Cell Signaling: Learning from ancestors

Applying ancestral sequence reconstruction techniques to protein kinases reveals the mutations that underlie different modes of activation.
  1. Suk ho Hong
  2. Neel H Shah  Is a corresponding author
  1. Columbia University, United States

A major goal of biological research is to understand the evolutionary histories of organisms and genes. One way to do this is to study biological entities that have become extinct, as this can provide insights into the form and function of their present-day descendants. This is perhaps best exemplified by paleogenetics and paleoproteomics research, where DNA and protein molecules from ancient biological samples are extracted and sequenced to help us better understand the evolutionary relationships between different species. Indeed, the analysis of biomolecules from our hominid ancestors has revealed significant new insights into the origins and diversity of our species (Warren, 2019). Unfortunately, DNA and proteins are degraded over time, which means that this approach can only be applied to evolutionary events from the past one million years.

Many large gene and protein families have evolved through rounds of gene duplication and functional specialization over hundreds of millions of years, which puts them beyond the reach of paleogenetics and paleoproteomics. How, then, might we use evolution to dissect the specialized properties of individual proteins in a family? One approach, called ancestral sequence reconstruction, involves using a statistical model to analyze the sequences of closely-related proteins from different organisms and generate plausible sequences for their ancestors (Hochberg and Thornton, 2017). Actual protein samples based on these sequences can then be made in the laboratory and compared to naturally occurring proteins.

Ancestral sequence reconstruction has been applied to a variety of protein families to understand, at the molecular level, how closely-related proteins have evolved distinct biochemical properties. For example, this method was previously used to examine how individual kinases – enzymes that modify other proteins through a process called phosphorylation – select different target molecules (Howard et al., 2014). Now, in eLife, the same group, led by Liam Holt at New York University (NYU) – including Dajun Sang as first author – reports on the application of ancestral sequence reconstruction to study the evolution of a subfamily of kinases called the MAP kinases (Sang et al., 2019).

Most eukaryotic organisms have hundreds of different protein kinases – humans have over 500 (Manning et al., 2002) – and many kinases need to be phosphorylated themselves in order to become active (Nolen et al., 2004). Some kinases can phosphorylate and activate themselves, through a process called autophosphorylation, while others are dependent on another kinase to be phosphorylated. Many members of the MAP kinase family autophosphorylate, but ERK1 and ERK2 (referred to as ERK1/2) cannot autophosphorylate efficiently. The molecular characteristics that prevent them from doing so were previously unknown.

Sang et al. compiled MAP kinase sequences from a variety of organisms and used ancestral reconstruction to predict the sequences of their common ancestors (Sang et al., 2019). They used standard biochemical techniques to produce proteins with the predicted sequences, and showed that the predicted common ancestor of ERK1/2 could not autophosphorylate efficiently, whereas other ancestral MAP kinases could (Figure 1). Two mutations were found when the sequence of the ERK1/2 common ancestor was compared to the sequences of the other ancestral MAP kinases. One was an amino acid substitution near the spine connecting different regions of the protein; the other was an amino acid deletion that shortened a flexible loop near the catalytic cleft in the kinases. Together, these two mutations suppress the ability of ERK1/2 and their common ancestor to autophosphorylate (Figure 1). Notably, Sang et al. – who are based at Memorial Sloan Kettering, the Icahn School of Medicine and Yale – also showed that reinserting the deleted amino acid in the flexible loop in human ERK1 relieved its dependence on other kinases for its activation in cells.

The evolution of different regulatory properties in MAP kinases.

A mock phylogenetic tree (left) shows the evolution of ERK1/2 and other MAP kinases. ERK1/2, and their common ancestor (dark blue) cannot efficiently activate themselves through autophosphorylation. More ancient ancestors in the MAP kinase family (light blue) are capable of efficient autophosphorylation. A cartoon diagram (right) highlights the structural properties that differentiate MAP kinases that are capable of autophosphorylation (light blue) from those that cannot autophosphorylate themselves efficiently (dark blue). All protein kinases have a two-lobe structure with a catalytic cleft in the middle. Different parts of the kinase are connected by a spine. The loop in front of the catalytic cleft has to shift position for the enzyme to become active. This is driven by phosphorylation of that loop, either by another kinase or through autophosphorylation (shown as pink residues in the inactive form of the enzyme becoming red residues in the active form, with a concomitant change in the shape of the loop). Sang et al. have identified two mutations that could explain why ERK1/2 and their common ancestor (bottom right, dark blue) are different from other MAP kinases (top right, light blue): i) they have a polar amino acid (yellow, bottom) rather than a hydrophobic amino acid (orange, top) at a site near the spine of the kinase; ii) a loop above the catalytic cleft is one amino acid shorter than in other MAP kinases. It is thought that these two mutations disrupt the geometry and flexibility of the catalytic cleft, altering the ability of the kinase to autophosphorylate.

To explain how these evolutionary sequence alterations resulted in a change in autophosphorylation ability, Sang et al. performed computer simulations of the internal motions of ERK2, with and without the ancestral insertion and substitution. These simulations revealed that the overall flexibility of ERK2 increased when it had ancestor-like sequence features. Sang et al. postulate that increased flexibility in the mutant kinase allows it to more readily adopt a shape compatible with autophosphorylation.

The two mutations reported in the latest work have intriguing implications for kinases in general. Alterations to the flexible loop have been observed in cancer-associated variants of several distantly related kinases (BRAF, HER2, and EGFR; Foster et al., 2016), and mutations at the spine-proximal position are associated with excessive activation and drug resistance in a variety of kinases (Azam et al., 2008). Although ERK1/2 are intimately embedded within oncogenic signaling pathways, mutations at these positions have not been found in those kinases in human cancers. Further analysis of kinase evolutionary history, juxtaposed with cancer genome sequencing, is likely to reveal other conserved mutational hotspots that have facilitated the evolution of divergent properties across protein kinases.


Article and author information

Author details

  1. Suk ho Hong

    Suk ho Hong is in the Department of Chemistry, Columbia University, New York, United States

    Competing interests
    No competing interests declared
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-6024-0685
  2. Neel H Shah

    Neel H Shah is in the Department of Chemistry, Columbia University, New York, United States

    For correspondence
    Competing interests
    No competing interests declared
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-1186-0626

Publication history

  1. Version of Record published: August 13, 2019 (version 1)


© 2019, Hong and Shah

This article is distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use and redistribution provided that the original author and source are credited.


  • 1,483
    Page views
  • 131
  • 0

Article citation count generated by polling the highest count across the following sources: Crossref, PubMed Central, Scopus.

Download links

A two-part list of links to download the article, or parts of the article, in various formats.

Downloads (link to download the article as PDF)

Open citations (links to open the citations from this article in various online reference manager services)

Cite this article (links to download the citations from this article in formats compatible with various reference manager tools)

  1. Suk ho Hong
  2. Neel H Shah
Cell Signaling: Learning from ancestors
eLife 8:e49976.
  1. Further reading

Further reading

    1. Biochemistry and Chemical Biology
    2. Neuroscience
    Jinli Geng, Yingjun Tang ... Xiaodong Liu
    Research Article Updated

    Dynamic Ca2+ signals reflect acute changes in membrane excitability, and also mediate signaling cascades in chronic processes. In both cases, chronic Ca2+ imaging is often desired, but challenged by the cytotoxicity intrinsic to calmodulin (CaM)-based GCaMP, a series of genetically-encoded Ca2+ indicators that have been widely applied. Here, we demonstrate the performance of GCaMP-X in chronic Ca2+ imaging of cortical neurons, where GCaMP-X by design is to eliminate the unwanted interactions between the conventional GCaMP and endogenous (apo)CaM-binding proteins. By expressing in adult mice at high levels over an extended time frame, GCaMP-X showed less damage and improved performance in two-photon imaging of sensory (whisker-deflection) responses or spontaneous Ca2+ fluctuations, in comparison with GCaMP. Chronic Ca2+ imaging of one month or longer was conducted for cultured cortical neurons expressing GCaMP-X, unveiling that spontaneous/local Ca2+ transients progressively developed into autonomous/global Ca2+ oscillations. Along with the morphological indices of neurite length and soma size, the major metrics of oscillatory Ca2+, including rate, amplitude and synchrony were also examined. Dysregulations of both neuritogenesis and Ca2+ oscillations became discernible around 2–3 weeks after virus injection or drug induction to express GCaMP in newborn or mature neurons, which were exacerbated by stronger or prolonged expression of GCaMP. In contrast, neurons expressing GCaMP-X were significantly less damaged or perturbed, altogether highlighting the unique importance of oscillatory Ca2+ to neural development and neuronal health. In summary, GCaMP-X provides a viable solution for Ca2+ imaging applications involving long-time and/or high-level expression of Ca2+ probes.

    1. Biochemistry and Chemical Biology
    2. Chromosomes and Gene Expression
    Radhika A Varier, Theodora Sideri ... Folkert Jacobus van Werven
    Research Article

    N6-methyladenosine (m6A) RNA modification impacts mRNA fate primarily via reader proteins, which dictate processes in development, stress, and disease. Yet little is known about m6A function in Saccharomyces cerevisiae, which occurs solely during early meiosis. Here we perform a multifaceted analysis of the m6A reader protein Pho92/Mrb1. Cross-linking immunoprecipitation analysis reveals that Pho92 associates with the 3’end of meiotic mRNAs in both an m6A-dependent and independent manner. Within cells, Pho92 transitions from the nucleus to the cytoplasm, and associates with translating ribosomes. In the nucleus Pho92 associates with target loci through its interaction with transcriptional elongator Paf1C. Functionally, we show that Pho92 promotes and links protein synthesis to mRNA decay. As such, the Pho92-mediated m6A-mRNA decay is contingent on active translation and the CCR4-NOT complex. We propose that the m6A reader Pho92 is loaded co-transcriptionally to facilitate protein synthesis and subsequent decay of m6A modified transcripts, and thereby promotes meiosis.