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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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The phosphoacceptor affects the +1 specificity of ICK. ICK kinase was incubated with 45 μM peptide and initial velocities measured.Bars show the log2 ratio of +1R/+1P initial velocities (V0R/V0P). Black and white bars indicate that ICK was incubated with peptides that contain serine and threonine respectively as phosphoacceptor. The gray bar indicates an equal mix of serine and threonine as phosphoacceptor. The lower gray bar is ratio data taken from the PSPL array in Figure 1—figure supplement 1 i.e. a ratio of phosphate incorporation into Y-A-X-X-X-X-X-S/T-R-X-X-X-A-G-K-K-biotin vs Y-A-X-X-X-X-X-S/T-P-X-X-X-A-G-K-K-biotin peptides, where S/T indicates an equal mixture of serine or threonine as phosphoacceptor and X indicates an equal mixture of all amino acids except C, S, or T at all other positions except +1 and the terminal linker amino acids.DOI:http://dx.doi.org/10.7554/eLife.04126.015
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fig4s1: The phosphoacceptor affects the +1 specificity of ICK. ICK kinase was incubated with 45 μM peptide and initial velocities measured.Bars show the log2 ratio of +1R/+1P initial velocities (V0R/V0P). Black and white bars indicate that ICK was incubated with peptides that contain serine and threonine respectively as phosphoacceptor. The gray bar indicates an equal mix of serine and threonine as phosphoacceptor. The lower gray bar is ratio data taken from the PSPL array in Figure 1—figure supplement 1 i.e. a ratio of phosphate incorporation into Y-A-X-X-X-X-X-S/T-R-X-X-X-A-G-K-K-biotin vs Y-A-X-X-X-X-X-S/T-P-X-X-X-A-G-K-K-biotin peptides, where S/T indicates an equal mixture of serine or threonine as phosphoacceptor and X indicates an equal mixture of all amino acids except C, S, or T at all other positions except +1 and the terminal linker amino acids.DOI:http://dx.doi.org/10.7554/eLife.04126.015

Mentions: In initial results comparing +1 specificities obtained from our positional scanning peptide library arrays to those from our ratiometric peptide assays, we noticed that ICK was an outlier among the mammalian RCKs: this kinase phosphorylated peptides with +1R and +1P equally in our ratiometric assay, while the arrays showed a clear +1P preference (albeit with detectable phosphorylation of the +1R peptide mixture, Figure 4, Figure 4—figure supplement 1). We had initially used peptides with only serine as a phosphoacceptor in our ratiometric assay, while the PSPL array peptides contained equal mixtures of serine and threonine. We therefore reasoned that the nature of the phosphoacceptor might influence the +1 specificity of kinases. To test this hypothesis, we analyzed additional peptide sets with equal mixes of serine and threonine, or with only threonine as the phosphoacceptor in our ratiometric assay. From these experiments, we found that, indeed, the phosphoacceptor affects +1 specificity: serine causes a shift towards +1R preference, while threonine causes a shift towards +1P preference (Figure 4, Figure 4—figure supplement 1). This dependence of +1 specificity on the phosphoacceptor is present in AncCMGI and is maintained in all ancestors and extant members of the IME2/RCK/LF4 family (Figures 3B and 4C, Figure 3—figure supplement 2).


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)

The phosphoacceptor affects the +1 specificity of ICK. ICK kinase was incubated with 45 μM peptide and initial velocities measured.Bars show the log2 ratio of +1R/+1P initial velocities (V0R/V0P). Black and white bars indicate that ICK was incubated with peptides that contain serine and threonine respectively as phosphoacceptor. The gray bar indicates an equal mix of serine and threonine as phosphoacceptor. The lower gray bar is ratio data taken from the PSPL array in Figure 1—figure supplement 1 i.e. a ratio of phosphate incorporation into Y-A-X-X-X-X-X-S/T-R-X-X-X-A-G-K-K-biotin vs Y-A-X-X-X-X-X-S/T-P-X-X-X-A-G-K-K-biotin peptides, where S/T indicates an equal mixture of serine or threonine as phosphoacceptor and X indicates an equal mixture of all amino acids except C, S, or T at all other positions except +1 and the terminal linker amino acids.DOI:http://dx.doi.org/10.7554/eLife.04126.015
© Copyright Policy - open-access
Related In: Results  -  Collection

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

fig4s1: The phosphoacceptor affects the +1 specificity of ICK. ICK kinase was incubated with 45 μM peptide and initial velocities measured.Bars show the log2 ratio of +1R/+1P initial velocities (V0R/V0P). Black and white bars indicate that ICK was incubated with peptides that contain serine and threonine respectively as phosphoacceptor. The gray bar indicates an equal mix of serine and threonine as phosphoacceptor. The lower gray bar is ratio data taken from the PSPL array in Figure 1—figure supplement 1 i.e. a ratio of phosphate incorporation into Y-A-X-X-X-X-X-S/T-R-X-X-X-A-G-K-K-biotin vs Y-A-X-X-X-X-X-S/T-P-X-X-X-A-G-K-K-biotin peptides, where S/T indicates an equal mixture of serine or threonine as phosphoacceptor and X indicates an equal mixture of all amino acids except C, S, or T at all other positions except +1 and the terminal linker amino acids.DOI:http://dx.doi.org/10.7554/eLife.04126.015
Mentions: In initial results comparing +1 specificities obtained from our positional scanning peptide library arrays to those from our ratiometric peptide assays, we noticed that ICK was an outlier among the mammalian RCKs: this kinase phosphorylated peptides with +1R and +1P equally in our ratiometric assay, while the arrays showed a clear +1P preference (albeit with detectable phosphorylation of the +1R peptide mixture, Figure 4, Figure 4—figure supplement 1). We had initially used peptides with only serine as a phosphoacceptor in our ratiometric assay, while the PSPL array peptides contained equal mixtures of serine and threonine. We therefore reasoned that the nature of the phosphoacceptor might influence the +1 specificity of kinases. To test this hypothesis, we analyzed additional peptide sets with equal mixes of serine and threonine, or with only threonine as the phosphoacceptor in our ratiometric assay. From these experiments, we found that, indeed, the phosphoacceptor affects +1 specificity: serine causes a shift towards +1R preference, while threonine causes a shift towards +1P preference (Figure 4, Figure 4—figure supplement 1). This dependence of +1 specificity on the phosphoacceptor is present in AncCMGI and is maintained in all ancestors and extant members of the IME2/RCK/LF4 family (Figures 3B and 4C, Figure 3—figure supplement 2).

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