Research ArticleBiochemistry

Engineering allosteric regulation in protein kinases

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Science Signaling  06 Nov 2018:
Vol. 11, Issue 555, eaar3250
DOI: 10.1126/scisignal.aar3250
  • Fig. 1 Regulatory diversity in the eukaryotic protein kinases.

    (A) Unanchored dendrogram of the human kinome, illustrating the diversity of the eukaryotic protein kinase superfamily and subfamilies. Individual subfamily members with functional mutations shown in a subsequent figure and included in table S7 are listed. TK, tyrosine kinase; TKL, TK like; STE, STE7/11/20; CK1, casein kinase 1; AGC, protein kinase A/G/C; CAMK, calmodulin kinase; CMGC, cyclin-dependent kinase (CDK)/MAPK/glycogen synthase kinase (GSK)/CDK-like kinase (CLK). (B) Allosteric regulatory sites from diverse kinases mapped to a single representative structure: yeast CDK Pho85 [Protein Data Bank (PDB): 2PK9, shown as space-filled surface]. Regulatory surfaces were identified by structural alignment of the kinase of interest to Pho85; all Pho85 positions within 4 Å of the interaction surface are colored. Color coding is the same as in (A). Bright yellow spheres indicate known phosphoregulatory sites.

  • Fig. 2 Alanine scan of acidic residues on the solvent-accessible surface of yeast MAPK Kss1.

    (A) Schematic of the Kss1-dependent yeast pheromone pathway. The αF mating pheromone binds to a G protein–coupled receptor (GPCR), leading to activation of a signaling cascade culminating at the MAPK Kss1. Kss1 then activates the Ste12 transcription factor to induce the mating transcriptional program, which can be monitored by fusing the promoter of the target gene AGA1 to a yellow fluorescent protein (YFP) reporter. (B) Ribbon diagram of a Kss1 homology model (30) with the 40 solvent-accessible Asp/Glu residues shown as spheres. All 40 positions were mutated individually to alanine to remove negative charge. The DFG motif and activation loop are indicated in light blue. (C) The 40 resulting yeast strains along with wild-type (WT) and kss1Δ controls were assayed for activation of the AGA1pr-YFP reporter by flow cytometry following treatment with 0, 0.01, 0.1, and 1 μM αF for 4 hours. Bars represent the average of the median YFP fluorescence from three biological replicates normalized to the untreated kss1Δ cells, and error bars are the SD of the biological replicates. Mutations at red and green positions resulted in significantly reduced or increased YFP expression [P < 0.05, as scored by one-way analysis of variance (ANOVA)] in response to at least two doses of αF, respectively. Yellow positions indicate that the mutation had no effect in this assay. The color coding is identical in (B).

  • Fig. 3 Engineering allosteric control of Kss1 by PKA phosphorylation.

    (A) Cartoon of the engineered PKA- and Kss1-dependent yeast pheromone pathway. In this schematic, Kss1 activation requires both activation loop phosphorylation by the upstream MAP2K Ste7 and phosphorylation by PKA at an allosterically coupled surface. To experimentally increase PKA activity, expression of constitutively activated RAS2G19V is induced by addition of estradiol, which, in turn, activates adenylate cyclase (AC) to generate cyclic adenosine monophosphate (cAMP) from ATP to activate PKA. (B) Kss1 mutants with PKA phosphorylation site consensus motifs introduced near position 8 (pka-X8, top) or position 70 (pka-X70, bottom) were assayed for expression of the AGA1pr-YFP reporter by flow cytometry after treatment with αF (0, 0.01, 0.1, or 1 μM) for 4 hours. Again, bars represent the average of the median YFP fluorescence from three biological replicates normalized to the untreated kss1Δ cells, and error bars are the SD of the biological replicates. “X” stands for the amino acid at position 8 or 70 as denoted under the bar graphs. The images below the bar graphs show morphology and expression of the AGA1pr-YFP reporter in yeast cells bearing the indicated Kss1 mutants in the presence of 1 μM αF following growth in the presence or absence of 20 nM estradiol. The percent cells shmooing is indicated and was quantified by counting cells in three 512 × 512 pixel frames in each condition. n > 40 cells for each sample. (C) 3×FLAG-tagged wild-type Kss1 and pka-X8 and pka-X70 mutants were immunoprecipitated from untreated cells or cells that had been treated with both 20 nM estradiol and 1 μM αF. IP eluates were analyzed by Western blotting for total Kss1 and Kss1 phosphorylated on its activation loop (Phospho act. loop) or at the engineered PKA site (Phospho pka site). Merged images show that all mutants can be phosphorylated on their activation loop in the presence of αF, but only pka-S8 and pka-S70 can be phosphorylated by PKA in the presence of estradiol.

  • Fig. 4 SCA of the eukaryotic protein kinases.

    The analysis was performed for two different multiple sequence alignments of the kinase catalytic domain: one specific to the CMGC kinases (635 sequences) and one containing 7128 kinases sampled across the kinome. (A) Histogram showing the distribution of pairwise sequence identities computed across all pairs of sequences in the CMGC alignment. (B) As described in (A) but for the kinome-wide alignment. Both alignments show a unimodal distribution with a mean pairwise sequence identity near ~25%. (C) Sector positions derived from the CMGC alignment (blue) or kinome-wide alignment (yellow) are distributed along the primary and secondary structure of the CMGC/MAPK ERK2. Subfamily-specific regions, such as the MAPK insert, are only part of the sector derived from the CMGC alignment. (D) Relationship between the sector and positional conservation (computed as the Kullback-Leibler relative entropy, Di) for both the CMGC and kinome-wide alignments. Sector positions are highlighted in blue or yellow for the CMGC and kinome-wide alignments, respectively. Red stars indicate highly conserved positions (defined as Di > 2.0 in the kinome-wide alignment). (E) Kinome-wide and CMGC-specific sectors (yellow and blue transparent surfaces, respectively) mapped on human ERK2 (gray ribbon) (PDB: 2ERK). Conserved positions are shown as red spheres.

  • Fig. 5 Association of Kss1 D/E mutations with conserved and coevolving positions.

    (A) Space-filling diagram of a Kss1 homology model (30). The CMGC sector, defined as positions that coevolve across the CMGC kinases, is indicated in blue. Acidic surface residues with a neutral, activating, or inactivating effect on kinase function upon mutation to alanine are shown as yellow, green, or red spheres, respectively. (B) Fisher’s exact table demonstrating statistically significant enrichment of acidic surface residues with a functional effect upon mutation at sector-connected positions. To be sector connected, a position must have at least one atom within 4 Å of the sector. Both a P value and estimated false discovery rate (FDR) are indicated. (C) Same as described in (B), but considering conservation-connected rather than sector-connected positions. The cutoff for conservation was chosen to give a similar number of positions as the sector. A detailed analysis of the effect of conservation and sector cutoffs is in the supplement (text S1, tables S5, S6, and S8). (D) Homology model of Kss1 illustrating the relationship of positions Asp8, Glu68, and Glu70 (cyan sticks) to the sector (blue surface). The kinase backbone is in gray cartoon; the activation loop and DFG active site motif are colored yellow. Residues proximal to Asp8, Glu68, and Glu70 are shown in gray sticks. (E and F) Close-up view of the region surrounding positions 8 (E) and 68 to 70 (F). Color coding as in (D).

  • Fig. 6 Modulation of Hog1 activation dynamics by introduction of PKA phosphorylation sites at sector edges.

    (A) Sector-connected D/E residues are indicated on a homology model of Hog1. (B) Schematic of the engineered PKA/Hog1 pathway. Note that the Sln1/Ypd1 branch of the endogenous HOG pathway is omitted for clarity. (C) The seven Hog1-pka yeast strains along with wild-type and hog1Δ controls were grown in the presence and absence of 20 nM estradiol and assayed for activation of the HOR2pr-GFP reporter by flow cytometry after treatment with 0.5 M NaCl for 2 hours. Data are the means of the median GFP fluorescence ± SD from three biological replicates normalized to the untreated hog1Δ cells. **P < 0.01 by one-way ANOVA. (D) Immunoblots probed with antibodies against FLAG and phospho-p38 to show total amounts of the Hog1-pka mutants and their respective abundance of activation loop phosphorylation under basal conditions and after treatment with 0.5 M NaCl for 15 min. (E) Quantification of Hog1-mKate nuclear localization dynamics in wild-type and Hog1-pka mutants. Cells were imaged under basal conditions and after treatment with 0.5 M NaCl for 5, 20, or 60 min. n > 50 cells for all mutants at all time points.

  • Fig. 7 Sector-connected surface sites are associated with function in multiple kinases.

    (A) The eukaryotic protein kinase superfamily-wide sector (blue spheres) mapped to the CMGC yeast kinase Pho85 (PDB: 2PK9, gray cartoon and surface). Red positions are sites collected from the literature known to alter kinase function when mutated in a functional study or human disease context (see table S7). (B) Fisher’s exact table demonstrating statistically significant enrichment of the functional mutations shown in (A) at sector-connected positions. (C) Fisher’s exact table demonstrating statistically significant enrichment of the functional mutations shown in (A) at conservation-connected positions. (D) Model for the evolution of regulatory diversity. Latent allosteric sites distributed across the protein surface (red circles) are connected to the active site via a protein sector (blue arrows). These sites are poised for the acquisition of new regulation via evolutionary, disease, or engineering processes. In any particular family member, only a subset of sites may be used, and the regulatory mechanism need not be conserved across homologs. PTM, posttranslational modification.

Supplementary Materials

  • www.sciencesignaling.org/cgi/content/full/11/555/eaar3250/DC1

    Text S1. The relationship between the sector, conservation, and allosteric hotspots.

    Text S2. Background on Kss1.

    Fig. S1. The effect of surface Asp/Glu-to-Ala mutations on Kss1 expression.

    Fig. S2. Experimental approach to introduce a PKA phosphorylation site that controls MAPK Kss1 activity.

    Fig. S3. The kinase sector encompasses the catalytic and regulatory spines.

    Fig. S4. ERK2 mutations within the kinase sector are enriched for loss of function.

    Fig. S5. The relationship of negatively charged surface positions to the kinome-wide eukaryotic protein kinase sector.

    Fig. S6. Phosphorylation, phenotype, and nuclear localization of Hog1-pka mutants.

    Table S1. Plasmids.

    Table S2. Yeast strains.

    Table S3. Comparison of Kss1 point mutations from the literature with our data and the CMGC sector.

    Table S4. Sector positions mapped to several representative kinase structures.

    Table S5. Statistical association between the sector, conservation, and ERK2 mutational data.

    Table S6. Statistical association between the sector, conservation, and KSS1 D/E surface mutations.

    Table S7. Functional kinase mutations sampled across the kinome.

    Table S8. Statistical association between the sector, conservation, and functional mutations sampled across a diversity of kinases.

    References (4259)

  • This PDF file includes:

    • Text S1. The relationship between the sector, conservation, and allosteric hotspots.
    • Text S2. Background on Kss1.
    • Fig. S1. The effect of surface Asp/Glu-to-Ala mutations on Kss1 expression.
    • Fig. S2. Experimental approach to introduce a PKA phosphorylation site that controls MAPK Kss1 activity.
    • Fig. S3. The kinase sector encompasses the catalytic and regulatory spines.
    • Fig. S4. ERK2 mutations within the kinase sector are enriched for loss of function.
    • Fig. S5. The relationship of negatively charged surface positions to the kinome-wide eukaryotic protein kinase sector.
    • Fig. S6. Phosphorylation, phenotype, and nuclear localization of Hog1-pka mutants.
    • Table S1. Plasmids.
    • Table S2. Yeast strains.
    • Table S3. Comparison of Kss1 point mutations from the literature with our data and the CMGC sector.
    • Table S4. Sector positions mapped to several representative kinase structures.
    • Table S5. Statistical association between the sector, conservation, and ERK2 mutational data.
    • Table S6. Statistical association between the sector, conservation, and KSS1 D/E surface mutations.
    • Table S7. Functional kinase mutations sampled across the kinome.
    • Table S8. Statistical association between the sector, conservation, and functional mutations sampled across a diversity of kinases.
    • References (4259)

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