Fig. 2: Identification of the dual specificity protein kinase MEK1 (MAP2K1) binding to MACC1 for tyrosine phosphorylation of MACC1. | Oncogene

Fig. 2: Identification of the dual specificity protein kinase MEK1 (MAP2K1) binding to MACC1 for tyrosine phosphorylation of MACC1.

From: The newly identified MEK1 tyrosine phosphorylation target MACC1 is druggable by approved MEK1 inhibitors to restrict colorectal cancer metastasis

Fig. 2

A Tandem MS-spectrum for a peptide derived from MACC1. The sequence of this peptide was verified by the presence of complementary b-ions (NH2 terminus-derived fragment ions) and y-ions (COOH terminus-derived fragment ions). B Identification of protein kinases interacting with MACC1 by quantitative proteomics. We performed two independent IPs of MACC1 from SW620 cells with high endogenous MACC1 expression employing two different MACC1 antibodies (four experiments), which were subjected to shot-gun MS. In total, 1203 proteins were identified, including 22 kinases (depicted). Highest signal intensities were measured for MEK1 (MAP2K1), a member of the dual specificity protein kinase family able to phosphorylate tyrosine and threonine residues. All other 21 kinases binding with lower intensities belong to the serine/threonine kinase family. C Validation of physical interaction of MACC1 and MEK1 was carried out in endogenously high MACC1-expressing SW620 cells (upper panel) and ectopically high MACC1-expressing SW480/MACC1-wt cells (lower panel). IP was performed for MACC1 and β-tubulin (control). Western blot was done using a MEK1 antibody. D Incubation of isolated MACC1-GFP from HEK293T cells with recombinant constitutively active MEK1 S218D S222D and γP32 ATP results in transfer of radioactive phosphate to MACC1 (each left panel). Non-labeled ATP completely out-competed labeled ATP (upper right panel). In the control reaction, cold ATP was omitted. The MEK1 inhibitor AZD6244 inhibited the transfer of radioactive phosphate to MACC1-GFP (lower right panel). In the control reaction, DMSO without inhibitor was added as solvent control. MACC1-GFP was isolated by nanotrap IP for GFP. One IP of 10-µg MACC1-GFP was incubated with 500-ng recombinant MEK1 S218D S222D at 37 °C. At indicated time points, 25% of the reaction was removed, supplemented with western blot loading buffer and boiled to stop the reaction. All samples were separated using polyacrylamide gel electrophoresis. The gels were dried and developed with imaging plates (BAS-IP MS 2340, Fujifilm, Japan). E, F To discriminate between MEK1 and MEK2, the unique peptides for each protein found during mass spectrometry were analyzed. There was a higher coverage (E) and intensity (F) for MEK1 compared to MEK2. G Predominant binding of MEK1 and not MEK2 shown by mass spectrometry analysis of peptide amounts and peptide coverage was confirmed with IPs enriching MACC1 from SW480/MACC1, SW620, and HEK293T/MACC1 cells. MEK1 and MEK2 were detected during immunoblotting with monoclonal antibodies specific for one isoform only from the same IPs.

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