Cell lines and cell culture
KBM7 cells (obtained from T. Brummelkamp) and KBM7 iCas9 cells (a gift from J. Zuber) were grown in IMDM (Thermo Fisher Scientific) supplemented with 10% heat-inactivated FBS (Sigma-Aldrich) and 1% penicillin–streptomycin (Gibco). RKO iCas9-GFP and iCas9-BFP (gifted by J. Zuber), K562 (purchased from ATCC) and NALM-6 (obtained from A. Villunger) cells were cultured in RPMI 1640 (Thermo Fisher Scientific) supplemented with 10% FBS and 1% penicillin–streptomycin. HEK293T lentiviral packaging cells (obtained from Clontech), HEK293T (purchased from ATCC) and Flp-In T-REx 293 (obtained from Invitrogen) cells were cultured in DMEM (Thermo Fisher Scientific) supplemented with 10% FBS and 1% penicillin–streptomycin.
For competitive Kinobead pull-downs, Jurkat, MCF7, K562, COLO-205 and MV-4-11 cells were cultured in RPMI 1640 medium (Biochrom) supplemented with 10% (v/v) FBS (Biochrom). SK-N-BE(2) cells were grown in DMEM/Ham’s F-12 (1:1) supplemented with 10% (v/v) FBS and OVCAR-8 cells were cultured in IMDM medium (Biochrom) supplemented with 10% (v/v) FBS.
Cell lines were cultured at 37 °C and 5% CO 2 in a humidified incubator and were regularly tested for mycoplasma contamination.
Plasmids and cloning
All plasmid preparation, unless specified otherwise, was performed in stable competent Escherichia coli (NEB) or, in the case of destination vectors, in One Shot ccdB Survival 2 T1R Competent Cells (Invitrogen) according to the manufacturer’s instructions.
The pLEX305-ccdB-Nluc-3×Flag luminescent reporter vector was generated as a destination vector starting from pLEX_305-ccdB-dTAG destination vector (Addgene, 91798) by restriction digest with AgeI and MluI and T4 DNA ligation (NEB) of a synthesized gene block (TWIST) containing the Nluc sequence and a C-terminal 3×Flag tag (5′-CGGGCAAAACCGGTGTCTTCACACTCGAAGATTTCGTTGGGGACTGGCGACAGACAGCCGGCTACAACCTGGACCAAGTCCTTGAACAGGGAGGTGTGTCCAGTTTGTTTCAGAATCTCGGGGTGTCCGTAACTCCGATCCAAAGGATTGTCCTGAGCGGTGAAAATGGGCTGAAGATCGACATCCATGTCATCATCCCGTATGAAGGTCTGAGCGGCGACCAAATGGGCCAGATCGAAAAAATTTTTAAGGTGGTGTACCCTGTGGATGATCATCACTTTAAGGTGATCCTGCACTATGGCACACTGGTAATCGACGGGGTTACGCCGAACATGATCGACTATTTCGGACGGCCGTATGAAGGCATCGCCGTGTTCGACGGCAAAAAGATCACTGTAACAGGGACCCTGTGGAACGGCAACAAAATTATCGACGAGCGCCTGATCAACCCCGACGGCTCCCTGCTGTTCCGAGTAACCATCAACGGAGTGACCGGCTGGCGGCTGTGCGAACGCATTCTGGCGGACTACAAGGACCACGACGGTGACTACAAGGACCACGACATCGACTACAAGGACGACGACGACAAGTAGTAAACGCGTTGACGATGG-3′).
To generate a destabilized version of the vector, an identical gene block was synthesized with the addition of the PEST sequence (Promega) 5′-AATTCTCACGGCTTTCCGCCTGAGGTTGAAGAGCAAGCCGCCGGTACATTGCCTATGTCCTGCGCACAAGAAAGCGGTATGGACCGGCACCCAGCCGCTTGTGCTTCAGCTCGATCAACGTC-3′ upstream of the stop codon. pENTR223 Gateway entry vectors for the kinases were obtained from Hahn/Root Labs Human Kinases ORF Kit59,60 (Addgene Kit, 1000000014) with the exception of FYN and MAPK4, which were purchased separately (BCCM, LMBP ORF81088-E05, LMBP ORF81100-B12). pRK5-HA-ubiquitin, pRK5-HA-ubiquitin_K48R and pRK5-HA-ubiquitin_K63R plasmids were provided by G. Versteeg. A pENTR221-GFP vector was generated by BP Gateway cloning (Invitrogen), starting from the PCR-amplified GFP sequence of pCAG-GFP61 (gifted by C. Cepko, Addgene, 11150) and insertion into the empty pDONR221 (Invitrogen, 12536017). Final luminescent reporter vectors were generated by LR Gateway cloning according to the manufacturer’s recommendations (Invitrogen, incubation was routinely run overnight at 25 °C before heat inactivation and transformation). The correct insert size was assessed by analytical digest and in-frame cloning was verified by sequencing (Microsynth).
Single point mutations, with the exception of BLK S5A and S6A, were generated from the respective pENTR223 plasmids using either the Q5 site-directed mutagenesis kit (primers are shown in Supplementary Table 1, method, SDM, NEB) or by Q5 (NEB) PCR amplification (primers are shown in Supplementary Table 1, method, PCR), followed by 1 h of DpnI digest (NEB) and direct transformation into DH5α E. coli (NEB).
Stability vectors were generated by digesting the previously published plasmid backbone pRRL_SFFV_empty_BFP_P2A_mCherry29 with SalI and BamHI, before insertion of the PCR-amplified kinase of interest using the NEBuilder HiFi DNA Assembly Master mix (NEB) according to the manufacturer’s instructions. BLK G2I, G2L, L3A, L3G, L3V, V4S, S5A and S6A stability reporter plasmids were generated analogously using the corresponding mutated primer pairs. Vectors for domain-swap experiments, truncated versions of the BLK stability reporter (with the exception of 1–7) and the ABL1(C464W) stability reporter were generated similarly by amplifying the respective DNA sequences from each kinase and performing two- or three-part assemblies. For the aforementioned truncated BLK version 1–7, oligos (Supplementary Table 3) were annealed, phosphorylated and ligated into the corresponding vector. Primers were designed using the NEBuilder Assembly tool (the sequences are provided in Supplementary Table 2).
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