Human dihydrofolate reductase is a substrate of protein kinase CK2α
Dihydrofolate reductase (DHFR) is a prominent molecular target in antitumor, antibacterial and antiprotozoan chemotherapies. Our in silico amino acid sequence and 3D structure analyses revealed the presence of several putative CK2 phosphorylation sites. Indeed, CK2α subunit phosphorylated DHFR in vitro. In order to identify phosphorylation site we used site-directed mutagenesis to obtain several DHFR mutants with predicted CK2-phosphorylable serine or threonine residues substituted with alanines. All enzyme forms were subjected to in vitro phosphorylation by CK2α subunit. The results pointed to serine 168. Mass spectrometry analyses revealed the presence of additional phosphoserine 145. Phosphorylation by CK2α of S145A mutant and lack of phosphorylation of S145A/S168A double mutant may indicate that S145 phosphorylation may occur only when serine 168 is already phosphorylated. The effect of these and other mutations on enzyme catalytic activity was also investigated.
Sample Processing Protocol
Homogenous wtDHFR was CK2- phosphorylated in reaction mixture containing 36.0 µM DHFR, 15 mM Tris- HCl pH=7,5, 15 mM MgCl2, 200 mM ATP. The reaction was initiated by addition of 8.1 nM CK2α subunit. The reaction was conducted at 37oC for 0-60 min and terminated by placing the samples on ice. Phosphorylated and unphosphorylated DHFR was subjected to MS analysis either as sample in solution or as band sample excised from the gel after resolving by SDS-PAGE. Proteins in gel bands were reduced with 100 mM DTT (for 30 minutes at 57°C), alkylated with 0.5 M iodoacetamide (45 min in a dark at room temperature) and digested overnight in 37°C with 10 ng/μl of trypsin in 25mM NH4HCO3 (sequencing Grade Modified Trypsin Promega V5111) by adding the enzyme directly to the reaction mixture. Peptides were eluted with 2% acetonitrile in presence of 0.1% TFA. The resulting peptide mixtures were applied to RP-18 pre-column (Waters, Milford, MA) using water containing 0.1% FA as a mobile phase and then transferred to a nano-HPLC RP-18 column (internal diameter 75 µM, Waters, Milford MA) using ACN gradient (0 – 35% ACN in 180 min) in the presence of 0.1% FA at a flow rate of 250 nl/min. The column outlet was coupled directly to the ion source of Orbitrap Velos (for band sample) or Q Exactive (for in solution sample) mass spectrometer (Thermo Electron Corp.,San Jose, CA) working in the regime of data-dependent MS to MS/MS switch and data were acquired in the m/z range of 300–2000. A three blank run ensuring absence of cross-contamination from previous samples preceded each analysis. Samples were measured in given sequence: first not phosphorylated sample then phosphorylated one.
Data Processing Protocol
Analysis of mass spectrometry data The acquired MS/MS data were preprocessed with MaxQuant softwere 220.127.116.11 and a search against the home made protein database (19.07.2017, 73112 sequences). The Mascot search parameters were as follows: mass tolerance for peptides: 20 ppm, mass tolerance for fragments: 0.1 Da, enzyme: Trypsin; missed cleavages: 2; variable modifications: Oxidation (M) Phosphorylation (STY); instrument: HCD; monoisotopic.
Dominik Cysewski, Institute of Biochemistry and Biophysics
Polish Academy of Sciences
Joanna Cieśla, Warsaw University of Technology, Faculty of Chemistry, 00-664 Warszawa, Noakowskiego 3, Poland ( lab head )
Skierka K, Wilamowski P, Wielechowska M, Cysewski D, Senkara E, Wińska P, Bretner M, Cieśla J. Human dihydrofolate reductase is a substrate of protein kinase CK2α. Biochem Biophys Res Commun. 2019 PubMed: 30961929