Journal: bioRxiv

Article Title: Proteome-wide screening for mitogen-activated protein kinase docking motifs and interactors

doi: 10.1101/2021.08.20.457142

Figure Lengend Snippet: (A) Domain structure of MKK6 and D-site variants. MKK6 D7 replaces the native p38-selective D-site with that from MKK7, which binds only to JNK. (A) Scheme showing the impact of replacing yeast MAPK pathway components with their human homologs on cell growth. (C) Assay showing growth inhibition of a hog1Δ pbs2Δ strain upon co-expression of p38α and the indicated MKK6 variants. Cells were grown in liquid culture, derepressed in raffinose media, and then spotted in 5-fold serial dilutions on solid media containing either galactose (to induce p38 expression) or glucose. (D) Growth assay of yeast co-expressing JNK1 and MKK6 variants conducted as in (C). (E) Scheme showing potential mechanisms for the growth rescue provided by expressing GST-D-site fusion proteins. (F) Effect of expressing cognate (MKK6, MEF2A) or non-cognate (MKK7) D-sites fused to GST on growth arrest mediated by p38α-MKK6 co-expression. Cells were grown and plated as in (C). (G) As (F) except with JNK1-MKK6 D7 co-expression along with cognate (MKK7, NFAT4) or non-cognate (MKK6) D-sites.

Article Snippet: Bacterial expression vectors for GST-JNK1 (pGEX4T1-3xFLAG-JNK1, Addgene #47574), GST-p38α, His 6 -MKK4, His 6 -MKK6 S207E/T211E (MKK6-EE), and constitutively active MEKK1 (MEKK-C) were previously reported ( , ).

Techniques: Inhibition, Expressing, Growth Assay

Journal: bioRxiv

Article Title: Proteome-wide screening for mitogen-activated protein kinase docking motifs and interactors

doi: 10.1101/2021.08.20.457142

Figure Lengend Snippet: (A) A schematic showing the workflow of the screen. (B) Waterfall plots showing the average Z-score for the enrichment/depletion rate of each D-site sequence variant from two independent screens against JNK1 (left) or p38α (right). WT MKK6 and MKK7 D-sites are highlighted. Read counts from Illumina sequencing runs are provided in Data file S1. (C) Scatter plot showing correlation of Z-scores between two replicate screens for JNK (left) and p38α (right). Blue, MKK6 variants; red, MKK7 variants. (D) Heatmaps showing the effect of each amino acid substitution to the MKK6 D-site in the p38α screen (left) and MKK7 D-site in the JNK screen (right). Values are normalized to the respective WT sequence (white), with red indicating enrichment and blue indicating depletion of the sequence from the population. Crossmarked boxes indicate sequences missing from the screening library. (E) Graph showing mean normalized enrichment scores of all double alanine substitutions to each position in the MKK7 D-site sequence in the JNK1 screen. The dotted line indicates value of the WT sequence. Error bars show SD for the 12 variants at that position. (F) Data for MKK6 double Ala variants in the p38α screen are plotted as in (E). (G) Crystal structures of JNK1 (tan) in complex with the MKK7 D-site (cyan) with the CD region and three hydrophobic pockets indicated (PDB entry 4UX9) . (H) As for panel (G), but showing p38α bound to the MKK6 D-site (PDB entry 5ETF) .

Article Snippet: Bacterial expression vectors for GST-JNK1 (pGEX4T1-3xFLAG-JNK1, Addgene #47574), GST-p38α, His 6 -MKK4, His 6 -MKK6 S207E/T211E (MKK6-EE), and constitutively active MEKK1 (MEKK-C) were previously reported ( , ).

Techniques: Sequencing, Variant Assay, Illumina Sequencing