Computational protocol: A novel reporter for real-time, quantitative imaging of AKT-directed K63-poly-ubiquitination in living cells

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[…] This reporter consists of a K63-linkage specific polyubiquitination target sequence of AKT (amino acid 2-19 of the PH domain harboring Lys8 and Lys14) [, , ]. Based on the fact that the selected short peptide of AKT is very specific and is present only in AKT1 (Entrez BLAST search), short peptides can be ubiquitinated in vitro [], and have surrogated for endogenous proteins in kinase reporters [, ], we chose this sequence for construction of the reporter. Several K63-linkage specific tandem ubiquitin interaction motif (tUIMs) with varying degrees of selectivity have been identified [–]. The tUIM with multiple NZF are known to possess remarkable preference for binding to K63-linked polyubiquitin chains []. We used ubiquitin interaction motifs (tUIM) from RAN-binding domain containing 1 (ZRANB1/Trabid; aa 2-200) containing three tandem Npl4 related zinc finger (NZF) motifs for construction of the reporter. These sequences are flanked by the N-terminus of luciferase (N-Luc, 4-354) at the carboxyl-terminus of the reporter while the C-terminus of luciferase (C-Luc, aa 358-544) is at the amino-terminus of the reporter. A c-DNA plasmid for ZRANB1 was purchased from Origin Technologies (pEZ-M12-ZRANB1, EX-H4597-M12). A seven amino acid long poly Alanine linker shown to increase the specificity of the synthetic UBD to K63-linkage specific chains [] was inserted between the UBD and the AKT substrate peptide sequence. The BTR reporter [] was re-cloned into the plasmid harboring the above mentioned luciferase fragments and was opened with NotI-HF and XmaI and cipped using calf intestinal alkaline phosphatase (New England Biolabs). The PCR fragment containing the linkers, NZF domain, poly A linker, AKT substrate peptide was also digested with NotI-HF and XmaI and ligated using the Quick ligation kit (Roche) and transformed into XL-10 Gold cells (Invitrogen) and spread on agar plates containing ampicillin (50 µg/mL). The following day eight colonies were picked, confirmed by restriction digestions, and by sequencing. The mutant reporter, K63UbR-MUT, was generated by substituting both Lysins within the PH domain with arginine (K8R, K14R) using a single primer mutagenesis protocol, with minor modifications as described earlier. The reporter plasmids will be freely available to researchers upon request to the corresponding authors.Initial model of the chimeric reporter was built using the I-TASSER server []. Manual modeling operations were performed in Coot [] and molecular graphics were generated in Pymol [] [Available at:]. [...] The in vitro ubiquitination sample of the WT reporter (K63UbR WT) was run on 4-12% Bis-Tis gel in clean conditions and stained using the MASS Spec compatible SimplyBlue SafeStain (Invitrogen). The protein samples were processed at the Proteomics Resource Facility of the Department of Pathology at the University of Michigan. Gel slice corresponding to (K63UbR) was destained with 30% methanol for 4 h. Upon reduction (10 mM DTT) and alklylation (65 mM 2-Chloroacetamide) of the cysteines, proteins were digested overnight with sequencing grade, modified trypsin (Promega). Resulting peptides were resolved on a nano-capillary reverse phase column (Acclaim PepMap C18, 2 micron, 25 cm, ThermoScientific) using a 1% acetic acid/acetonitrile gradient at 300 nl/min and directly introduced in to Orbitrap Fusion tribrid mass spectrometer (Thermo Scientific, San Jose, CA). MS1 scans were acquired at 120 K resolution. Data-dependent high-energy C-trap dissociation MS/MS spectra were acquired with top speed option (3 sec) following each MS1 scan (relative CE ∼32%). Proteins were identified by searching the MS/MS spectra against human protein database (UniProtKB; 4-16-2015) appended with chimeric K63UbR sequence using Proteome Discoverer (v2.1, Thermo Scientific). Search parameters included MS1 mass tolerance of 20 ppm and fragment tolerance of 0.2 Da; two missed cleavages were allowed; carbamidimethylation of cysteine was considered fixed modification and oxidation of methionine, di-glycine remnant on lysine were considered as potential modifications. Percolator algorithm was used for discriminating between correct and incorrect identifications. Proteins/peptides that were identified with <1% false discovery rate (FDR) were retained. […]

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