperature. The primers delivered a BamHI at the 5’ end and an EcoRI at the 3’ end (bold in sequences above), allowing to clone the PCR product upon respective digests into pGEX-2T vector (Smith and Johnson, 1988; PMID: 3047011) for bacterial expression of the GST-BTD fusion protein from transformed E. coli UT580.

(B) BTD-GST protein expression and purification

Proteins were purified for use in kinase assays using a GST tag from E. coli UT580. E. coli UT580 cells transformed with BTD-pGEX-2T were inoculated in 50-ml of LB-Amp medium and incubated overnight at 37°C and 210 rpm. The overnight culture was transferred to 450 ml of LB-Amp medium and incubated until an OD₅₈₀ of 0.5 at 37°C and 210 rpm. Protein expression was induced by adding 1 mM IPTG (Isopropyl β-D-1-thiogalactopyranoside; Carl Roth GmbH, Karlsruhe). The culture was then incubated overnight at 18°C and 210 rpm. 

Purification was performed using Glutathione Sepharose 4B (GE Healthcare, Little Chalfont, United Kingdom) according to standard protocols (Harper and Speicher, 2013; PMID: 20978970). Firstly, 2 ml of the Sepharose was rehydrated in 20 ml of MT-PBS (150 mM NaCl; 16 mM Na₂HPO₄; 16 mM NaH₂PO₄) for one hour. The Sepharose beads were then centrifuged at 4°C, 3000 rpm for one minute, washed twice with MT-PBS, and subsequently stored refrigerated in 2 mL of MT-PBS. The overnight culture was centrifuged in large centrifuge tubes at 4°C, 5000 rpm for 10 minutes. The sediment was resuspended in 10 ml of ice-cold MT-PBS, and a protease inhibitor tablet (Roche Diagnostics, Mannheim, Germany) was added. Cell lysis was performed with a French Press OneShot 20 KPSI (I&L Biosystems, Königswinter, Germany) cell disruptor at 1.35 bar. The resulting cell lysate was transferred to a 30 ml glass tube containing 1 ml of 10% Triton X-100, mixed and centrifuged for five minutes at 4°C and 10,000 rpm. The supernatant was added to the prepared Sepharose beads and incubated for 45 minutes at 8°C on a shaker. The beads were then washed three times with 40 ml of MT-PBS; each wash being separated by a centrifuge step at 4°C at 3.000 rpm for one minute. Eventually, the sediment was resuspended in 10 ml of MT-PBS and loaded onto the empty Poly-Prep disposable plastic columns (Bio-Rad GmbH, Feldkirchen; 7311550EDU). Once the Sepharose had settled, the column was washed with 10 ml of MT-PBS, and the fusion protein was then eluted with 10 ml of a 5 mM glutathione solution (Carl Roth GmbH, Karlsruhe) in 50 mM Tris-HCl (pH 8.0). The eluate containing the GST-BTD peptide was collected in 500 μl fractions and stored at -20°C until further use. Protein content and quality of the fractions were tested by using an aliquot on a PAGE. Protein concentration was determined by a Bradford assay (Bio-Rad GmbH, Feldkirchen; 5000006)

(C) KinaseFinder assay

Kinase assays were performed by ProQinase, Freiburg (now: Reaction Biology Europe, see: https://www.reactionbiology.com). To obtain the required 3 ml of a 2 mg/ml solution of the purified GST-BTD fusion protein several fractions of eluate were combined and concentrated by dialysis to the final concentration of approximately 2 mg/ml in 50 mM HEPES (pH 7.5) (Carl Roth GmbH, Karlsruhe). Shipment was on dry ice. ProQinase then performed a kinase activity assay using 245 human Ser/Thr kinases in 96-well plates using the buffer corresponding to each kinase and [³³γ-P]-ATP for one hour at 30°C. The reaction was then stopped with 10% H₃PO₄ solution, and the reaction mixture was transferred to a 96-well filter plate, dried, and washed three times with 150 mM H₃PO₄, followed by a wash step with 100% ethanol. After drying the plates for 30 minutes at 40°C, 50 μl of a scintillator was added to each well, and the incorporation of ³³Pi (counts per minute) was read using a microplate scintillation reader. 

Potential kinases using BTD as substrate were identified the following way: first kinase activity (A) was determined, second background activity (B) subtracted (i.e. the mean value of two measurements of the substrate without kinase), and third the value divided by the mean value of autophosphorylation (C) of the respective kinase. A ratio value >1 (A-B/C) was considered as significant.