Phosphorylated and nonphosphorylated serine and threonine residues evolve at different rates in mammals

Abstract

Protein phosphorylation plays an important role in the regulation of protein function. Phosphorylated residues are generally assumed to be subject to functional constraint, but it has recently been suggested from a comparison of distantly related vertebrate species that most phosphorylated residues evolve at the rates consistent with the surrounding regions. To resolve the controversy, we infer the ancestral phosphoproteome of human and mouse to compare the evolutionary rates of phosphorylated and nonphosphorylated serine (S), threonine (T), and tyrosine (Y) residues. This approach enables accurate estimation of evolutionary rates as it does not assume deep conservation of phosphorylated residues. We show that phosphorylated S/T residues tend to evolve more slowly than nonphosphorylated S/T residues not only in disordered but also in ordered protein regions, indicating evolutionary conservation of phosphorylated S/T residues in mammals. Thus, phosphorylated S/T residues tend to be subject to stronger functional constraint than nonphosphorylated residues regardless of the protein regions in which they reside. In contrast, phosphorylated Y residues evolve at similar rates as nonphosphorylated ones. We also find that the human lineage has gained more phosphorylated T residues and lost fewer phosphorylated Y residues than the mouse lineage. The cause of the gain/loss imbalance remains a mystery but should be worth exploring.

FIG. 1.

Reconstruction of ancestral phosphorylated residues. (a) Phylogenetic tree with branch length proportional to the number of expected substitutions per amino acid position estimated by PAML, using a concatenated alignment of 3,526 groups of human–mouse–dog–opossum orthologous phosphoproteins. The scale bar stands for 0.02 expected substitutions per site in the aligned regions. The oval denotes the common ancestor of human, dog, and mouse. (b) Classification of ancestral residues using serine as an example. Sp, Sn, S, Ns, and SE, respectively, stands for “serine residue that is predicted to be phosphorylated,” “serine residue that is not predicted to be phosphorylated,” “serine residue whose phosphorylation status is unknown,” “non-serine residue,” and “serine residue that is experimentally validated as phosphorylated.” A (B) is a conserved (one-change) ancestral phosphorylated residue, with a human or mouse descendant residue experimentally validated as phosphorylated; C (D) is a conserved (one-change) ancestral nonphosphorylated residue, with neither the human nor the mouse descendant residue experimentally validated as phosphorylated; E (F) is a conserved (one-change) ancestral phosphorylated residues, with neither the human nor the mouse descendant residue experimentally validated as phosphorylated; G (H) is a conserved (one-change) ancestral nonphosphorylated residue, with a human or mouse descendant residue experimentally validated as phosphorylated.

FIG. 2.

Structural characteristics of phosphorylated residues (P), not predicted as phosphorylated residues (NP) and not known to be phosphorylated in either human or mouse (NP-A) residues. (a) Proportions (%) of P, NP, and NP-A residues in predicted disordered regions. (b) Proportions (%) of P, NP, and NP-A residues with high surface accessibility.

FIG. 3.

Rates of change of phosphorylated residues (P) and of residues not predicted as phosphorylated (NP). The rates of change of P-Disordered (P residues in disordered regions), NP-Disordered (NP residues in disordered regions), P-Ordered (P residues in ordered regions), and NP-Ordered (NP residues in ordered regions). A connector linking two bars indicates that the difference between the two bars is significant at P < 0.05 (by Fisher's exact test).

FIG. 4.

Gains and losses of phosphorylated residues. (a) The ratios of gains and losses of phosphorylated residues in the human and mouse lineages. A connector linking two bars indicates that the difference is significant at P < 0.05 (by Fisher's exact test). (b) The normalized ratios of (a). The ratios were normalized by dividing the proportions in supplementary table S3 (Supplementary Material online) by the branch lengths of the human and mouse lineages in fig. 1a, respectively.