Peptide Substrates and Chemical Probes for Kinase Assays

The polypeptide substrate used for PDK1 was T308tide (KTFCGTPEYLAPEVRR; > 75% purity). The peptide substrate used for SGK and Akt was KK-Crosstide (KKGRPRTSSFAEG). Other polypeptides used were PIFtide (REPRILSEEEQEMFRDFDYIADWS), a peptide derived from the PRK2 HM (a PDK1 substrate), and substrates GS-022 (RRRQFSLRRKAK), and GS-023 (RRRQFSLRRKA-K(5-FAM). Peptides used in AlphaScreen interaction assays were biotin-PIFtide (biotin- REPRILSEEEQEMFRDFDYIADWS) and biotin-GS-022 (biotin-RRRQFSLRRKAK). Peptides were synthesized by Pepscan. PS210 was synthesized by us and characterized previously (29 (link), 30 (link)) and PS653 was from Maybridge (32 (link)). Biotin-C6-PIP₃ was from Echelon Biosciences. IP₅ (64 (link)), scyllo-IP₅ (64 (link)), 2-O-(2-aminoethyl)-IP₅ (AMR1474) (43 (link)), 2-O-butyryl-IP₅ (HYG7) (65 (link)) and 2-O-benzoyl-IP₅ (HYG8) (66 (link)) were synthesized and characterized as previously reported. IP₆ was obtained commercially (Sigma) and further purified by gradient elution from a Q-Sepharose Fast-Flow resin and isolated as the triethylammonium salt before use. The syntheses and characterization of 2-O-acetyl-IP₅ (HYG6) and 2-O-butyl-IP₅ (HYG14) are described in the Supplementary Materials. All inositol phosphates and derivatives were fully characterized by ¹H, ¹³C and ³¹P NMR spectroscopy and used as their triethylammonium salts. Antibodies were purchased from Cell Signaling (#13038, #5642) and secondary antibodies from LI-COR (926-68022 and 926-32213). AlphaScreen beads were purchased from Perkin Elmer (6760619, 6765301, 6760106). SYPRO-Orange 5000x was purchased from Invitrogen™ (S6650). Valsartan was purchased from Sigma (SML0142) and des(oxopentyl)valsartan from Carbosynth. Complete protease inhibitor cocktail tablets were from Roche. Protein concentration was determined using Bio-Rad Protein Assay Dye Reagent Concentrate (#50000069). Ni-NTA was from Jena Bioscience and glutathione sepharose resin was from GE Healthcare. SNAP-Cell® TMR-Star was purchased from NEB (#S9105). Human embryonic kidney (HEK) 293 cells (ATCC) were cultured in Dulbecco’s modified Eagle’s medium containing 10% fetal bovine serum and PenStrep. Mammalian tissue culture materials were from Greiner and JETBiofil. Insect cell expression system and all the insect cell related material were from Invitrogen (Thermo Fisher Scientific). DNA constructs used for transient transfection were purified from bacteria using a Qiagen plasmid Maxi kit.

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Sacerdoti M., Gross L.Z., Riley A.M., Zehnder K., Ghode A., Klinke S., Anand G.S., Paris K., Winkel A., Herbrand A., Godage H.Y., Cozier G.E., Süß E., Schulze J.O., Pastor-Flores D., Bollini M., Cappellari M.V., Svergun D., Gräwert M.A., Aramendia P.F., Leroux A.E., Potter B.V., Camacho C.J, & Biondi R.M. (2023). Modulation of the substrate specificity of the kinase PDK1 by distinct conformations of the full-length protein. Science signaling, 16(789), eadd3184.

Publication 2023

Antibodies Assay Bacteria Biotin Cell Cultured medium Derivatives Dna constructs Eagle Embryonic Fetal bovine serum Glutathione Human Inositol phosphates Insect Kidney Mammalian Nmr spectroscopy Pdk1 Peptides Plasmid Polypeptides Protease inhibitor Protein Ps210 Rad protein Resin Salts Sepharose Syntheses Tissue Transfection Transient Valsartan

Corresponding Organization : German Cancer Research Center

Other organizations : Fundación Instituto Leloir, Consejo Nacional de Investigaciones Científicas y Técnicas, Pennsylvania State University, European Molecular Biology Laboratory, University of Buenos Aires

Top 5 similar protocols

Variable analysis

independent variables

Peptide substrates: T308tide, KK-Crosstide, PIFtide, GS-022, GS-023, biotin-PIFtide, biotin-GS-022
Small molecules: PS210, PS653, Valsartan, des(oxopentyl)valsartan
Inositol phosphates and derivatives: IP5, scyllo-IP5, 2-O-(2-aminoethyl)-IP5 (AMR1474), 2-O-butyryl-IP5 (HYG7), 2-O-benzoyl-IP5 (HYG8), 2-O-acetyl-IP5 (HYG6), 2-O-butyl-IP5 (HYG14), IP6

dependent variables

PDK1 substrate phosphorylation
SGK and Akt substrate phosphorylation
Protein-protein interactions (measured by AlphaScreen assay)

control variables

Peptide purity (> 75%)
Protein concentration determination method (Bio-Rad Protein Assay Dye Reagent Concentrate)
Cell line (HEK 293)
Cell culture media and conditions
DNA constructs (purified from bacteria using Qiagen plasmid Maxi kit)
Antibodies (purchased from Cell Signaling and LI-COR)
AlphaScreen beads (purchased from Perkin Elmer)
SYPRO-Orange 5000x (purchased from Invitrogen™)
Protease inhibitor cocktail (Complete from Roche)
Affinity resins (Ni-NTA and glutathione sepharose)
Fluorescent dye (SNAP-Cell® TMR-Star from NEB)

positive controls

Biotin-PIFtide and biotin-GS-022 peptides used in AlphaScreen interaction assays

negative controls

Not explicitly mentioned

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