All here generated material is freely available from the authors upon request including the vectors pHGGWA-CYCD3;1, pHMGWA-SDS, pHMGWA-CYCB1;2, pCDFDuet-StrepIII-CDKA;1-GST-Cak1, pCDFDuet-StrepIII-CDKB1;1-GST-Cak1, pCDFDuet-StrepIII-CDKB2;2-GST-Cak1, and pCDFDuet-StrepIII-CDKA;1-GST-CDKF;1 (Table1).
All manipulations were performed according standard molecular biological procedures. The sequences of all primers used in this study can be found in Additional file2: Table S1.
StrepIII-CDKA;1 - CDKB1;1 - CDKB2;2
To obtain Strep-tag III (StrepIII) sequence, oligo nucleotides HH309 and HH310, HH311 and HH312, respectively, were annealed in 1X NEBuffer 3 (NEB) and phosphorylated by T4 polynucleotide kinase in 1X T4 DNA ligase buffer (Thermo scientific) and ligated into Xho I - Hind III sites of pBluescript SK + (Stratagene). To conjugate attB1 recombination site and TEV protease recognition sequence at 5′ and 3′, respectively, of StrepIII, PCR was performed with primers HH323 and HH324. TEV protease recognition sequence and attB2 recombination site were introduced at 5′ and 3′, respectively, of CDKA;1 by PCR with primers HH266 and HH268. The resulting PCR products were fused by PCR with primers HH323 and HH95, then recombined into pDONR223 by using LR Clonase II (Invitrogen) to obtain pDNOR223-StrepIII-CDKA;1. The same strategies were taken to get pDNOR223-StrepIII-CDKB1;1 and pDNOR223-StrepIII-CDKB2;2.
GST-Cak1 was amplified by sequential PCR using pGEX3C-cdk2-GST-Cak1 as a template. First, to inactivate the Nco I recognition site in Cak1, GST-Cak1 was amplified into two parts with primers HH393 and HH376 and primers HH383 and HH394 (Table1). To fuse the two PCR products, PCR was performed with primers HH383 and HH376, and the products were digested with Bgl II and Xho I and ligated into Bgl II - Xho I sites of pCDFDuet-1 (Novagen) to obtain pCDFDuet-Cak1. To conjugate GST-tag to CDKF;1, GST and CDKF;1 cDNAs were amplified with primers HH667 and HH670, HH669 and HH668, respectively. The resulting PCR products were fused by PCR with primers HH667 and HH668. PCR products were digested with Nde I and Xho I and ligated into Nde I - Xho I sites of pCDFDuet-1 to obtain pCDFDuet-CDKF;1. A StrepIII-tagged CDKA;1, CDKB1;1 or CDKB2;2 linked by a TEV protease recognition sequence were amplified by primers HH395 and HH396, HH395 and HH397 or HH398 and HH401, respectively. For CDKA;1 and CDKB1;1, PCR products were digested with Nco I and Not I and ligated into Nco I - Not I sites of pCDFDuet-Cak1 and pCDFDuet-CDKF;1; for CDKB2;2, PCR products were cloned into Nco I - Not I sites of pCDFDuet-Cak1 and pCDFDuet-CDKF;1 by SLIC.
HisGST-CYCD3;1, HisMBP-SDS, CYCB1;2
pDONR221-CYCD3;1 was kindly provided by Lieven De Veylder. SDS cDNA was kindly provided by Petra Bulankova and Karel Riha. To introduce attB recombination sites to SDS, PCR was performed with primers HH509 and HH510, then HH347 and HH348, sequentially. To introduce attB recombination sites to CYCB1;2, PCR was performed with primers HH339 and HH340, then HH347 and HH348, sequentially on the CYCB1;2 cDNA[43, 44] The resulting PCR products were recombined into pDNOR223 and then recombined into a destination vector pHGGWA or pHMGWA by using Gateway technology (Invitrogen) to fuse a HisGST- or HisMBP-tag, respectively.
pDNOR201-Wee1 and pDNOR201-CDKF;1 were kindly provided by Annika K. Weimer and Stefan Pusch, respectively. Recombination reaction were performed between the entry clone and a destination vector pHGGWA to fuse a HisGST-tag, resulting in pHGGWA-Wee1 and pHGGWA-CDKF;1.
To express and purify GST-AtRBR1-His6, first pGEX-4 T-1 (GE Healthcare) was amplified with primers HH294 and HH295 and self-ligated to introduce a hexa-histidine sequence (pGEX-His
). A full-length AtRBR1 was amplified with primers HH194 and HH293. PCR products were digested with Bam HI and Xho I, and ligated into Bam HI - Xho I sites of pGEX-His
pDNOR201-KRP6 were kindly provided by Stefan Pusch. To express and purify HisGST-KRP6, a recombination reaction were performed between the entry clone and a destination vector pHGGWA to fuse a HisGST-tag, resulting in pHGGWA-KRP6.
Phosphorylation of CDKA;1 in E. Coli
CDKA;1 proteins were produced in E. coli co-expressing either GST-fused Wee1, Cak1 or CDKF;1. The two proteins were co-expressed by transforming an E. coli SoluBL21 strain (AMS Biotechnology) with plasmids, pCDFDuet-CDKA;1 and pHGGWA-Wee1 or pHGGWA-CDKF;1; for co-expression of CDKA;1 and Cak1, pCDFDuet-CDKA;1-Cak1 was used. Cells were grown in LB medium containing appropriate antibiotics to OD600 of 0.6 at 37°C. The production of proteins was induced by adding IPTG to 1 mM, cells were then cultured for 3 h at 37°C before being harvested by centrifugation. Cell pellets were boiled in 1X SDS-PAGE sample buffer (62.5 mM Tris-Cl, pH 6.8, 2%(w/v) SDS, 10%(v/v) glycerol, 1% ß-mercaptoethanol, 0.005% bromophenol blue) and the supernatant were conducted to the protein blotting. After separating the proteins on a 10% SDS-PAGE gel (SDS-PAGE running buffer (25 mM Tris, 192 mM glycine, 0.1%(w/v) SDS)), proteins were transferred onto a polyvinylidenfluorid membrane (Millipore) in a modified towbin buffer (SDS-PAGE running buffer containing 15%(v/v) methanol) with a wet blotting system (Bio-rad). To detect pTyr15 and pThr161, membrane was blocked with protein-free T20 (TBS) blocking buffer (Thermo scientific) and probed with a 1:5000 and 1:2000 dilution of phospho-cdc2 (Tyr15) (10A11) (Cell signaling) and phospho-cdc2 (Thr161) (Cell signaling), respectively as primary antibodies in protein-free T20 (TBS) blocking buffer and a 1:100000 dilution of horseradish peroxidase-conjugated anti-rabbit antibody (GE Healthcare) as a secondary antibody in 1%(w/v) non-fat dry milk in TBST. To detect StrepIII- and GST-tagged proteins, membrane was blocked with 5%(w/v) non-fat dry milk in TBST and probed with a 1:250000 and 1:20000 dilution of Strep-Tactin HRP and anti-GST HRP (GE Healthcare), respectively. In the case of StrepIII-tagged protein detection, biotin blocking buffer (IBA) was added at 1:1000 dilution prior to the Sterp-Tactin HRP incubation. Enhanced chemoluminescent detection was performed with HRP substrate (Millipore).
Preparation of cyclin/CDK complexes in E. Coli
CDK-cyclin complexes were produced in E. coli co-expressing GST-fused Saccharomyces cerevisiae Cak1. All three proteins were co-expressed by transforming an E. coli SoluBL21 strain with the two plasmids, pCDFDuet-1, containing a StrepIII-CDK and GST-Cak1, and pHGGWA or pHMGWA, containing the respective cyclin. Cells were grown in 50 ml of LB medium containing 50 μg/ml ampicillin and 50 μg/ml spectinomycin at 37°C to OD600 of 0.6, and incubated for another 30 min at 18°C. The production of proteins was induced by adding IPTG to 0.3 mM, cells were then cultured overnight at 18°C before being harvested by centrifugation.
The cell pellet was re-suspended in 2.5 ml Ni-NTA binding buffer (50 mM NaH2PO4, 100 mM NaCl, 10%(v/v) glycerol, 25 mM imidazole, pH 8.0.) and lysed by sonication. After addition of Triton X-100 to 0.2%(w/v), the cell slurry was incubated with gentle agitation for 20 min at 4°C then centrifuged at 10,000 × g for 40 min at 4°C. The supernatant was passed through a 0.45 μm filter. The cleared lysates were applied onto a Econopack column (Bio-rad) packed with 300-μl Ni-NTA resin (Qiagen), which was washed sequentially with 3 ml Ni-NTA binding buffer, and eluted with 600 μl Ni-NTA elution buffer (50 mM NaH2PO4, 100 mM NaCl, 10%(v/v) glycerol, 225 mM imidazole, pH 8.0.). The buffer was exchanged to kinase buffer (50 mM Tris–HCl, pH 7.5, 10 mM MgCl2, 1 mM EGTA) containing protease inhibitors cocktail (Roche) and Phos-STOP (Roche) with a PD MiniTrap G-25 column (GE Healthcare). Purified kinases were frozen in liquid N2 and stored at −80°C until use.
Preparation of the substrates
Histone H1 was purchased from Millipore. E. coli BL21-AI (Invitrogen) cells were transformed with the plasmid, pGEX-AtRBR1-His
or pHGGWA-KRP6 and grown until OD600 = 0.6 at 37°C. The culture was transferred to 18°C and grown for 30 min. The production of the fusion protein was induced by adding 0.3 mM IPTG and 0.2%(w/v) L-arabinose overnight at 18°C. Cells were harvested by centrifugation and re-suspended in phosphate-buffered saline (PBS) buffer (140 mM NaCl, 2.7 mM KCl, 10.1 mM Na2HPO4, 1.8 mM KH2PO4, pH 7.3), and lysed by sonication. After addition of Triton X-100 to 0.2%(w/v), the cell slurry was incubated at 4°C then clarified by centrifugation. The supernatant was passed through a column packed with Glutathione-agarose (Sigma), which was washed sequentially with PBS, and eluted with GST elution buffer (50 mM Tris–HCl, pH 8.0, 10 mM Glutathione). The eluate was sequentially purified with a column packed with Ni-NTA resin. GST-AtRBR1-His6 was eluted with Ni-NTA elution buffer and the buffer was exchanged to kinase buffer with a PD-10 column (GE Healthcare). Substrates were aliquoted and frozen in liquid N2 and stored at −80°C until use.
CDK-cyclin complexes were processed for kinase assays as described previously with Histone H1, GST-AtRBR1-His6 or GST-KRP6 as a substrate. Loading of equal amounts of CDKA;1, CDKB1;1 and CDKB2;2 was assured by quantifying them by western blot with Strep-Tactin HRP.