BACKGROUND
erks are the effector kinases of the ras/raf/mek/erk signaling pathway involved in multiple essential cell processes such as proliferation  <cit> , differentiation  <cit> , survival and memory formation  <cit> .
abnormal activation of this cascade leads to pathologies such as cancer  <cit>  or cognitive impairments  <cit> .
since the discovery of two erk isoforms in mammals, erk <dig>  and erk <dig>  in  <dig>  <cit> , numerous researchers have strived to understand their respective roles.
while some cancers have been associated with isoforms of erk cascade members such as b-raf for melanoma  <cit> , the putative differential involvement of either erk isoform in cancer or any disease remains unknown.
erk <dig> and erk <dig> are expressed ubiquitously in mammals where both display the same kinase specific activity in vitro  <cit>  and share a highly similar 3d structure .
furthermore in mammals, both translocate to the nucleus upon stimulation by cell surface receptors  <cit>  and while they do share at least  <dig> interactors, no isoform-specific substrates have been identified  <cit> .
indeed, erk <dig> and erk <dig> share  <dig> out of  <dig> amino acids that have been demonstrated to directly interact with substrates  <cit> , the sole difference being a conservative substitution: leucine155erk <dig> into isoleucine175erk <dig> .
erk <dig> is regarded by many as essential due to the embryonic lethality of erk <dig> knock-out mice , whereas mice lacking erk <dig> are viable and fertile suggesting a dispensable role of erk <dig>  <cit> .
similarly, some studies based on sirna mediated-invalidations suggest specific functions .
however, targeted erk <dig> and/or erk <dig> gene disruption in mice organs can evoke redundancy  <cit> .
in mouse fibroblasts we showed that solely si-rna mediated erk <dig> knock-down reduced cell proliferation by itself, however when erk <dig> levels were clamped down, erk <dig> knock-down became effective at reducing cell proliferation.
hence, we and others have hypothesized that the apparent dominant role of erk <dig> is only due to its higher expression rather than functional differences  <cit> .
to date, the controversial question of erks differential function versus redundancy has not been successfully addressed.
here we show by a combined approach based on erk1/ <dig> sequence evolution and erk1/ <dig> protein expression across vertebrates, that while some tetrapods express both erk <dig> and erk <dig> proteins, others have lost the erk <dig> gene and others express either only erk <dig> or only erk <dig> at detectable levels despite having both genes.
hence our results strongly suggest that erk1/ <dig> can act interchangeably, a conclusion strengthened by the observation that amino-acids required for function are invariant in erk <dig> and erk <dig>
RESULTS
all mammals tested to date express both erk <dig> and erk <dig> from eutherians  to the marsupial opossum .
to search clues for specific functions or functional redundancy of erk isoforms, we studied protein expression of erk isoforms in parallel with the evolution of their sequences in all key vertebrate clades.
we chose to study protein expression in brains to allow comparison across very divergent species and gain high sensitivity since in mammals, this organ has the greatest level of erk expression.
<cit> .
to ensure detection specificity we performed western blots incubated with anti-erk and anti-phospho-erk antibodies.
in vertebrates, erk <dig> and erk <dig> are highly conserved, thus allowing various antibodies to recognize evolutionarily distant erks.
furthermore, the epitope recognizing doubly-phosphorylated erk is conserved from sea anemones to humans, contributing to unambiguous protein identification .
as a proof of principle for this approach, we can readily detect a single erk protein with three distinct anti-phospho erk antibodies and one antibody targeting total-erk in hagfish , the most evolutionary divergent vertebrate specie compared to mammals .fig.
<dig> erk <dig> and erk <dig> protein appear first in bony vertebrates .
western-blot analysis of samples from brain, spinal cord or cultured cells: immunoblots were incubated with polyclonal anti-erk antibody  or anti-phosphoerk antibody .
taxonomic names are listed in additional file  <dig>  predicted mw sizes of vertebrate erks range from  <dig>  kda for lamprey’s erk to  <dig>  for zebrafish’s erk <dig>  sc: spinal cord; ce: cerebellum; op: optic lobe.
positions of erk <dig>  erk <dig>  phospho-erk <dig>  and phospho-erk <dig>  are indicated on the sides
one erk is expressed in agnathans and chondrichthyes
lamprey  belongs to the same agnathan clade as hagfish and possesses only a single erk gene within its genome  <cit>  and we detected only one erk in lamprey’s brain extracts .
phylogenetic analysis of hagfish and lamprey erks sequences highlight the similarity of the single erk from these species .fig.
<dig> evolution of vertebrate erk sequences.
a maximium-likelihood phylogenetic analysis of erk nucleotide and  derived erk amino-acid sequences, at key nodes of vertebrate evolution.
erk1/erk <dig> and erk2/erk <dig> phylogenies are indicated by red and blue branches respectively.
key taxonomic clades for this manuscript  are indicated by brackets.
accession numbers and common names are listed in additional file  <dig>  branch length is indicative of degree of sequence divergences.
the order of species in the derived trees is noted to vary slightly between different topological methods and whether it is calculated using protein or nucleotide sequences.
given that all vertebrate erk sequences are highly conserved, minor variations in tree placement can be expected
the next major clade in the vertebrate lineage, cartilaginous fishes  are constituted of three sub-clades: chimeras, sharks and rays.
among chimeras, the genome of the elephant shark  is sequenced and has only a single erk  <cit> .
to extend this result to the two other classes of chondrichthyes we first analyzed erk expression in brains.
we detected a sole erk isoform in the brain of winter skate , medulla of the small-spotted catshark  and spinal cord of the spiny dogfish .
using degenerated primers targeting teleost erk <dig> and erk <dig>  we also amplified and cloned a single erk from winter skate  and spiny dogfish , both of which share a high sequence identity  to elephant shark erk.
we conclude that members of the three clades of chondrichthyes express a single erk.
the single erk of chondrichthyes is likely to be erk <dig>  firstly because its sequence segregates within erk <dig> group by three independent topological methods , and the bootstrap values of the branches of fig.
<dig> that place chondrichthyes erk into the erk <dig> group support this finding.
secondly, the size of the erk gene from elephant shark is large , similar to the large size of teleost and mammal erk <dig> genes and opposite of the extremely compact erk <dig> genes present in vertebrates as it will be described at the end of the result section .
erk <dig> and erk <dig> from all bony vertebrates arose from a single duplication event
all teleosts genomes sequenced so far  possess two erk genes; and we readily detect two erk proteins from brain extracts of two representatives of the same teleost clade: dicentrarchus labrax and hemichromis bimaculatus .
bichirs are extant representatives of the most ancient branch of ray-finned fishes, diverging from the rest of actinopterygii prior to the whole genome duplication specific of teleosts  <cit> .
therefore erk isoform identification in bichir is critical for determining erk <dig> and erk <dig> emergence in bony fish.
we analyzed brain extracts from bichir  and detected two erk isoforms  and we further cloned two erk genes from p. senegalus.
phylogenetic analysis of bichir erk isoforms revealed that they already fully segregate at gene and protein levels as erk <dig> and erk <dig> as classified in teleosts and tetrapods .
this demonstrates duplicate erk1/ <dig> emergence at least  <dig> mya prior to the divergence of the bichirs from primitive bony vertebrates  <cit> .
furthermore, in human and zebrafish genomes ypel <dig> gene is located downstream of erk1/mapk <dig> gene in the same orientation than erk1; and ypel <dig> is located downstream of erk2/mapk <dig> in the same orientation than erk2; ypel <dig> and ypel <dig> being also isoforms therefore the synteny of isoforms is conserved from fish to mammals between these two loci, which is further indicative of a single duplication event that generated erk <dig> and erk <dig> in vertebrates.
erk <dig> gene has been lost independently in two tetrapod clades
western blot studies and gene sequencing revealed that several extant members of the three tetrapod clades express both erk <dig> and erk2: turtles for the sauropsid clade fig.
3a), axolotl for the amphibian clade  and mouse for the mammalian clade .
however, only one erk was detected in western blots of protein extracts from african clawed frog  and chicken .
the search of exons in fully sequenced genomes combined with the screening of est databases reveals that the erk <dig> gene is absent from the genomes of xenopus tropicalis , chicken   <cit>  and  <dig> other recently published genomes of neo-gnathae .
furthermore, erk <dig> protein expression is not detectable in the brain of the paleo-gnathae nandu , and no erk <dig> gene was identified in the genome of the paleo-gnathae ostrich .
these observations suggest that erk <dig> has been lost in all bird lineages and in some amphibians.
as summarized in table  <dig>  only erk <dig> gene has been lost in some tetrapods and all chondrichthyes.fig.
<dig> erk <dig> is not detected in snakes and lizards.
a, b western-blot analysis performed as in fig.
<dig> incubated with a polyclonal anti-erk antibody  or anti-phosphoerk antibody .
a brain extracts from snakes, lizards, crocodile and turtles .
control brain extracts from cattle.
co: cortex, ce: cerebellum, me: medulla, ol: olfactory bulb, pi: pineal gland, w: whole brain.
note that degraded bands are observed in turtle e. orbicularis likely due postmortem sampling.
b extracts from lizard embryo fibroblasts were stimulated or not  one hour in presence of fcs alone  or fcs + phosphatase inhibitor navo4— to activate erks
mus musculus
bos taurus
cricetulus griseus
oryctolagus cuniculus
monodelphis domestica
ornithorhynchus anatinus
gallus gallus
o
rhea americana
o
crocodylus niloticus
thamnophis elegans
pantherophis guttatus
pituophis catenifer sayi
anolis carolinensis
anolis sagrei
tarentola mauritanica
trachemys scripta elegans
emys orbicularis
xenopus laevi
o
ambiostoma mexicanum
latimeria chalumnae
dicentrarchus labrax
hemichromis bimaculatus
polypterus senegalus
squalus acanthias
o
scyliorhinus canicula
o
raja ocellata
o
genomes and est databases were scrutinized for the presence of erk1/ <dig> exons, if gene is present: ✓ if gene is absent: o
presence of proteins readily detected on western blots in this work is indicated: ✓
nd not determined
in reptiles either one or both erks are expressed
both erk proteins are expressed in turtle brain, however crocodiles appear to express only erk <dig> protein in the same organ .
by contrast, we could only detect erk <dig> protein in snakes and lizards .
side-by-side loading of cerebellum extracts from lizard, crocodile and turtle confirm these observations .
furthermore, an apparent lack of erk <dig> was observed in brain extracts from all squamate species tested:  <dig> lizards,  <dig> snakes and one gecko .
the two reptile genomes sequenced so far possess erk <dig> and erk <dig> ; therefore to confirm the surprising absence of erk <dig> protein in the model animal for this clade, a. carolinensis  <cit> , we first confirmed the absence of erk <dig> expression in an array of a. carolinensis adult tissues .
we then derived primary lizard embryo fibroblasts  from mid-gestation embryos.
even when erk was hyper-stimulated by serum in presence of orthovanadate to increase detection with anti-phospho erk antibodies, only a single erk1-sized band was observed .
to unambiguously assign protein identity and remove the possibility that two erk proteins are present and co-migrate, lefs were transfected with pools of three independent sirna sequences targeting erk <dig> or erk <dig>  at minimum, each sirna displays  <dig> differences with the non-targeted isoform to ensure isoform-specificity .
efficiency and isoform specificity of each sirna pool was verified by measuring erk1/ <dig> mrna levels post-transfection .
three days post-transfection, the expression level of a. carolinensis erk protein was consistently lowered by sirna targeting erk <dig> whereas sirna targeting erk <dig> had no effect .
therefore, we suggest with a strong degree of evidence that erk <dig> is the only protein isoform detected in anole lizard, and more generally in squamates.fig.
<dig> only erk <dig> protein and erk <dig> mrna are expressed significantly in lizards.
a sirna sequences targeting anole lizard  erk <dig> mrna  and anole lizard erk <dig> mrna .
in red, the nucleotides that differ when both erk <dig> and erk <dig> isoforms are aligned.
b, c sirna pools targeting erk <dig> or erk <dig> or control-sirna were transfected three days prior to mrna harvesting.
b absolute quantification of erk <dig> mrna  or erk <dig> mrna  after sirna transfection, in a. sagrei and a. carolinensis lizard embryo fibroblasts .
erk <dig> or erk <dig> mrna quantities were determined relatively to quantities of linearized plasmids harboring either a. carolinensis erk <dig> or erk <dig> cdnas as described in materials and methods.
to ease comparison between erk <dig> and erk <dig>  data are expressed as percentage of sicontrol quantities.
bars represent mean +/− s.d.
n =  <dig>  c western-blot analysis of samples from two independent transfections loaded on the same gel   fluorescence quantification of western blot.
d number of erk <dig> and erk <dig> mrna molecules per 25 ng of total rna from reptile samples: lizard , crocodile , turtle  and mouse nih3t <dig> cells.
bars represent means +/− s.d.
n =  <dig>  firefly luciferase activity driven by mouse and anole lizard erk promoters .
normalization by co-transfection of a plasmid expressing renilla luciferase driven by the thymidine kinase promoter.
transfection was performed in a. carolinensis fibroblasts  or mouse nih3t <dig> fibroblasts .
bars represent means +/− s.d.
n = 4
this raises the interesting question of why erk <dig> is not detected in squamates.
quantitative measurements of erk1/ <dig> mrna expression reveal that erk <dig> mrna expression is 12- to 24-fold higher than erk <dig> in a. carolinensis tissues .
this markedly contrasts with juvenile crocodile brain where erk <dig> mrna expression is 8-fold greater than erk <dig> and with mouse nih3t <dig> cells where erk <dig> mrna expression is 3- to 4-fold greater than erk <dig>  conversely, in adult turtle brain, erk <dig> and erk <dig> mrnas are expressed at similar levels .
in all these animals, protein expression of erk isoforms correlates well with mrna expression therefore lizards express undetectable erk <dig> protein due to minimal erk <dig> mrna expression.
we then compared opposite mammalian versus squamate erks expression by cloning mouse and anole lizard erk <dig> and erk <dig> promoters upstream of the luciferase reporter gene .
in cells from these organisms, transient transfection revealed that mouse erk <dig> promoter is much weaker than mouse erk <dig> promoter; conversely a. carolinensis erk <dig> promoter is markedly stronger than a. carolinensis erk <dig> promoter .
hence, the low expression of erk <dig> in mouse and low expression of erk <dig> in anole lizard seem to originate from the weakness of their respective promoters.
the difference of strength between mouse erk promoters is larger than the protein ratio observed in the same cells; therefore further research is needed to understand the individual contribution of promoters, rna regulation and protein stability to establish the final erk1/erk <dig> protein ratio.
erk <dig> evolve faster, but amino-acids required for kinase function are invariant in erk <dig> and erk2
in order to compare erks nucleotide and erks protein sequence evolution, we generated phylogenetic trees.
we found that erk nucleic sequences displayed similar branch lengths for erk <dig> and erk2; however, at the protein level, the branch lengths were markedly longer for erk <dig> proteins than for erk <dig> proteins  indicating that the rate of evolution at the gene level is similar between erk <dig> and erk <dig> but the rate of protein evolution is higher for erk <dig> than erk <dig>  this trend was confirmed by showing in table  <dig> that the synonymous mutation rates  of vertebrate erk <dig> and erk <dig> sequences are nearly identical  whereas the non-synonymous rate of mutation  is more elevated for erk <dig>  than erk <dig> .
indeed, erk <dig> was shown to display the lowest evolution rate of vertebrate map kinases  <cit> .
to scrutinize if differences of evolutionary rates between erk <dig> and erk <dig> is ongoing, we restricted our analysis on mammalian erk1/ <dig> pairs from the same organism, which also ensures that they underwent the same evolutionary pressure.
we show that the ka/ks ratio is extremely low along the mammalian erk <dig> sequence, which is typically the case during purifying selection .
erk <dig> is also under strong purifying selection along the sequence but not as much as erk <dig>  confirming that evolution of mammalian erk <dig> sequences is still greater than erk <dig> sequences .
this is confirmed by the global calculation of ka and ks ratios for  <dig> mammalian erks, which reveal that erk <dig> ka/ks ratio is  <dig>  fold higher than erk2’s .
by calculating shannon entropy along the sequences of all available pairs of full-length mammalian erk1/ <dig> we show that nucleotides divergences are widespread along the sequences of both erks , whereas mammalian erk <dig> proteins sequences diverge markedly more than erk <dig>  especially at the n- and c-terminal ends away from the kinase core .
on the 3d structure of human erk <dig>  the amino acids that diverge from consensus in at least 5 % of  <dig> mammalian erk1s were highlighted.
apart from ile <dig>  all amino-acids are localized at the surface of the molecule and ileu <dig> is replaced only with a valine which is a conservative change .
more importantly, all these divergent amino-acids are localized on the back of the kinase, distally from the catalytic cleft of the kinase and docking domains of erk for substrates and interactors .
hence this faster evolution of erk <dig> occurs at positions neutral for function.table  <dig> erk1s protein sequences evolve faster than erk2s’, not their nucleotide sequences
the synonymous substitution rates  and the non-synonymous substitution rates  of vertebrate erks and mammalian erks were calculated in mega <dig> as described in methods
fig.
<dig> only amino-acid neutral for function diverge among mammalian erks.
a ratio of non-synonymous rate  over synonymous rate  for all available erk <dig> and erk <dig> pairs of mammalian gene sequences .
multiple sequence alignment was generated and ka/ks values were then computed at each codon.
abscissa: distance from start atg codon.
the 5′ end of the sequences  could not be analyzed because some sequences were incomplete.
b still images were generated for 3d pdb structure 4qtb  displaying the following highlighted positions: amino-acids interacting with substrates forming the dejl motif are colored in blue, light blue for docking groove and darker blue for acidic patch; amino-acids interacting with substrates via the def domain  are colored in green; the threonine and tyrosine phosphorylated upon activation by mek are colored in white and the “face of the kinase” is circled by a dashed white line.
amino-acids that diverge among at least 5 % of  <dig> mammalian erk1s are colored in yellow .
i) front-side of the kinase where atp-transfer occurs in the catalytic cleft between the two lobes ii) left-side of the kinase iii) back-side of the kinase iv) right-side of the kinase
discussion
the present study reveals that erk <dig> and erk <dig> identities are well separated in tetrapods, indicative that they arose from a single duplication event.
the synteny between erk isoforms with ypel isoforms is conserved at least in human and zebrafish indicating that a segment of chromosome has duplicated.
furthermore, genome segments encompassing human erk <dig> and human erk <dig> display patterns of synteny with a single amphioxus linkage group  <cit> .
taken together, these elements are indicative that erk <dig> and erk <dig> likely originated from the vertebrate-specific whole genome duplications .
prior to bony fish, all vertebrates express only one erk.
considering that two rounds of wgd occurred prior to divergence of gnathostome lineages  <cit> , we conclude that paralogous erk duplicates were lost in chondrichthyes.
the resolution of phylogenetic analysis in these deep vertebrate branches does not allow to classify the single erk of agnathans as erk <dig> or erk <dig>  however extant chondrichthyes seem to express the erk <dig> isoform, a conclusion confirmed by the large size of the erk gene of elephant shark fish, a unique characteristic of erk <dig> genes in all bony vertebrates.
it has been hypothesized that gene duplicates are usually lost during evolution after a wgd if no new function appear among paralogs  <cit> .
the predominant loss of erk <dig> protein expression from cartilaginous fishes to birds, and lack of detectable erk <dig> expression in crocodiles mimics the lack of major phenotype of mice invalidated for the erk <dig> gene.
this could suggest that erk <dig> carries ancestral and essential erk function and therefore its expression is required in vertebrates.
at the protein level, however the results presented here within the reptile clade shatter this hypothesis since we demonstrate that erk <dig> is not detected in squamates.
the identities of lizard erk isoforms are kept since the sequence of erk <dig> from the lizard a. carolinensis is very similar to the sequence of mouse erk <dig>  and amino acid position that diverge between lizard erk <dig> and human erk <dig> are all localized distally from the face of the kinase .
similarly lizard erk <dig> sequence is 91 % identical to that of mouse erk <dig> and all divergent amino acids are located distally from the face of the kinase, except the conservative substitutions serine135-humanerk <dig> for threonine132-anoliserk <dig> and glutamic-acid194-humanerk <dig> for aspartic-acid191-anoliserk <dig> .
hence, in squamates, erk <dig> and erk <dig> have kept their identity but only erk <dig> is significantly expressed.
though undetectable relatively to erk <dig> by our methods, squamates’ erk <dig> is certainly expressed since it is still under selective pressure to remain free of nonsense mutations.
similarly, in crocodiles erk <dig> protein is undetectable relative to erk <dig> and its sequence remains fully functional however, it is unlikely to play a specific role since in the same clade of archosaurians since birds have lost erk <dig> altogether.
the extinction of erk <dig> protein expression in crocodiles may even be a premise to the loss of erk <dig> gene in birds since birds diverged later than crocodiles from their common ancestor.
we propose that both erks contribute to reach a global threshold concentration which maintains selective pressure on isoforms’ functionality.
even in adults, a minimum threshold of erk is required to sustain vertebrate life   <cit> .
considering that in vertebrates expressing both functional erk proteins, erk quantity can be overwhelmingly provided by erk <dig> or by erk <dig>  our results suggest that erk <dig> and erk <dig> are functionally redundant.
in mammals, two gene loci may increase the ability to regulate exquisitely the quantity of erk during all life stages.
during the revision of this manuscript, meloche and co-workers showed that mice completely lacking erk <dig> expression were rescued only when the erk <dig> was overexpressed, suggesting a full redundancy of erk1/ <dig> proteins during development up to normal reproduction  <cit> .
erk <dig> genes are lost more often than erk2’s in vertebrates and mammalian erk1s evolve faster than erk2s’ which raises the possibility that these two observations are linked.
in multicellular organisms three independent criteria determine the evolution rate of proteins  <cit> : gene essentiality, expression level  <cit>  and gene compactness  <cit> .
the faster evolution rate of erk <dig> cannot be attributed to differences in gene essentiality since so far no differences in functions have been demonstrated between erk <dig> and erk <dig> and because tetrapods express either one or both isoforms.
despite being a minor determinant of protein evolution in multicellular organisms, low protein expression of erk <dig> could play a role in the faster evolution rate of erk <dig> since erk <dig> is expressed usually at lower levels than erk <dig> .
more strikingly, the difference in gene compactness between mammalian erks is tremendous, erk <dig> gene being in average  <dig> fold smaller than erk <dig> .
several authors have correlated small gene size with faster evolution rate, by decreasing the probability of recombination between exons to purify mutations  compared to crocodile  and turtle .
furthermore squamates’ erk <dig> and erk <dig> proteins seem to evolve at similar rate since there is no branch length bias in the phylogenetic analysis, unlike for all other bony vertebrates .
hence there seem to be a correlation between lower differences of erk gene sizes in squamate and lower difference between erks protein evolution rates.
the availability of many squamate genomes is needed to confirm this trend.
hence, except in squamates that present erk genes of similar sizes, a larger gene size may explain the erk <dig> slower protein evolution, and specific regulation of erk <dig> expression derived from the larger gene size, may explain the observed erk <dig> prevalence in vertebrates.fig.
<dig> in vertebrates, erk <dig> genes are larger than erk <dig> genes.
genomes from vertebrates were screened for the presence of the full length sequences of erk genes, and then the size from atg to stop codon was calculated in kilobases.
in teleosts and mammals, animals for which both erk <dig> and erk <dig> genes sequences are available were preferred, only one pair of mammalian erks is not from the same animal.
full erk <dig> gene sequence of coelacanth  is not available.
for teleosts, the erk genes of t. fubripes and t. nigroviridis were not taken into account due to their known extreme genome compactness that would skew the size distribution  their erk <dig> gene size being  <dig> and  <dig>  kb respectively and their erk <dig> gene size being  <dig>  and 10 kb respectively).
for teleosts and mammals, the average sizes of the genes are written on the upper left side of the graphical bars, and the p-value that evaluates the statistical significance of the difference between erk <dig> and erk <dig> gene sizes is indicated.
for reptiles, the fold difference between the sizes of erk <dig> genes and erk <dig> genes is indicated on top of the bars with an arrow.
silhouettes are from http://phylopic.org/
CONCLUSIONS
our study suggests that erk <dig> and erk <dig> are functionally redundant in tetrapods since either one or both isoforms are expressed in animals from the same sauropsidian clade .
erk <dig> proteins evolve faster than erk <dig> proteins, even when the study was restricted to the mammalian clade; however amino acids diverge only at position neutral for function hence we propose that the origin of this difference originates from genomic factors such as the large gene size difference, but not from functional differences.
it remains to be determined whether the large gene size differences may also explain why erk <dig> genes are lost more readily than erk2’s.
when evaluating erk1/ <dig> invalidations, observed phenotypes should now be linked to the quantitative amount of total-erk reduction, not to functional differences among isoforms.
erks being the most expressed proteins of this signaling cascade, future research should aim at understanding the regulation and role of total erk quantity in cells.

 erks are the effector kinases of the ras/raf/mek/erk signaling pathway involved in multiple essential cell processes such as proliferation, differentiation, survival and memory formation. abnormal activation of this cascade leads to pathologies such as cancer or cognitive impairments. since the discovery of two erk isoforms in mammals, erk and erk in, numerous researchers have strived to understand their respective roles. while some cancers have been associated with isoforms of erk cascade members such as b-raf for melanoma, the putative differential involvement of either erk isoform in cancer or any disease remains unknown. erk and erk are expressed ubiquitously in mammals where both display the same kinase specific activity in vitro and share a highly similar 3d structure. furthermore in mammals, both translocate to the nucleus upon stimulation by cell surface receptors and while they do share at least interactors, no isoform-specific substrates have been identified. indeed, erk and erk share out of amino acids that have been demonstrated to directly interact with substrates, the sole difference being a conservative substitution: leucine155erk into isoleucine175erk. erk is regarded by many as essential due to the embryonic lethality of erk knock-out mice, whereas mice lacking erk are viable and fertile suggesting a dispensable role of erk. similarly, some studies based on sirna mediated-invalidations suggest specific functions. however, targeted erk and/or erk gene disruption in mice organs can evoke redundancy. in mouse fibroblasts we showed that solely si-rna mediated erk knock-down reduced cell proliferation by itself, however when erk levels were clamped down, erk knock-down became effective at reducing cell proliferation. hence, we and others have hypothesized that the apparent dominant role of erk is only due to its higher expression rather than functional differences. to date, the controversial question of erks differential function versus redundancy has not been successfully addressed. here we show by a combined approach based on erk1/ sequence evolution and erk1/ protein expression across vertebrates, that while some tetrapods express both erk and erk proteins, others have lost the erk gene and others express either only erk or only erk at detectable levels despite having both genes. hence our results strongly suggest that erk1/ can act interchangeably, a conclusion strengthened by the observation that amino-acids required for function are invariant in erk and erk  all mammals tested to date express both erk and erk from eutherians to the marsupial opossum. to search clues for specific functions or functional redundancy of erk isoforms, we studied protein expression of erk isoforms in parallel with the evolution of their sequences in all key vertebrate clades. we chose to study protein expression in brains to allow comparison across very divergent species and gain high sensitivity since in mammals, this organ has the greatest level of erk expression.. to ensure detection specificity we performed western blots incubated with anti-erk and anti-phospho-erk antibodies. in vertebrates, erk and erk are highly conserved, thus allowing various antibodies to recognize evolutionarily distant erks. furthermore, the epitope recognizing doubly-phosphorylated erk is conserved from sea anemones to humans, contributing to unambiguous protein identification. as a proof of principle for this approach, we can readily detect a single erk protein with three distinct anti-phospho erk antibodies and one antibody targeting total-erk in hagfish, the most evolutionary divergent vertebrate specie compared to mammals.fig. erk and erk protein appear first in bony vertebrates. western-blot analysis of samples from brain, spinal cord or cultured cells: immunoblots were incubated with polyclonal anti-erk antibody or anti-phosphoerk antibody. taxonomic names are listed in additional file predicted mw sizes of vertebrate erks range from  kda for lamprey’s erk to for zebrafish’s erk sc: spinal cord; ce: cerebellum; op: optic lobe. positions of erk erk phospho-erk and phospho-erk are indicated on the sides one erk is expressed in agnathans and chondrichthyes lamprey belongs to the same agnathan clade as hagfish and possesses only a single erk gene within its genome and we detected only one erk in lamprey’s brain extracts. phylogenetic analysis of hagfish and lamprey erks sequences highlight the similarity of the single erk from these species.fig. evolution of vertebrate erk sequences. a maximium-likelihood phylogenetic analysis of erk nucleotide and derived erk amino-acid sequences, at key nodes of vertebrate evolution. erk1/erk and erk2/erk phylogenies are indicated by red and blue branches respectively. key taxonomic clades for this manuscript are indicated by brackets. accession numbers and common names are listed in additional file branch length is indicative of degree of sequence divergences. the order of species in the derived trees is noted to vary slightly between different topological methods and whether it is calculated using protein or nucleotide sequences. given that all vertebrate erk sequences are highly conserved, minor variations in tree placement can be expected the next major clade in the vertebrate lineage, cartilaginous fishes are constituted of three sub-clades: chimeras, sharks and rays. among chimeras, the genome of the elephant shark is sequenced and has only a single erk. to extend this result to the two other classes of chondrichthyes we first analyzed erk expression in brains. we detected a sole erk isoform in the brain of winter skate, medulla of the small-spotted catshark and spinal cord of the spiny dogfish. using degenerated primers targeting teleost erk and erk we also amplified and cloned a single erk from winter skate and spiny dogfish, both of which share a high sequence identity to elephant shark erk. we conclude that members of the three clades of chondrichthyes express a single erk. the single erk of chondrichthyes is likely to be erk firstly because its sequence segregates within erk group by three independent topological methods, and the bootstrap values of the branches of fig.  that place chondrichthyes erk into the erk group support this finding. secondly, the size of the erk gene from elephant shark is large, similar to the large size of teleost and mammal erk genes and opposite of the extremely compact erk genes present in vertebrates as it will be described at the end of the result section. erk and erk from all bony vertebrates arose from a single duplication event all teleosts genomes sequenced so far possess two erk genes; and we readily detect two erk proteins from brain extracts of two representatives of the same teleost clade: dicentrarchus labrax and hemichromis bimaculatus. bichirs are extant representatives of the most ancient branch of ray-finned fishes, diverging from the rest of actinopterygii prior to the whole genome duplication specific of teleosts. therefore erk isoform identification in bichir is critical for determining erk and erk emergence in bony fish. we analyzed brain extracts from bichir and detected two erk isoforms and we further cloned two erk genes from p. senegalus. phylogenetic analysis of bichir erk isoforms revealed that they already fully segregate at gene and protein levels as erk and erk as classified in teleosts and tetrapods. this demonstrates duplicate erk1/ emergence at least mya prior to the divergence of the bichirs from primitive bony vertebrates. furthermore, in human and zebrafish genomes ypel gene is located downstream of erk1/mapk gene in the same orientation than erk1; and ypel is located downstream of erk2/mapk in the same orientation than erk2; ypel and ypel being also isoforms therefore the synteny of isoforms is conserved from fish to mammals between these two loci, which is further indicative of a single duplication event that generated erk and erk in vertebrates. erk gene has been lost independently in two tetrapod clades western blot studies and gene sequencing revealed that several extant members of the three tetrapod clades express both erk and erk2: turtles for the sauropsid clade fig. 3a), axolotl for the amphibian clade and mouse for the mammalian clade. however, only one erk was detected in western blots of protein extracts from african clawed frog and chicken. the search of exons in fully sequenced genomes combined with the screening of est databases reveals that the erk gene is absent from the genomes of xenopus tropicalis, chicken and other recently published genomes of neo-gnathae. furthermore, erk protein expression is not detectable in the brain of the paleo-gnathae nandu, and no erk gene was identified in the genome of the paleo-gnathae ostrich. these observations suggest that erk has been lost in all bird lineages and in some amphibians. as summarized in table  only erk gene has been lost in some tetrapods and all chondrichthyes.fig. erk is not detected in snakes and lizards. a, b western-blot analysis performed as in fig.  incubated with a polyclonal anti-erk antibody or anti-phosphoerk antibody. a brain extracts from snakes, lizards, crocodile and turtles. control brain extracts from cattle. co: cortex, ce: cerebellum, me: medulla, ol: olfactory bulb, pi: pineal gland, w: whole brain. note that degraded bands are observed in turtle e. orbicularis likely due postmortem sampling. b extracts from lizard embryo fibroblasts were stimulated or not one hour in presence of fcs alone or fcs + phosphatase inhibitor navo4— to activate erks mus musculus bos taurus cricetulus griseus oryctolagus cuniculus monodelphis domestica ornithorhynchus anatinus gallus gallus o rhea americana o crocodylus niloticus thamnophis elegans pantherophis guttatus pituophis catenifer sayi anolis carolinensis anolis sagrei tarentola mauritanica trachemys scripta elegans emys orbicularis xenopus laevi o ambiostoma mexicanum latimeria chalumnae dicentrarchus labrax hemichromis bimaculatus polypterus senegalus squalus acanthias o scyliorhinus canicula o raja ocellata o genomes and est databases were scrutinized for the presence of erk1/ exons, if gene is present: ✓ if gene is absent: o presence of proteins readily detected on western blots in this work is indicated: ✓ nd not determined in reptiles either one or both erks are expressed both erk proteins are expressed in turtle brain, however crocodiles appear to express only erk protein in the same organ. by contrast, we could only detect erk protein in snakes and lizards. side-by-side loading of cerebellum extracts from lizard, crocodile and turtle confirm these observations. furthermore, an apparent lack of erk was observed in brain extracts from all squamate species tested: lizards, snakes and one gecko. the two reptile genomes sequenced so far possess erk and erk; therefore to confirm the surprising absence of erk protein in the model animal for this clade, a. carolinensis, we first confirmed the absence of erk expression in an array of a. carolinensis adult tissues. we then derived primary lizard embryo fibroblasts from mid-gestation embryos. even when erk was hyper-stimulated by serum in presence of orthovanadate to increase detection with anti-phospho erk antibodies, only a single erk1-sized band was observed. to unambiguously assign protein identity and remove the possibility that two erk proteins are present and co-migrate, lefs were transfected with pools of three independent sirna sequences targeting erk or erk at minimum, each sirna displays differences with the non-targeted isoform to ensure isoform-specificity. efficiency and isoform specificity of each sirna pool was verified by measuring erk1/ mrna levels post-transfection. three days post-transfection, the expression level of a. carolinensis erk protein was consistently lowered by sirna targeting erk whereas sirna targeting erk had no effect. therefore, we suggest with a strong degree of evidence that erk is the only protein isoform detected in anole lizard, and more generally in squamates.fig. only erk protein and erk mrna are expressed significantly in lizards. a sirna sequences targeting anole lizard erk mrna and anole lizard erk mrna. in red, the nucleotides that differ when both erk and erk isoforms are aligned. b, c sirna pools targeting erk or erk or control-sirna were transfected three days prior to mrna harvesting. b absolute quantification of erk mrna or erk mrna after sirna transfection, in a. sagrei and a. carolinensis lizard embryo fibroblasts. erk or erk mrna quantities were determined relatively to quantities of linearized plasmids harboring either a. carolinensis erk or erk cdnas as described in materials and methods. to ease comparison between erk and erk data are expressed as percentage of sicontrol quantities. bars represent mean +/− s.d. n =  c western-blot analysis of samples from two independent transfections loaded on the same gel fluorescence quantification of western blot. d number of erk and erk mrna molecules per 25 ng of total rna from reptile samples: lizard, crocodile, turtle and mouse nih3t cells. bars represent means +/− s.d. n =  firefly luciferase activity driven by mouse and anole lizard erk promoters. normalization by co-transfection of a plasmid expressing renilla luciferase driven by the thymidine kinase promoter. transfection was performed in a. carolinensis fibroblasts or mouse nih3t fibroblasts. bars represent means +/− s.d. n = 4 this raises the interesting question of why erk is not detected in squamates. quantitative measurements of erk1/ mrna expression reveal that erk mrna expression is 12- to 24-fold higher than erk in a. carolinensis tissues. this markedly contrasts with juvenile crocodile brain where erk mrna expression is 8-fold greater than erk and with mouse nih3t cells where erk mrna expression is 3- to 4-fold greater than erk conversely, in adult turtle brain, erk and erk mrnas are expressed at similar levels. in all these animals, protein expression of erk isoforms correlates well with mrna expression therefore lizards express undetectable erk protein due to minimal erk mrna expression. we then compared opposite mammalian versus squamate erks expression by cloning mouse and anole lizard erk and erk promoters upstream of the luciferase reporter gene. in cells from these organisms, transient transfection revealed that mouse erk promoter is much weaker than mouse erk promoter; conversely a. carolinensis erk promoter is markedly stronger than a. carolinensis erk promoter. hence, the low expression of erk in mouse and low expression of erk in anole lizard seem to originate from the weakness of their respective promoters. the difference of strength between mouse erk promoters is larger than the protein ratio observed in the same cells; therefore further research is needed to understand the individual contribution of promoters, rna regulation and protein stability to establish the final erk1/erk protein ratio. erk evolve faster, but amino-acids required for kinase function are invariant in erk and erk2 in order to compare erks nucleotide and erks protein sequence evolution, we generated phylogenetic trees. we found that erk nucleic sequences displayed similar branch lengths for erk and erk2; however, at the protein level, the branch lengths were markedly longer for erk proteins than for erk proteins indicating that the rate of evolution at the gene level is similar between erk and erk but the rate of protein evolution is higher for erk than erk this trend was confirmed by showing in table  that the synonymous mutation rates of vertebrate erk and erk sequences are nearly identical whereas the non-synonymous rate of mutation is more elevated for erk than erk. indeed, erk was shown to display the lowest evolution rate of vertebrate map kinases. to scrutinize if differences of evolutionary rates between erk and erk is ongoing, we restricted our analysis on mammalian erk1/ pairs from the same organism, which also ensures that they underwent the same evolutionary pressure. we show that the ka/ks ratio is extremely low along the mammalian erk sequence, which is typically the case during purifying selection. erk is also under strong purifying selection along the sequence but not as much as erk confirming that evolution of mammalian erk sequences is still greater than erk sequences. this is confirmed by the global calculation of ka and ks ratios for mammalian erks, which reveal that erk ka/ks ratio is fold higher than erk2’s. by calculating shannon entropy along the sequences of all available pairs of full-length mammalian erk1/ we show that nucleotides divergences are widespread along the sequences of both erks, whereas mammalian erk proteins sequences diverge markedly more than erk especially at the n- and c-terminal ends away from the kinase core. on the 3d structure of human erk the amino acids that diverge from consensus in at least 5 % of mammalian erk1s were highlighted. apart from ile all amino-acids are localized at the surface of the molecule and ileu is replaced only with a valine which is a conservative change. more importantly, all these divergent amino-acids are localized on the back of the kinase, distally from the catalytic cleft of the kinase and docking domains of erk for substrates and interactors. hence this faster evolution of erk occurs at positions neutral for function.table erk1s protein sequences evolve faster than erk2s’, not their nucleotide sequences the synonymous substitution rates and the non-synonymous substitution rates of vertebrate erks and mammalian erks were calculated in mega as described in methods fig. only amino-acid neutral for function diverge among mammalian erks. a ratio of non-synonymous rate over synonymous rate for all available erk and erk pairs of mammalian gene sequences. multiple sequence alignment was generated and ka/ks values were then computed at each codon. abscissa: distance from start atg codon. the 5′ end of the sequences could not be analyzed because some sequences were incomplete. b still images were generated for 3d pdb structure 4qtb displaying the following highlighted positions: amino-acids interacting with substrates forming the dejl motif are colored in blue, light blue for docking groove and darker blue for acidic patch; amino-acids interacting with substrates via the def domain are colored in green; the threonine and tyrosine phosphorylated upon activation by mek are colored in white and the “face of the kinase” is circled by a dashed white line. amino-acids that diverge among at least 5 % of mammalian erk1s are colored in yellow. i) front-side of the kinase where atp-transfer occurs in the catalytic cleft between the two lobes ii) left-side of the kinase iii) back-side of the kinase iv) right-side of the kinase discussion the present study reveals that erk and erk identities are well separated in tetrapods, indicative that they arose from a single duplication event. the synteny between erk isoforms with ypel isoforms is conserved at least in human and zebrafish indicating that a segment of chromosome has duplicated. furthermore, genome segments encompassing human erk and human erk display patterns of synteny with a single amphioxus linkage group. taken together, these elements are indicative that erk and erk likely originated from the vertebrate-specific whole genome duplications. prior to bony fish, all vertebrates express only one erk. considering that two rounds of wgd occurred prior to divergence of gnathostome lineages, we conclude that paralogous erk duplicates were lost in chondrichthyes. the resolution of phylogenetic analysis in these deep vertebrate branches does not allow to classify the single erk of agnathans as erk or erk however extant chondrichthyes seem to express the erk isoform, a conclusion confirmed by the large size of the erk gene of elephant shark fish, a unique characteristic of erk genes in all bony vertebrates. it has been hypothesized that gene duplicates are usually lost during evolution after a wgd if no new function appear among paralogs. the predominant loss of erk protein expression from cartilaginous fishes to birds, and lack of detectable erk expression in crocodiles mimics the lack of major phenotype of mice invalidated for the erk gene. this could suggest that erk carries ancestral and essential erk function and therefore its expression is required in vertebrates. at the protein level, however the results presented here within the reptile clade shatter this hypothesis since we demonstrate that erk is not detected in squamates. the identities of lizard erk isoforms are kept since the sequence of erk from the lizard a. carolinensis is very similar to the sequence of mouse erk and amino acid position that diverge between lizard erk and human erk are all localized distally from the face of the kinase. similarly lizard erk sequence is 91 % identical to that of mouse erk and all divergent amino acids are located distally from the face of the kinase, except the conservative substitutions serine135-humanerk for threonine132-anoliserk and glutamic-acid194-humanerk for aspartic-acid191-anoliserk. hence, in squamates, erk and erk have kept their identity but only erk is significantly expressed. though undetectable relatively to erk by our methods, squamates’ erk is certainly expressed since it is still under selective pressure to remain free of nonsense mutations. similarly, in crocodiles erk protein is undetectable relative to erk and its sequence remains fully functional however, it is unlikely to play a specific role since in the same clade of archosaurians since birds have lost erk altogether. the extinction of erk protein expression in crocodiles may even be a premise to the loss of erk gene in birds since birds diverged later than crocodiles from their common ancestor. we propose that both erks contribute to reach a global threshold concentration which maintains selective pressure on isoforms’ functionality. even in adults, a minimum threshold of erk is required to sustain vertebrate life. considering that in vertebrates expressing both functional erk proteins, erk quantity can be overwhelmingly provided by erk or by erk our results suggest that erk and erk are functionally redundant. in mammals, two gene loci may increase the ability to regulate exquisitely the quantity of erk during all life stages. during the revision of this manuscript, meloche and co-workers showed that mice completely lacking erk expression were rescued only when the erk was overexpressed, suggesting a full redundancy of erk1/ proteins during development up to normal reproduction. erk genes are lost more often than erk2’s in vertebrates and mammalian erk1s evolve faster than erk2s’ which raises the possibility that these two observations are linked. in multicellular organisms three independent criteria determine the evolution rate of proteins: gene essentiality, expression level and gene compactness. the faster evolution rate of erk cannot be attributed to differences in gene essentiality since so far no differences in functions have been demonstrated between erk and erk and because tetrapods express either one or both isoforms. despite being a minor determinant of protein evolution in multicellular organisms, low protein expression of erk could play a role in the faster evolution rate of erk since erk is expressed usually at lower levels than erk. more strikingly, the difference in gene compactness between mammalian erks is tremendous, erk gene being in average fold smaller than erk. several authors have correlated small gene size with faster evolution rate, by decreasing the probability of recombination between exons to purify mutations compared to crocodile and turtle. furthermore squamates’ erk and erk proteins seem to evolve at similar rate since there is no branch length bias in the phylogenetic analysis, unlike for all other bony vertebrates. hence there seem to be a correlation between lower differences of erk gene sizes in squamate and lower difference between erks protein evolution rates. the availability of many squamate genomes is needed to confirm this trend. hence, except in squamates that present erk genes of similar sizes, a larger gene size may explain the erk slower protein evolution, and specific regulation of erk expression derived from the larger gene size, may explain the observed erk prevalence in vertebrates.fig. in vertebrates, erk genes are larger than erk genes. genomes from vertebrates were screened for the presence of the full length sequences of erk genes, and then the size from atg to stop codon was calculated in kilobases. in teleosts and mammals, animals for which both erk and erk genes sequences are available were preferred, only one pair of mammalian erks is not from the same animal. full erk gene sequence of coelacanth is not available. for teleosts, the erk genes of t. fubripes and t. nigroviridis were not taken into account due to their known extreme genome compactness that would skew the size distribution their erk gene size being and  kb respectively and their erk gene size being and 10 kb respectively). for teleosts and mammals, the average sizes of the genes are written on the upper left side of the graphical bars, and the p-value that evaluates the statistical significance of the difference between erk and erk gene sizes is indicated. for reptiles, the fold difference between the sizes of erk genes and erk genes is indicated on top of the bars with an arrow. silhouettes are from http://phylopic.org/  our study suggests that erk and erk are functionally redundant in tetrapods since either one or both isoforms are expressed in animals from the same sauropsidian clade. erk proteins evolve faster than erk proteins, even when the study was restricted to the mammalian clade; however amino acids diverge only at position neutral for function hence we propose that the origin of this difference originates from genomic factors such as the large gene size difference, but not from functional differences. it remains to be determined whether the large gene size differences may also explain why erk genes are lost more readily than erk2’s. when evaluating erk1/ invalidations, observed phenotypes should now be linked to the quantitative amount of total-erk reduction, not to functional differences among isoforms. erks being the most expressed proteins of this signaling cascade, future research should aim at understanding the regulation and role of total erk quantity in cells. 
our results demonstrate that tetrapods can live by expressing either one or both erk isoforms, supporting the notion that erk1/ act interchangeably. two erk isoforms have been described, erk and erk2; erk being regarded by many as essential due to the embryonic lethality of erk knock-out mice, whereas mice lacking erk are viable and fertile. to investigate this question we used a novel approach based on comparing the evolution of erk isoforms’ sequences and protein expression across vertebrates. erk isoforms appeared by a single gene duplication at the onset of vertebrate evolution at least mya. we suggest that future erk research should shift towards understanding the role and regulation of total erk quantity, especially in light of newly described erk gene amplification identified in tumors. finally, phylogenetic analysis revealed that erk amino acids sequences evolve faster than erk2’s likely due to genomic factors, including a large difference in gene size, rather than from functional differences since amino acids essential for function are kept invariant. we gathered and cloned erk and erk coding sequences and we examined protein expression of isoforms in brain extracts in all major clades of vertebrate evolution. to demonstrate the unexpected squamates’ lack of erk expression, we targeted each erk isoform in lizard primary fibroblasts by specific sirna-mediated knockdown. substrate recognition sites and catalytic cleft are nearly invariant in all vertebrate erks further suggesting functional redundancy. the controversial question of why we have two erks and whether they have differential functions or display functional redundancy has not yet been resolved.
 the ras/raf/mek/erk signaling pathway is involved in essential cell processes and it is abnormally activated in ~30 % of cancers and cognitive disorders. two erk isoforms have been described, erk and erk2; erk being regarded by many as essential due to the embryonic lethality of erk knock-out mice, whereas mice lacking erk are viable and fertile. the controversial question of why we have two erks and whether they have differential functions or display functional redundancy has not yet been resolved.  to investigate this question we used a novel approach based on comparing the evolution of erk isoforms’ sequences and protein expression across vertebrates. we gathered and cloned erk and erk coding sequences and we examined protein expression of isoforms in brain extracts in all major clades of vertebrate evolution. for the first time, we measured each isoforms’ relative protein level in phylogenetically distant animals using anti-phospho antibodies targeting active erks. we demonstrate that squamates, despite having both genes, do not express erk protein whereas other tetrapods either do not express erk protein or have lost the erk gene. to demonstrate the unexpected squamates’ lack of erk expression, we targeted each erk isoform in lizard primary fibroblasts by specific sirna-mediated knockdown. we also found that undetectable expression of erk in lizard is compensated by a greater strength of lizard’s erk promoter. finally, phylogenetic analysis revealed that erk amino acids sequences evolve faster than erk2’s likely due to genomic factors, including a large difference in gene size, rather than from functional differences since amino acids essential for function are kept invariant.  erk isoforms appeared by a single gene duplication at the onset of vertebrate evolution at least mya. our results demonstrate that tetrapods can live by expressing either one or both erk isoforms, supporting the notion that erk1/ act interchangeably. substrate recognition sites and catalytic cleft are nearly invariant in all vertebrate erks further suggesting functional redundancy. we suggest that future erk research should shift towards understanding the role and regulation of total erk quantity, especially in light of newly described erk gene amplification identified in tumors. electronic supplementary material the online version of this article contains supplementary material, which is available to authorized users. issue-copyright-statement© the author 2015 