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Ancestral resurrection reveals evolutionary mechanisms of kinase plasticity.

Howard CJ, Hanson-Smith V, Kennedy KJ, Miller CJ, Lou HJ, Johnson AD, Turk BE, Holt LJ - Elife (2014)

Bottom Line: Mutant analysis revealed that a variable residue within the kinase catalytic cleft, DFGx, modulates +1 specificity.Expansion of Ime2 kinase specificity by mutation of this residue did not cause dominant deleterious effects in vivo.Tolerance of cells to new specificities likely enabled the evolutionary divergence of kinases.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, United States.

ABSTRACT
Protein kinases have evolved diverse specificities to enable cellular information processing. To gain insight into the mechanisms underlying kinase diversification, we studied the CMGC protein kinases using ancestral reconstruction. Within this group, the cyclin dependent kinases (CDKs) and mitogen activated protein kinases (MAPKs) require proline at the +1 position of their substrates, while Ime2 prefers arginine. The resurrected common ancestor of CDKs, MAPKs, and Ime2 could phosphorylate substrates with +1 proline or arginine, with preference for proline. This specificity changed to a strong preference for +1 arginine in the lineage leading to Ime2 via an intermediate with equal specificity for proline and arginine. Mutant analysis revealed that a variable residue within the kinase catalytic cleft, DFGx, modulates +1 specificity. Expansion of Ime2 kinase specificity by mutation of this residue did not cause dominant deleterious effects in vivo. Tolerance of cells to new specificities likely enabled the evolutionary divergence of kinases.

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Raw data and selectivity values for full positional scanning peptide arrays of AncLF4 and AncICK.(A) Raw PSPL result for maximum likelihood reconstructions of AncNgru and AncICK. (B) Averaged, quantified selectivity values for two replicate runs of the kinases shown in (A). Data were collected and processed as in Figure 1—figure supplements 1 and 2.DOI:http://dx.doi.org/10.7554/eLife.04126.012
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fig3s1: Raw data and selectivity values for full positional scanning peptide arrays of AncLF4 and AncICK.(A) Raw PSPL result for maximum likelihood reconstructions of AncNgru and AncICK. (B) Averaged, quantified selectivity values for two replicate runs of the kinases shown in (A). Data were collected and processed as in Figure 1—figure supplements 1 and 2.DOI:http://dx.doi.org/10.7554/eLife.04126.012

Mentions: In order to learn the trajectory by which kinase specificity at the +1 position evolved, we reconstructed ancestral kinases within the CMGC group at multiple evolutionary time points before and after the +1 specificity change from proline to arginine was presumed to have occurred (Figure 3A). These kinases were assayed using consensus peptide substrates with identical sequence except for having either proline (red) or arginine (blue) at the +1 position. The log-ratio of arginine/proline preference from this assay is plotted in Figure 3B. Because the +1 specificity could be dependent on the surrounding sequence context present in the consensus peptide substrates, we also characterized the full primary specificities of AncLF4 and AncICK kinases by PSPL arrays (Figure 3, Figure 3—figure supplement 1). While the specificity for arginine at the −3 position and proline at the −2 position was conserved among these ancestors, we observed significant variation in their relative preference for +1R vs +1P (Figure 3B,C; Figure 3, Figure 3—figure supplement 1). As described above, AncCMGI appears to have preferred +1P substrates. On the phylogenetic branch leading to the common ancestor of the IME2/RCK/LF4 group (i.e., AncNgru), the +1 specificity relaxed to equally accommodate both +1R and +1P (+1PR). This hybrid specificity was conserved in the LF4 lineage and also on the branch leading to AncICK. Evolution after AncICK, however, proceeded along two divergent evolutionary paths. Namely, the specificity reverted to the ancestral +1P-preferring state along the branch leading to the mammalian RCK kinases (ICK and MAK). In contrast, the specificity shifted to +1R in the fungal lineage leading to the ancestor of the IME2 kinases (i.e., AncIME2). AncIME2 had a moderate preference for +1R, and this preference is maintained in other fungal IME2 ancestors, becoming more pronounced in the ancestor of Yarrowia lipolytica (AncYlip). These results are summarized in their phylogenetic context in Figure 3A and are robust to statistical uncertainties about the reconstructed ancestral sequences, although the degree of +1 proline selectivity was slightly lower in AncCMGI-B1 and higher in AncCMGI-B2 (see ‘Materials and methods’, Figure 3, Figure 3—figure supplement 2).10.7554/eLife.04126.011Figure 3.The substrate peptide +1 specificity evolved from proline in AncCMGI to arginine in S. cerevisiae Ime2 via an expanded specificity intermediate.


Ancestral resurrection reveals evolutionary mechanisms of kinase plasticity.

Howard CJ, Hanson-Smith V, Kennedy KJ, Miller CJ, Lou HJ, Johnson AD, Turk BE, Holt LJ - Elife (2014)

Raw data and selectivity values for full positional scanning peptide arrays of AncLF4 and AncICK.(A) Raw PSPL result for maximum likelihood reconstructions of AncNgru and AncICK. (B) Averaged, quantified selectivity values for two replicate runs of the kinases shown in (A). Data were collected and processed as in Figure 1—figure supplements 1 and 2.DOI:http://dx.doi.org/10.7554/eLife.04126.012
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4228266&req=5

fig3s1: Raw data and selectivity values for full positional scanning peptide arrays of AncLF4 and AncICK.(A) Raw PSPL result for maximum likelihood reconstructions of AncNgru and AncICK. (B) Averaged, quantified selectivity values for two replicate runs of the kinases shown in (A). Data were collected and processed as in Figure 1—figure supplements 1 and 2.DOI:http://dx.doi.org/10.7554/eLife.04126.012
Mentions: In order to learn the trajectory by which kinase specificity at the +1 position evolved, we reconstructed ancestral kinases within the CMGC group at multiple evolutionary time points before and after the +1 specificity change from proline to arginine was presumed to have occurred (Figure 3A). These kinases were assayed using consensus peptide substrates with identical sequence except for having either proline (red) or arginine (blue) at the +1 position. The log-ratio of arginine/proline preference from this assay is plotted in Figure 3B. Because the +1 specificity could be dependent on the surrounding sequence context present in the consensus peptide substrates, we also characterized the full primary specificities of AncLF4 and AncICK kinases by PSPL arrays (Figure 3, Figure 3—figure supplement 1). While the specificity for arginine at the −3 position and proline at the −2 position was conserved among these ancestors, we observed significant variation in their relative preference for +1R vs +1P (Figure 3B,C; Figure 3, Figure 3—figure supplement 1). As described above, AncCMGI appears to have preferred +1P substrates. On the phylogenetic branch leading to the common ancestor of the IME2/RCK/LF4 group (i.e., AncNgru), the +1 specificity relaxed to equally accommodate both +1R and +1P (+1PR). This hybrid specificity was conserved in the LF4 lineage and also on the branch leading to AncICK. Evolution after AncICK, however, proceeded along two divergent evolutionary paths. Namely, the specificity reverted to the ancestral +1P-preferring state along the branch leading to the mammalian RCK kinases (ICK and MAK). In contrast, the specificity shifted to +1R in the fungal lineage leading to the ancestor of the IME2 kinases (i.e., AncIME2). AncIME2 had a moderate preference for +1R, and this preference is maintained in other fungal IME2 ancestors, becoming more pronounced in the ancestor of Yarrowia lipolytica (AncYlip). These results are summarized in their phylogenetic context in Figure 3A and are robust to statistical uncertainties about the reconstructed ancestral sequences, although the degree of +1 proline selectivity was slightly lower in AncCMGI-B1 and higher in AncCMGI-B2 (see ‘Materials and methods’, Figure 3, Figure 3—figure supplement 2).10.7554/eLife.04126.011Figure 3.The substrate peptide +1 specificity evolved from proline in AncCMGI to arginine in S. cerevisiae Ime2 via an expanded specificity intermediate.

Bottom Line: Mutant analysis revealed that a variable residue within the kinase catalytic cleft, DFGx, modulates +1 specificity.Expansion of Ime2 kinase specificity by mutation of this residue did not cause dominant deleterious effects in vivo.Tolerance of cells to new specificities likely enabled the evolutionary divergence of kinases.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, United States.

ABSTRACT
Protein kinases have evolved diverse specificities to enable cellular information processing. To gain insight into the mechanisms underlying kinase diversification, we studied the CMGC protein kinases using ancestral reconstruction. Within this group, the cyclin dependent kinases (CDKs) and mitogen activated protein kinases (MAPKs) require proline at the +1 position of their substrates, while Ime2 prefers arginine. The resurrected common ancestor of CDKs, MAPKs, and Ime2 could phosphorylate substrates with +1 proline or arginine, with preference for proline. This specificity changed to a strong preference for +1 arginine in the lineage leading to Ime2 via an intermediate with equal specificity for proline and arginine. Mutant analysis revealed that a variable residue within the kinase catalytic cleft, DFGx, modulates +1 specificity. Expansion of Ime2 kinase specificity by mutation of this residue did not cause dominant deleterious effects in vivo. Tolerance of cells to new specificities likely enabled the evolutionary divergence of kinases.

Show MeSH