Critical Assessment of Genome Interpretation

Stability and catalytic efficiency of mitogen-activated protein kinase 3

Challenge: MAPK3

Variant data: registered users only

Last updated: 12 October 2021

This challenge is closed. 

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Summary 

MAPK3 (ERK1) is active as serine/threonine kinase in the Ras-Raf-MEK-ERK signal transduction cascade that regulates cell proliferation, transcription, differentiation, and cell cycle progression. MAPK3 is activated by phosphorylation which occurs with strict specificity by MEK1/2 on Thr202 and Tyr204, and may also act as a transcriptional repressor independent of its kinase activity. A library of twelve missense variants selected from the COSMIC database was assessed by near and far-UV circular dichroism and intrinsic fluorescence spectra to determine thermodynamic stability at different concentrations of denaturant. These data were used to calculate a ΔΔG_H20 value; i.e., the difference in unfolding free energy ΔG_H20 between each variant and the wildtype protein, both in phosphorylated and unphosphorylated forms. The challenge is to predict these two ΔΔG_H20 values and the catalytic efficiency (kcat/km)^mut/(kcat/km)^wt, as determined by a fluorescence assay, of the phosphorylated form for each MAPK3 variant.

Background 

Human MAP kinases (Mitogen-activated protein kinases), also known as extracellular signal-regulated kinases (ERKs), are proteins involved in the integration of multiple biochemical signals and in several cellular processes ranging from transcription regulation and development, to differentiation and proliferation (Lavoie et al., 2020). MAPK3 is activated through its phosphorylation by upstream kinases (Roskoski, 2012). Upon activation, MAPK3 translocates to the nucleus to phosphorylate specific nuclear targets. Independently of its kinase activity, MAPK3 may act as a transcriptional repressor and has been identified as a moonlighting protein based on these multiple distinct functions (Lu et al., 2018).

These functions may vary depending on the cellular context (Raman et al., 2007) and include cell growth, adhesion, survival and differentiation through transcriptional regulation, translation, and cytoskeletal rearrangements. Around 160 putative protein substrates have been described for MAP kinases. The substrates localized in the nucleus seem to be responsible for the stimulation and regulation of transcription, while the substrates localized in cytosol and organelles are responsible for translation, mitosis and apoptosis and may take part in the checkpoint of spindle assembly. Owing to their biological importance, protein kinases represent an important target of biomedical research and of a large part of drug discovery research (Moret et al., 2020 ).

The single amino acid variants included in the MAPK3 challenge were selected from the hundreds of variants with high mutation rates detected in tumor samples reported in the COSMIC (Catalog of Somatic Mutations in Cancer) database. These are somatic variants distributed along the protein sequence which could additionally be expressed as a soluble recombinant protein in E. coli.

Experiment 

Unphosphorylated human MAPK3 (ERK1) variants were  obtained with specific mutagenesis primers by PCR, using wildtype protein as a template. Wildtype proteins and mutants are then expressed in E. coli cells Rosetta with phosphatase and purified by affinity chromatography by a 2‐ml prepacked His trap column (GE Healthcare, Chicago, IL).      

The phosphorylated (active) form of MAPK3 wildtype and variants were obtained by using the plasmid pGEX-KG-MEKR4F (the active mutant of MEK1) plasmid (kindly provided by Professor M. Cobb from Southwestern University, TX, USA) for the co-expression of the wildtype and the mutants. MAPK3 wildtype and variants were expressed as N-terminally His-tagged proteins in E. coli cells BL21(DE3) and purified by ion exchange and affinity chromatography by a 2‐ml prepacked His trap column (GE Healthcare, Chicago, IL) .

This protocol introduces heterogeneity in the protein mixture, with up to one or two more phosphorylation sites being potentially generated, in addition to the two canonical sites (Thr202, Tyr204). To check the MAPK3 phosphorylation, we performed western blot analysis with the Antibody cat. #44-680G in IHC (Invitrogen). 

The thermodynamic stability was measured at increasing denaturant concentration by monitoring the spectral changes (far-UV circular dichroism and intrinsic fluorescence emission) induced by the denaturant. The variation of unfolding free energy (ΔG) is calculated fitting the spectral changes at zero denaturant concentration (Walters et al., 2009) (ΔG_H2O). The data are the average of at least three replicates on different purification batches. 

The catalytic activity of the MAPK3 wild-type and variants is determined by a fluorescence assay as described in Shults et al. (2003, 2005, 2006). This method is based on Chelation-Enhanced Fluorescence (ChEF) upon substrate peptide phosphorylation by the kinase.

Prediction challenge

For each variant, participants are asked to predict the ΔΔG_H20 value for the phosphorylated and unphosphorylated forms.  The ΔΔG_H20 is the difference in unfolding free energy (ΔG_H20) between the mutant and wildtype proteins, in kcal/mol. Furthermore, the prediction of the ratio between the catalytic efficiency [r_kcat/km = (kcat/km)^mut/(kcat/km)^wt] of the mutant and the wildtype.

The experimental free energy change values (ΔG_H20) of the wildtype protein in phosphorylated and unphosphorylated forms fitted with a two and three-states curves are the following: