BACKGROUND
extracellular signal-regulated kinase  <dig>  is a member of the mitogen-activated protein kinase  family of kinases activated in response to numerous growth factors and cytokines, leading to phosphorylation and functional regulation of downstream targets.
erk <dig> has been linked to more than  <dig> different substrates whose phosphorylation by erk <dig> is orchestrated by coordination of signaling networks through common binding to so-called scaffold proteins  <cit> .
the definition of a scaffold protein was recently refined and their identification as such suggested from qualities of multivalent binding, non-catalytic placeholders, and bidirectional process control  <cit> .
several scaffold proteins have been described for the mapks such as kinase suppressor of ras   <cit> , jnk-interacting protein   <cit> , iq motif containing gtpase activating protein  <dig>   <cit> , and β-arrestin  <cit> , which interact with members of the mapk cascade, providing multivalency, spatial concentration, and/or signaling fidelity.
however, although mapks are known to regulate the action of several membrane proteins and receptors, none of these scaffold proteins are themselves membrane proteins, requiring additional mechanisms for colocalization of the scaffold protein, the membrane protein, as well as the kinases.
moreover, most of the available molecular insights are from structures of kinases in complex with folded domains or with small peptides of the scaffold proteins, and details regarding scaffolding by non-globular proteins are lacking.
mapks are s/t kinases that interact with targets and regulators via two types of domains, d-domains and f-sites .
d-domains, also known as docking sites for erk and jnk, lxl  domains, or kinase interaction motifs  have the canonical sequence of 2– <dig> basic residues , spaced by 1– <dig> residues to a hydrophobic motif ΦxΦ, where Φ is generally v, l, or i  <cit> .
d-domains are found in mapk substrates such as the transcription factor elk- <dig> and p <dig> ribosomal s kinase , as well as in other mapk targets  <cit> .
in erk <dig>  d-domains interact with the d-domain recognition site also known as the cd/ed  domain, located more than 10 Å from the active site  <cit> .
the f-site recruitment site in erk <dig> is much less studied and incompletely understood.
it binds to f-sites, also called def -domains with the canonical fxfp sequence  <cit> .
f-sites allow for aromatic residues at the p <dig>  and p <dig> positions   <cit> , and f-sites have been reported in substrates such as elk- <dig>   <cit>  and c-fos   <cit> , and within the nucleoporin fg-repeats   <cit> .
so far the only structure available of an f-site recruitment site-interacting protein is that of erk <dig> in complex with the 15 kda phosphoprotein enriched in astrocytes , which notably lacks any of the above-mentioned motifs  <cit> .
the plasma membrane na+/h+ exchanger  <dig>  is a major regulator of ph and volume in essentially all cells studied.
furthermore, nhe <dig> is involved in the regulation of cell proliferation, survival, motility, and other essential physiological processes, and its dysregulation contributes importantly to major human malignancies, including cancer and cardiovascular diseases  <cit> .
numerous hormones and growth factors acting via receptor tyrosine kinases or gtp-binding protein-coupled receptors can elicit posttranslational regulation of nhe <dig> .
the mapks erk1/ <dig>  p <dig> mapk, and c-jun n-terminal kinase  are widely implicated in nhe <dig> regulation , and direct phosphorylation of human  nhe <dig> by erk1/ <dig> was previously proposed based on 32p measurements  <cit>  and mass spectrometry  <cit> .
conversely, nhe <dig> has been reported to regulate signaling through regulation of erk1/ <dig> and p <dig> mapk activity , and yeast two-hybrid screens have suggested the interaction of nhe <dig> with several members of the mapk hierarchy  <cit> .
however, with the exception of the interaction with b-raf  <cit> , evidence from mammalian systems is lacking, and the possible sites of nhe1-mapk interaction, its structural details, and possible functional consequences are unexplored.
we recently showed by pondr and disopred predictions, as well as by nuclear magnetic resonance  spectroscopy and other biophysical techniques, that the distal ~  <dig> residues of the hnhe <dig> c-terminal intracellular domain , containing most of the known nhe <dig> phosphorylation sites, are intrinsically disordered   <cit> .
to our knowledge, no studies have yet addressed the mechanisms through which mapks interact with id proteins , although about one third of all proteins in higher eukaryotes contain significant id regions   <cit> , and id is abundant in cellular signalling  <cit> , scaffolding  <cit> , as well as in mapks themselves  <cit> .
here, we demonstrate that hnhe <dig> acts as an erk <dig> membrane protein scaffold in vivo that is necessary for erk <dig> activation via direct interactions, and we show that loss of scaffolding by hnhe <dig> leads to decreased erk <dig> activation.
using nmr spectroscopy we show that nhe <dig> scaffolds inactive  erk <dig> in a “shuffle complex” that involves a d-domain and two non-canonical f-sites.
we characterize the order and kinetics of both previously reported and novel erk2-mediated phosphorylations of hnhe <dig> in vitro.
our findings provide a molecular mechanism for the widely recognized and functionally important scaffolding function of hnhe <dig>  and give mechanistic insight into the regulation of erk <dig> activity by the intrinsically disordered hnhe1cdt.
RESULTS
the recently suggested links between hnhe <dig> and erk1/ <dig> prompted us to investigate whether erk1/ <dig> and nhe <dig> directly interact in a cellular context.
using ap- <dig> cells  stably expressing full-length wt hnhe <dig>  we asked if hnhe <dig> and erk1/ <dig> engage in a direct interaction in vivo.
interaction was assessed using in situ proximity ligation assay , which interrogates close interaction  between proteins .
as seen, the detection of multiple pla puncta when cells were incubated with both nhe <dig> and erk1/ <dig> antibodies revealed the presence of hnhe1-erk1/ <dig> complexes in ap- <dig> wt hnhe <dig> cells , compared to a much lower signal in negative controls incubated with one antibody only .
data from multiple experiments are quantified in fig.
1c, demonstrating that the pla signal is significantly greater in nhe1-erk antibody-labelled cells than in negative controls.
thus, these data show that hnhe <dig> and erk1/ <dig> directly interact in the cell.fig.
<dig> direct interaction between erk <dig> and nhe <dig> in vivo and in silico.
a proximity ligase assay carried out in ap- <dig> wt hnhe <dig> cells treated with nhe <dig> and erk1/ <dig> primary antibodies.
proximity ligase signal appears as red dots and the merge image highlights the interaction between nhe <dig> and erk.
f-actin was stained with phalloidin <dig>  scale bars represent 10 μm.
data are representative of three independent replicates.
b proximity ligase assay carried out in ap- <dig> wt hnhe <dig> cells.
as a negative control, cells were only treated with nhe <dig> primary antibodies and the appearance of the red dots indicates unspecific binding.
f-actin was stained with phalloidin <dig>  scale bars represent 10 μm.
data are representative of three independent replicates.
c quantification of pla data was carried out in imagej.
pla signal from at least ten different image areas in each experiment were counted by particle analysis and the average pla signal per cell was plotted in the bar graph.
data are representative of three independent replicates.
d erk1/ <dig> docking motifs, d-domain and f-site.
Φ indicates hydrophobic amino acid residues , and x any other amino acid.
e overall nhe <dig> topology and localization of putative d-domains and erk <dig> phosphorylation sites in the hnhe1cdt.
the positions of identified d-domains as well as predicted p-phosphorylation sites are indicated by stars.
insert: alignment of the consensus erk <dig> phosphorylation sites in hnhe1; p sites indicated by grey background.
f alignment of hnhe <dig> d-domains to known d-domains.
the consensus hydrophobic and positively charged residues are highlighted in yellow and blue, respectively.
g sequence conservation of putative d-domains in nhe1cdt in various species.
d <dig>  d <dig>  and d <dig> are indicated with grey bars above the alignment.
the solid horizontal line separates tetrapods  from teleosts   <cit>
the disordered tail of hnhe <dig> interacts with iaerk2
scrutinizing the intracellular domain of nhe <dig> by in silico methods for potential erk1/ <dig> interaction sites identified three potential d-domains in the intrinsically disordered region   <cit> , 706– <dig> , 732– <dig> , and 676– <dig>   .
the previously described conserved tv-box is part of the d3-domain  <cit> , and this d-domain is the only one conserved throughout nhe <dig> evolution .
since erk <dig> and erk <dig> are 84 % identical by sequence and share many if not all functions  <cit> , and erk <dig> is the more widely studied enzyme of the two, we focused in vitro studies on erk <dig>  to discriminate between the roles of each d-domain, we investigated the interaction between the disordered tail of nhe <dig> ) and recombinantly produced human iaerk <dig> by nmr spectroscopy, which previously had provided insight into the transient structure and conserved regions of hnhe1cdt  <cit>  .
first, we measured perturbations of chemical shifts and peak intensities of the hnhe1cdt wt arising from addition of iaerk <dig> to a 1: <dig> molar ratio using 15n,1h-hsqc spectra .
signals from all residues of the d3-domain disappeared, and decreased intensities and chemical shift perturbations were observed in the c-terminal neighbouring residues, suggesting this domain engages in the interaction.
additionally, two phe residues in the distal end of hnhe1cdt 809– <dig>  as well as a phe residue within a potential substrate site, 778– <dig>  were highly perturbed.
importantly, although the latter resembles a substrate site, both are reminiscent of the canonical erk f-site motif, suggesting several interaction sites between hnhe1cdt and iaerk <dig>  additional yet minor perturbations were observed in the linking regions between these sites, which were partially caused by slight ph variations .
since some residues in d <dig> are unassigned due to their overlap in the nmr spectra, and perturbations were observed close to this site , we cannot exclude d <dig> to also contribute to the interaction.
lastly, as escherichia coli expression of erk <dig> can lead to autophosphorylation of y <dig>  <cit> , we assessed the level of erk <dig> autophosphorylation by native page, showing that more than 80 % of erk <dig> is non- and less than 20 % mono-phosphorylated .
to ensure that this had no effect on the interaction with nhe1cdt we fully dephosphorylated erk <dig> with the tyr phosphatase heptp and re-analysed the interaction, which gave an identical binding profile , in accordance with the low activity of the erk <dig> mono-phosphorylated state  <cit> .
in conclusion, nhe1cdt interact with iaerk <dig> through multiple contact sites involving the d3-domain, as the dominating d-domain, and two f-sites .fig.
<dig> inactive erk <dig> interacts preferably with the d3-domain and two f-sites of hnhe1cdt.
a position of transient helices , tv- and lsd-boxes , predicted d-domains and f-sites , as well as erk <dig> phosphorylation sites  in the hnhe1cdt.
b combined chemical shift perturbations Δδ of wt hnhe1cdt by iaerk <dig> interaction.
c
15n,1h-hsqc peak intensity ratios of hnhe1cdt wt in the presence/absence of iaerk <dig>  d difference in relaxation rates Δr
<dig> between hnhe1cdt and nhe1cdt:iaerk <dig>  e–g
15n,1h-hsqc peak intensity ratios of e hnhe1cdt d3-axa, f hnhe1cdt f1-a, and g hnhe1cdt f2-aa in the presence/absence of iaerk <dig>  h internally urea referenced secondary cα chemical shifts  of wt hnhe1cdt identify the presence of several transient helices .
● indicate the position of prolines, □ unassigned residues, and blue box severe peak overlap
we next investigated whether interfering with any of these contact sites would affect the interactions and first exploited the knowledge that mapk interaction is severely perturbed by mutations of ΦxΦ to axa in d-domains  <cit> .
hence, we constructed axa variants of all three d-domains alone and in combination, both in the full-length hnhe <dig>  for cellular studies and in hnhe1cdt  for in vitro studies.
in d3-axa, chemical shift perturbations in the two f-sites upon erk <dig> addition were preserved in the interaction with iaerk <dig>  whereas no perturbations were observed in the axa-mutated d3-domain, implying that this site is important for the interaction .
no notable effects of d1- and d2-axa substitutions were observed , arguing against their involvement in the interactions.
this data also indicated that the f-sites interacted with iaerk <dig> independently of the d-domain.
to assess this further the f-sites were individually mutated by substituting ftp778– <dig> with atp778– <dig>  and fp811– <dig> with aa811– <dig> .
both f-site variants showed strongly decreased chemical shift perturbations at the mutation sites upon erk <dig> addition, leaving the other f-site and the d3-domain unaffected .
this conclusively identified all three sites as erk <dig> interaction sites.
further, substitutions at each site left the other sites unaffected, indicating that these regions of hnhe <dig> interact independently with iaerk <dig>
nhe <dig> does not fold upon binding to erk <dig> but may be a flexible wrapper
to further address how hnhe1cdt interacted with iaerk <dig> we analysed the complex by size exclusion chromatography  and compared elution profiles with those of the individual proteins .
since hnhe1cdt is an idr, it has a larger hydrodynamic radius than iaerk, and hnhe1cdt thus eluted first from the column.
subtracting individual runs from that of the mixture revealed a broad peak with an elution volume smaller than that of iaerk <dig>  yet larger than that of hnhe1cdt, suggesting that hnhe1cdt folds or wraps around iaerk <dig>  circular dichroism  spectroscopic analyses , as well as nmr chemical shift analyses , did not indicate folding upon binding formation of significant secondary structure, suggesting that hnhe1cdt forms a relatively extended structure around iaerk <dig>  to substantiate this conclusion, we recorded 15n transverse relaxation rates of the unbound  and the iaerk2-bound hnhe1cdt , and analysed their differences .
since d <dig> residues broadened beyond detection in the complex, their r <dig> values could not be extracted.
however, for residues interacting with iaerk <dig>  a significant increase in r <dig> rates is expected compared to those of hnhe1cdt alone, due to the larger radius of gyration of the complex or due to chemical exchange between different states.
indeed, residues from both f-sites had substantially larger r <dig> rates in the complex compared to hnhe1cdt alone, and many residues between these sites were also affected, although not to the same extent.
this result supports the sec results and suggests a substantial interaction area between the two proteins.
the loss of nmr signal upon binding imposes a challenge for obtaining affinity constants.
although nmr peak intensities are only directly proportional to the populations when all states can be accounted for, they may provide apparent upper-limit affinities.
from a titration of hnhe1cdt with iaerk <dig>  global fitting of intensity changes using residues ltv684– <dig> of the d3-domain gave a kdapp of 16 ± 2 μm, while the other interaction sites had relatively lower affinities with apparent affinities, kdapp of 86 ± 26 μm  and 69 ± 14 μm  .
attempts to substantiate this and obtain stoichiometries by isothermal titration calorimetry or microscale thermophoresis were unsuccessful, due to limited stability of concentrated erk <dig>  as well as adhesion of erk <dig> to the capillary tubing.
instead, to determine the stoichiometry of the interaction we used non-denaturing mass spectrometry, allowing the non-covalent complex to be captured at low micromolar concentration.
deconvolution of the spectrum using in-house software  <cit>  revealed three main charge state envelopes, corresponding to hnhe1cdt , iaerk <dig> , and from the 1: <dig> complex  .
a minor population of iaerk <dig> dimers was also detected , most likely due to the presence of a hexahistidine-tag, as noted  <cit> .
these data unequivocally showed that hnhe1cdt and iaerk <dig> form a 1: <dig> complex, fully in-line with the perturbation of the relaxation times and the size exclusion data.
collectively, these data indicated that nhe1cdt binds to erk <dig> with medium affinity, through at least three independent contact sites forming a 1: <dig> complex.fig.
<dig> native mass spectrum  and molecular model of the hnhe1cdt:iaerk <dig> complex.
a non-denaturing mass spectrum of the hnhe1-erk <dig> complex, acquired from 200 mm ammonium acetate.
deconvolution was performed using the unidec software and the individual charge series fits are displayed below, corresponding to nhe <dig> , erk <dig> , nhe1:erk <dig> complex , and minor contribution of erk <dig> dimer .
b proposed model for the hnhe1-erk <dig> interactions.
the hnhe1cdt interacts with erk <dig> at three sites, exploiting a d-domain and two f-sites .
at the d-domain recognition site, k <dig>  l <dig>  and v <dig> of the hnhe1cdt d3-domain, as well as d <dig>  d <dig>  y <dig>  and h <dig> of the iaerk <dig> d-domain recognition site are shown as sticks
finally, we used the nmr chemical shifts and relaxation data together with known structures of iaerk <dig> complexes to model the hnhe1cdt-iaerk <dig> interaction .
when modelled at the d-domain recognition site of iaerk <dig>  each individual d-domain  when bound to the d-domain recognition site allowed for either of the f-sites  to reach the f-site recognition site.
in each case the model predicted long id linkers between the binding sites, which were long enough to allow nhe <dig> to wrap around iaerk <dig>  this is in accordance with the nmr data and supports their uncoupled behaviour.
collectively, these data suggest that hnhe1cdt could interact with iaerk <dig> in a tripartite 1: <dig> interaction exploiting a d-domain  and two f-sites.
erk <dig> phosphorylates hnhe1cdt at six consensus sites in a distinct order and with different kinetics
in silico analysis predicted six canonical erk <dig> substrate sites in hnhe1cdt , several of which were previously shown to be phosphorylated in vivo  <cit>  and in vitro  <cit> .
we therefore used active erk <dig>  and time-resolved  nmr spectroscopy to investigate whether hnhe1cdt could be phosphorylated by aerk <dig> in vitro, and how the individual d-domains and f-sites might contribute to this.
addition of catalytic amounts of aerk <dig> to 15n-labelled hnhe1cdt resulted in changes in chemical shifts diagnostic of specific s/t-phosphorylation events  <cit>  .
from nmr assignments of the fully phosphorylated state of hnhe <dig>  we identified all six sites  to be phosphorylated in a distinct temporal order and with different rate constants .
phosphorylated  residues s693p and t779p appeared first and simultaneously.
after a lag phase, when s693p and t779p had essentially reached saturation, s785p, s723p, s726p, and s771p appeared in close succession .
s771p was the last to appear and did not reach saturation in the time frame of the experiment.
two-state behaviour was observed for s <dig> and t <dig> , whereas phosphorylation of s <dig> and s <dig> showed more complex behaviour with interlinked rates.
this can be inferred from the observation of two intermediates, i.e.
phosphorylation of the neighbour in the self-unphosphorylated state, and vice versa .
to determine if s <dig> and t <dig> acted as priming sites for subsequent phosphorylations of the four other sites, these were mutated to alanines in two different variants  and the phosphorylation kinetics repeated.
the same order of phosphorylation was observed , which indicated that the relative order of phosphorylation depended solely on substrate specificity and/or recognition domain affinity, and that neither s <dig> or t <dig> acted as priming sites.
in summary, these results demonstrate complex behaviour of hnhe1cdt phosphorylation by aerk <dig>  with a defined order, and additional intermediate states.
s <dig> and t <dig> were the first and most reactive aerk <dig> phosphorylation sites in hnhe1cdt, but did not act as priming sites, followed by s <dig> , then s <dig> simultaneously with s <dig> , followed by dually phosphorylated s <dig> and s <dig>  and lastly s <dig> fig.
<dig> active erk <dig> phosphorylates six p-sites in hnhe1cdt in a distinct order and with different kinetics.
a
15n,1h-hsqc spectra of unphosphorylated  and erk <dig> phosphorylated hnhe1cdt wt .
addition of catalytic amounts of aerk <dig> to 15n-labelled hnhe1cdt resulted in diagnostic changes in chemical shifts evident of specific s/t-phosphorylation events.
b zoom on s/t and sp/tp regions of a. red labels/arrows indicate phosphorylation-induced peak shifts, and blue arrows/labels peak shifts of neighbouring residues sensing phosphorylation.
c time courses of hnhe1cdt phosphorylations and order of peak appearances.
d phosphorylation time courses.
zoom i–v on boxes in b. peaks of unphosphorylated  and phosphorylated states  simultaneously disappear/appear with time, in a distinct order.
dashed lines encircle multiple peaks of the same residue reporting on distinct phosphorylation states.
stars indicate position of intermediates.
e relative positions of erk <dig> phosphorylation sites  to d-domains and the f-sites in hnhe1cdt f changes of peak intensities with time reported on the phosphorylation rates at the individual sites.
s <dig> and t <dig> were the first and most reactive aerk <dig> phosphorylation sites in hnhe1cdt, followed by s <dig> , then s <dig> simultaneously with s <dig> , followed by dually phosphorylated s <dig> and s <dig>  and lastly s <dig>  g kinetics of t <dig> phosphorylation shown by peak disappearance of d <dig> and v <dig> concomitant with peak appearance for v777* and t779p.
apparent rate constants can be extracted by fitting the unphosphorylated disappearing peak or the appearing phosphorylated peak of either the phosphorylated residue or close neighbours, and should, for a two-state reaction, be the same irrespective of which peak is used for fitting.
two-state behaviour was observed for s <dig> and t <dig>  h–j phosphorylation of s <dig> and s <dig> showed more complex behaviour with interlinked rates.
these residues are so close that a neighbouring phosphorylation event would influence their chemical shifts, leading to the observation of intermediates, i.e.
phosphorylation of the neighbour in the self-unphosphorylated state, and vice versa .
the observation of two intermediates i <dig> and i <dig> suggested parallel phosphorylation of s <dig> and s <dig>  both peaks of the dually unphosphorylated state disappeared with faster rates than the final peaks from the dually phosphorylated state appeared.
thus, the apparent phosphorylation rates of each site were highly dependent on the phosphorylation state of the neighbour, i.e.
k
<dig> and k
<dig> as well as k
<dig> and k
<dig> are not identical.
i <dig> appeared first with a rate similar to the fast decay of the unphosphorylated states , whereas i <dig> appeared concomitantly with the dually phosphorylated state but much slower  and with low intensities.
thus, although both orders of phosphorylation were observed, the main path was via intermediate i <dig>  i.e.
phosphorylation of s <dig> first, followed by phosphorylation of s <dig>  k the weak peaks reporting on the unphosphorylated states of s <dig> and s <dig> initially gained intensity before starting to decrease due to phosphorylation .
as peak intensities are strongly dependent on dynamics, this observed increase may result from altered dynamics caused by the nearby phosphorylation of t <dig>  l apparent rate constants for the individual phosphorylation sites and the effect of d-domain and f-site mutations.
docking site mutations do not change the order of phosphorylation events, yet modulate the individual rates in a distant dependent manner.
based on single measurements and standard deviations from the exponential fits, the apparent rates are significantly different except for s <dig> wt compared to d3-axa, s <dig> wt compared to f2-aa, and s <dig> wt compared to d1d2- <dig>
d-domains play differential roles in scaffolding and activation
we subsequently asked whether the d-domains and f-sites affected the nhe <dig> phosphorylation pattern in vitro, as well as the scaffolding function in vivo.
all sites were analysed in vitro using the axa mutations of the d-domains  and alanine mutations of the f-sites, and were analysed together with wt hnhe <dig> using the same batch of aerk.
for all variants, all six s/t-sites were phosphorylated in the same order as in the wt hnhe <dig>  but with altered kinetics .
analysing those data is not trivial.
since s <dig> and s <dig>  as well as s <dig>  t <dig>  and s <dig> are close in sequence, they sense the phosphorylation state of the neighbour, leading to intermediate states with altered dynamics and/or chemical environment.
the t <dig> phosphorylation site overlaps with the f1-site, so the effect of f <dig> mutation may be either due to the loss of the interaction or due to the altered intrinsic affinity of the phosphosite.
also, the slow phosphorylation sites s <dig> and s <dig> may be affected by altered kinetics of the fast sites due to simple intramolecular substrate competition.table  <dig> apparent rate constants, k
app for hnhe1cdt phosphorylation by aerk <dig>  and effect of d-domain and f-site mutations
k
s723/s726a
afitting of s <dig> and s <dig> individually would require fitting to biexponentials due to their extensive crosstalk, which in turn reduces fitting accuracy.
therefore, only decrease of the s <dig> peak intensity was fitted, which reports on both s <dig> and s <dig> phosphorylation due to their crosstalk
in detail, s <dig> phosphorylation was not affected by the d3-axa mutation, but was slowed down by the d1d2- <dig> and f1-a variants, and accelerated by the most distant f2-aa variant.
similarly, s723/s <dig> phosphorylations were slowed down by all mutations except the most distant one, f2-aa that again led to accelerated rates.
t <dig> phosphorylation was slowed down by all mutations, and s <dig> was strongly decreased by both f-site mutants.
taken together, mutations of the d-domains and f-sites affected phosphorylation in a distance dependent manner.
in three cases  the rate of phosphorylation went up, suggesting that the presence of this site was inhibitory.
in all three cases this occurred for the site furthest away from the mutation, reflecting competition between sites.
this suggests that the d-domains and f-sites are not mandatory for phosphorylation, but rather exert regulatory roles, and that each site uses the most optimal erk <dig> interaction site to become phosphorylated.
to determine whether the d-domain and f-site variants affected hnhe1-erk <dig> interaction in a cellular context we used immunofluorescence analysis, which revealed that hnhe <dig> localized predominantly to the plasma membrane region of ap- <dig> cells, although some intracellular hnhe <dig> labeling was also seen , fully consistent with previous reports  <cit>  and with the pla studies .
stimulation with epidermal growth factor  induced distinct plasma membrane ruffles, to which hnhe <dig> clearly localized, in congruence with its role in cell motility  <cit> , and similar to the localization of nhe <dig> to prolactin-induced ruffles, which we recently demonstrated  <cit> .
erk1/ <dig> localized diffusely in the cytosol and nucleus, as well as in plasma membrane regions, where it partially co-localized with hnhe <dig> .
in a cellular context we refrained from using the f1-variant, as the phenylalanine is part of a phosphorylation site , thus complicating the interpretations.
similar to wt hnhe <dig>  all hnhe <dig> variants localized predominantly to the plasma membrane region, suggesting that the mutations did not affect nhe <dig> membrane targeting .
compared to cells expressing wt hnhe <dig>  co-localization with erk1/ <dig> was unaltered both under basal and stimulated conditions in cells expressing hnhe1-d1d2- <dig> , suggesting that d <dig> and d <dig> are not important for in vivo scaffolding and in congruence with their role in phosphorylation .
interestingly, whereas stimulation by egf tended to increase co-localization in cells expressing wt hnhe <dig> and d1d2- <dig>  the opposite was true in cells expressing the d3-axa, d1d2d3- <dig>  and f2-aa-variants .
hence, in these variants, a loss of colocalization was seen, which suggests that scaffolding as well as the dynamics of the hnhe1-erk <dig> interaction are dependent on both the d3-domain and the f2-site.
these data fully support the in vitro nmr data and highlight both the d3-domain and f2-site as important for scaffolding.fig.
<dig> nhe <dig> co-localizes with erk <dig> shown by immunofluorescence.
a immunofluorescence images of ap- <dig> cells  treated or not for 15 min with egf .
merged images were zoomed to highlight the co-localization of erk1/ <dig> and nhe <dig> .
all other variants are shown in additional file 6: figure s <dig>  data are representative of three independent biological replicates.
b representative line scans across membrane areas of images as in a. line scans were performed using olympus image analysis software.
the figure shows the pixel intensities at each wavelength over the line shown, in the absence and presence of egf as shown.
data are examples based on analysis of at least  <dig> cells in three to four independent replicates per condition.
c summary of line scan analysis data for all variants.
the figure shows the percentage of cells with nhe1-erk1/ <dig> co-localization in both membranes, based on the experiments illustrated in b. data are shown as mean percentage with sem error bars, based on analysis of at least  <dig> cells in three to five independent replicates per condition.
§§ and §§§, p <  <dig>  and p <  <dig> , respectively, relative to ap1 + egf; * p <  <dig>  relative to own control in absence of egf.
two-way anova, Šídák’s multiple comparisons test
nhe <dig> regulates erk <dig> phosphorylation status in a cellular context
to address whether nhe <dig> regulates erk <dig> activity, we next assessed erk1/ <dig> activation by determining relative t202/y <dig> -t185/y <dig>  phosphorylation, corresponding to erk1/ <dig> activation.
untransfected ap- <dig> cells, or ap- <dig> cells stably expressing wt hnhe <dig>  were exposed to egf  to induce erk1/ <dig> activity .
as seen, despite the known low level of egf receptor expression in chinese hamster ovary -derived cells such as ap- <dig> cells, stimulation of cells expressing wt hnhe <dig> with egf evoked a modest but significant increase in erk1/ <dig> phosphorylation at these sites, i.e.
erk1/ <dig> activation.
importantly, the presence of wt hnhe <dig> was necessary for detectable activation of erk1/ <dig> by egf.
this suggests that hnhe <dig> is important for erk1/ <dig> activation in this context.
to assess whether the interaction-site mutations would affect nhe1-regulated erk <dig> activity, we exposed untransfected ap- <dig> cells or ap- <dig> cells expressing wt and variant hnhe1s, to egf and determined erk1/ <dig> activity .
compared to cells expressing wt hnhe <dig>  cells expressing hnhe1-d3-axa, hnhe1-d1d2d3- <dig>  or hnhe1-f2-aa variants exhibited a significant decrease in relative egf-induced erk1/ <dig> phosphorylation .
these data show that hnhe <dig> is important for egf-induced erk1/ <dig> activation in a cellular context, in a manner dependent on its d3-domain and f2-site.
these data further support that this at least in part reflects a scaffolding role of hnhe <dig> involving the three independent low affinity sites.
notably, the effects of the mutations on erk1/2-nhe <dig> co-localization and erk1/ <dig> activity are comparable if not identical, and mirrors the interaction monitored by nmr spectroscopy.fig.
<dig> nhe <dig> regulates the phosphorylation status of cellular erk1/ <dig>  erk1/ <dig> phosphorylation in untransfected ap- <dig> cells or ap- <dig> cells expressing wt and variant hnhe <dig>  as indicated.
cells were stimulated or not with human recombinant egf  for 15 min.
a representative immunoblots of p-erk1/ <dig> -t185/y <dig>  phosphorylation) and total erk1/ <dig> under the conditions shown.
the arrows indicate erk <dig>  and erk <dig> , respectively.
b summary data from three to eight independent biological replicates per condition.
blots were scanned, and band intensities quantified using un-scan-it graph digitizer software , as described in the “methods”.
quantified data are shown as means with sem error bars, normalized to those in untransfected, unstimulated ap- <dig> cells.
*, **, and ***, p <  <dig> , p <  <dig> , and p <  <dig> , respectively, compared to untransfected control; #, ##, and ###, p <  <dig> , p <  <dig> , and p <  <dig> , respectively, compared to the same cells in absence of egf.
two-way anova, holm-Šídák’s multiple comparisons test
discussion
nhe <dig> activity after various stimuli is regulated by erk1/ <dig>  and the nhe <dig> c-terminal tail is directly phosphorylated by erk <dig> in vitro  <cit>  and in vivo  <cit> .
vice versa, nhe <dig> can regulate erk1/ <dig> activity , yet molecular details and mechanistic understanding of their interaction have been lacking.
in conjunction with yeast two-hybrid studies suggesting interaction of nhe <dig> with mapks  <cit> , these studies led us to hypothesize that nhe <dig> and erk <dig> engage in direct physical interaction.
supporting this hypothesis, we report here that erk <dig> and nhe <dig> interact directly in vivo as well as in vitro, and that erk <dig> phosphorylates multiple sites in hnhe1cdt.
based on nmr analyses, in conjunction with various combinations of d-domain and f-site mutations, we suggest that hnhe1cdt scaffolds iaerk <dig> and that they interact in a non-cooperative modular manner that involves a d-domain  and two f-sites.
nmr titrations revealed the affinity of the d3-domain to be in the low micromolar range, in agreement with known d-domain affinities  <cit> .
d-domain and f-site mutations did not prevent erk2-mediated hnhe <dig> phosphorylation in vitro, but altered its kinetics.
in vivo, hnhe <dig> and erk1/ <dig> co-localized at the plasma membrane in a manner sensitive to erk1/ <dig> stimulation, and mutations in the hnhe <dig> d3-domain and f-site altered erk1/ <dig> activity.
thus, a central conclusion of this work is that nhe <dig> and erk <dig> directly interact and engage in physical and functional reciprocal interactions.
this provides a novel molecular framework for understanding previous reports of both nhe1-mediated scaffolding and regulation of erk1/ <dig>  <cit>  and erk1/2-mediated phosphorylation of nhe <dig>  <cit> .
the organization of idps as “flexible wrappers” has previously been suggested as a general scaffolding mechanism  <cit> , involved in bipartite interactions, for example of ste <dig> with the yeast mapk fus <dig>  <cit>  and of p21cip <dig> with cyclin-dependent kinases  <cit> .
upon identification of the f-site, a bipartite modular, non-cooperative recognition system was originally suggested for kinases with the d-domain and f-site acting independently or in combination  <cit> .
the tripartite  interaction between hnhe1cdt and iaerk <dig> uncovered here extends this concept.
for hnhe <dig>  the multi-site interaction may function in a manner analogous, but not identical, to a fuzzy complex  <cit> , where several binding sites are at play concomitantly, yet with none of the interactions seemingly affecting each other.
the “hot potato hypothesis” was originally suggested by perham in  <dig> to describe the handover of substrates and intermediates in multi-enzyme complexes  <cit> .
indeed, the non-cooperative, multi-site scaffold interaction between hnhe <dig> and erk <dig> may function similarly to holding a hot potato; the sites do not cooperate to increase the affinity, but will “shuffle” dynamically with sites being sometimes off, sometimes on, and hence we term this type of scaffold interaction a “shuffle complex” .fig.
<dig> scaffolding of erk <dig> by the intrinsically disordered nhe1cdt via a “shuffle complex”.
suggested topology of the hnhe1cdt-iaerk <dig> complex indicating the tripartite binding mode between hnhe1cdt and human iaerk <dig>  a–b as nhe <dig> functions as a dimer in the cell, one or both nhe1s in a dimer can shuffle an erk1/ <dig>  c alternatively, one nhe <dig> binds erk1/ <dig>  whereas the other nhe <dig> in the dimer can scaffold other members of the signalling complex.
alternating conformations are indicated encompassing the individual sites and combinations
nhe <dig> is, to the best of our knowledge, the first membrane protein described to scaffold members of the mapk pathway, spanning all four levels of the mapk hierarchy  <cit> .
many soluble scaffold proteins acting together with, for example, g protein-coupled receptors  and growth factor receptors, have been described, but detailed interaction data have not provided insight into how scaffolding and regulation are coupled.
in the cell, meks, phosphatases, and substrates all compete for the d-domain recognition site on erk <dig>  <cit> , and it is currently not known how nhe <dig> interacts with the other mapk members, including mek, and assembles a signalling complex.
as nhe <dig> acts as a dimer in vivo  <cit> , we propose that upon release of the d3-domain from one nhe <dig> monomer due to competition with mek, the shuffle complex organization keeps erk <dig> in place via scaffolding by d-domains of the other nhe <dig> subunit in the nhe <dig> dimer, or by the f-sites .
furthermore, in a potential cellular complex, the remaining d-domains as well as f-sites will be available for further scaffolding of other members of the mapk hierarchy , as suggested by yeast two-hybrid screens, potentially mek, although this remains to be explored.
nhe <dig> is the first example of an id substrate of erk <dig> for which detailed interaction data now exist, and to the best of our knowledge, no other idp or idr has to date been experimentally linked to erk <dig> phosphorylation or scaffolding.
it has been noted that the d-domain of the tyrosine-phosphatase ptp-sl resides in a region with high disorder propensity  <cit> .
disorder predictions of the nuclear pore protein tpr  show its erk2-interacting f-site to reside in an idr, similar to the nucleoporin fg-repeat regions and to hnhe1cdt.
thus, it appears that the f-site recruitment site interaction may be frequently exploited by idps.
however, it remains to be seen whether the multi-site shuffle interaction is a novel canonical idp/idr-erk <dig> interaction mode or if it is a unique scaffolding function specific to nhe <dig>
all six putative erk <dig> phosphorylation sites of hnhe1cdt were phosphorylated by aerk <dig> in vitro .
previous in vivo phosphoproteomics have mapped phosphorylation at five of these sites, yet without identification of the responsible kinases, and with no information on the sequence of individual phosphorylation events  <cit> .
further, some of these sites, i.e.
s <dig>  t <dig>  and s <dig>  were previously identified upon in vitro phosphorylation of nhe <dig> by erk <dig>  <cit> .
no non-canonical phosphorylation was detected in the present study, in contrast with previous reports identifying s <dig>  s <dig>  and s <dig> as erk-dependent nhe <dig> phosphorylation sites  <cit> .
while it is possible that additional complexity may be introduced in the in vivo setting, our data underscore the major advantage of nmr for direct identification of phosphorylation sites.
physiological roles have been proposed for all six phosphorylations, although their interplay and dynamics have never previously been assessed.
s <dig> and s <dig>  were reported to be phosphorylated by p <dig> mapk in murine pro-b-cells  <cit> , and phosphorylation of s <dig> was suggested to mediate apoptosis-induced alkalinization by nhe <dig>  <cit> .
based on studies of nhe <dig> mutants expressed in nhe1-deficient cho cells, s <dig> was reported to mediate erk-dependent nhe <dig> activation  <cit> , and later, s <dig>  t <dig>  and s <dig>  but not s <dig>  s <dig>  or s <dig>  which were assigned roles in erk-dependent nhe <dig> phosphorylation after sustained acidosis in renal cells  <cit> .
in amphiuma erythrocytes, phosphorylation of residues corresponding to s <dig> and s <dig>  were detected by ms, where s <dig>  was constitutively phosphorylated  <cit> .
the precise downstream effects of these phosphorylations are not currently known, but they are likely to both impact nhe <dig> structural dynamics and hence activity, and to contribute to the scaffolding role of nhe <dig> in regulation of erk, hence fine-tuning cellular erk signaling.
timing of signalling events is crucial for many cellular functions, and phosphorylation events that are interdependent or distributive with very different rate constants are possible ways of controlling signal duration and strength.
indeed, it has been suggested that such temporally ordered phosphorylations serve as platforms for signal integration  <cit> .
our findings provide evidence for a distinct temporal order of erk <dig> phosphorylation of hnhe1cdt with the occurrence of specific intermediates.
these intermediates could function as tightly regulated docking sites or thresholds that convert graded signals to switch-like responses  <cit> .
such dynamics in erk <dig> signalling have been observed to affect the half-life of an erk <dig> downstream effector, the transcription factor c-fos  <cit> .
timed phosphorylation events in hnhe1cdt may therefore similarly partake in control of the erk <dig> signal duration.
the hnhe1cdt has other confirmed phosphorylation sites than those demonstrated in the present study, and many more putative ones  <cit> , several of which are close to the erk <dig> interaction sites, for example s <dig>  phosphorylated by rsk  <cit> .
these sites may mediate interactions with other binding partners, introducing additional layers of complexities, for example of pathway crosstalk.
the close proximity of the primary s <dig> and t <dig> phosphorylation sites to the d3-domain and f1-site, respectively, suggests regulatory role for the interaction of hnhe1cdt with, and phosphorylation by, erk <dig>  analogously, the first phosphorylation events in hnhe1cdt  may change the binding mode and/or dynamics with erk <dig> in a way that regulates the phosphorylation of succeeding sites, although we showed that they do not act as priming sites.
the effects of hnhe <dig> variants on the phosphorylation kinetics support the hypothesis that all sites are at play within the shuffle complex, where the sites closest to the phosphorylation site are exploited for interaction with erk <dig>  the current data does not allow us to evaluate whether all six sites are phosphorylated during nhe <dig> activation.
some sites, and most likely the slowest ones observed here, may only react under certain physiological conditions.
we hypothesize that such conditional phosphorylations could be important for a rheostatic regulation of both erk <dig> and nhe <dig>  consequently, the results of this work open a series of new questions, both regarding the generality of shuffle complexes in scaffolding by idps, but also concerning the functional roles and spatial and temporal interconnectivity of the six identified phosphorylation sites in hnhe <dig>
CONCLUSIONS
in this work we have demonstrated that the intrinsically disordered region of hnhe <dig> acts as a membrane scaffold engaging erk <dig> in a multi-site shuffle complex.
we show that the interaction is recapitulated in vivo, and that co-regulation of hnhe <dig> and erk <dig> manifests in distinct in vivo effects on erk1/ <dig> activity and in vitro effects on hnhe <dig> phosphorylation.
our work provides a molecular mechanism for the important scaffolding function of nhe <dig>  and characterizes a direct interaction between the intrinsically disordered hnhe1cdt and erk <dig>  leading to hnhe1cdt phosphorylation and regulation of erk1/ <dig> activity.

 extracellular signal-regulated kinase is a member of the mitogen-activated protein kinase family of kinases activated in response to numerous growth factors and cytokines, leading to phosphorylation and functional regulation of downstream targets. erk has been linked to more than different substrates whose phosphorylation by erk is orchestrated by coordination of signaling networks through common binding to so-called scaffold proteins. the definition of a scaffold protein was recently refined and their identification as such suggested from qualities of multivalent binding, non-catalytic placeholders, and bidirectional process control. several scaffold proteins have been described for the mapks such as kinase suppressor of ras, jnk-interacting protein, iq motif containing gtpase activating protein, and β-arrestin, which interact with members of the mapk cascade, providing multivalency, spatial concentration, and/or signaling fidelity. however, although mapks are known to regulate the action of several membrane proteins and receptors, none of these scaffold proteins are themselves membrane proteins, requiring additional mechanisms for colocalization of the scaffold protein, the membrane protein, as well as the kinases. moreover, most of the available molecular insights are from structures of kinases in complex with folded domains or with small peptides of the scaffold proteins, and details regarding scaffolding by non-globular proteins are lacking. mapks are s/t kinases that interact with targets and regulators via two types of domains, d-domains and f-sites. d-domains, also known as docking sites for erk and jnk, lxl domains, or kinase interaction motifs have the canonical sequence of 2– basic residues, spaced by 1– residues to a hydrophobic motif ΦxΦ, where Φ is generally v, l, or i. d-domains are found in mapk substrates such as the transcription factor elk- and p ribosomal s kinase, as well as in other mapk targets. in erk d-domains interact with the d-domain recognition site also known as the cd/ed domain, located more than 10 Å from the active site. the f-site recruitment site in erk is much less studied and incompletely understood. it binds to f-sites, also called def -domains with the canonical fxfp sequence. f-sites allow for aromatic residues at the p and p positions, and f-sites have been reported in substrates such as elk- and c-fos, and within the nucleoporin fg-repeats. so far the only structure available of an f-site recruitment site-interacting protein is that of erk in complex with the 15 kda phosphoprotein enriched in astrocytes, which notably lacks any of the above-mentioned motifs. the plasma membrane na+/h+ exchanger is a major regulator of ph and volume in essentially all cells studied. furthermore, nhe is involved in the regulation of cell proliferation, survival, motility, and other essential physiological processes, and its dysregulation contributes importantly to major human malignancies, including cancer and cardiovascular diseases. numerous hormones and growth factors acting via receptor tyrosine kinases or gtp-binding protein-coupled receptors can elicit posttranslational regulation of nhe. the mapks erk1/ p mapk, and c-jun n-terminal kinase are widely implicated in nhe regulation, and direct phosphorylation of human nhe by erk1/ was previously proposed based on 32p measurements and mass spectrometry. conversely, nhe has been reported to regulate signaling through regulation of erk1/ and p mapk activity, and yeast two-hybrid screens have suggested the interaction of nhe with several members of the mapk hierarchy. however, with the exception of the interaction with b-raf, evidence from mammalian systems is lacking, and the possible sites of nhe1-mapk interaction, its structural details, and possible functional consequences are unexplored. we recently showed by pondr and disopred predictions, as well as by nuclear magnetic resonance spectroscopy and other biophysical techniques, that the distal ~  residues of the hnhe c-terminal intracellular domain, containing most of the known nhe phosphorylation sites, are intrinsically disordered. to our knowledge, no studies have yet addressed the mechanisms through which mapks interact with id proteins, although about one third of all proteins in higher eukaryotes contain significant id regions, and id is abundant in cellular signalling, scaffolding, as well as in mapks themselves. here, we demonstrate that hnhe acts as an erk membrane protein scaffold in vivo that is necessary for erk activation via direct interactions, and we show that loss of scaffolding by hnhe leads to decreased erk activation. using nmr spectroscopy we show that nhe scaffolds inactive erk in a “shuffle complex” that involves a d-domain and two non-canonical f-sites. we characterize the order and kinetics of both previously reported and novel erk2-mediated phosphorylations of hnhe in vitro. our findings provide a molecular mechanism for the widely recognized and functionally important scaffolding function of hnhe and give mechanistic insight into the regulation of erk activity by the intrinsically disordered hnhe1cdt.  the recently suggested links between hnhe and erk1/ prompted us to investigate whether erk1/ and nhe directly interact in a cellular context. using ap- cells stably expressing full-length wt hnhe we asked if hnhe and erk1/ engage in a direct interaction in vivo. interaction was assessed using in situ proximity ligation assay, which interrogates close interaction between proteins. as seen, the detection of multiple pla puncta when cells were incubated with both nhe and erk1/ antibodies revealed the presence of hnhe1-erk1/ complexes in ap- wt hnhe cells, compared to a much lower signal in negative controls incubated with one antibody only. data from multiple experiments are quantified in fig. 1c, demonstrating that the pla signal is significantly greater in nhe1-erk antibody-labelled cells than in negative controls. thus, these data show that hnhe and erk1/ directly interact in the cell.fig. direct interaction between erk and nhe in vivo and in silico. a proximity ligase assay carried out in ap- wt hnhe cells treated with nhe and erk1/ primary antibodies. proximity ligase signal appears as red dots and the merge image highlights the interaction between nhe and erk. f-actin was stained with phalloidin scale bars represent 10 μm. data are representative of three independent replicates. b proximity ligase assay carried out in ap- wt hnhe cells. as a negative control, cells were only treated with nhe primary antibodies and the appearance of the red dots indicates unspecific binding. f-actin was stained with phalloidin scale bars represent 10 μm. data are representative of three independent replicates. c quantification of pla data was carried out in imagej. pla signal from at least ten different image areas in each experiment were counted by particle analysis and the average pla signal per cell was plotted in the bar graph. data are representative of three independent replicates. d erk1/ docking motifs, d-domain and f-site. Φ indicates hydrophobic amino acid residues, and x any other amino acid. e overall nhe topology and localization of putative d-domains and erk phosphorylation sites in the hnhe1cdt. the positions of identified d-domains as well as predicted p-phosphorylation sites are indicated by stars. insert: alignment of the consensus erk phosphorylation sites in hnhe1; p sites indicated by grey background. f alignment of hnhe d-domains to known d-domains. the consensus hydrophobic and positively charged residues are highlighted in yellow and blue, respectively. g sequence conservation of putative d-domains in nhe1cdt in various species. d d and d are indicated with grey bars above the alignment. the solid horizontal line separates tetrapods from teleosts the disordered tail of hnhe interacts with iaerk2 scrutinizing the intracellular domain of nhe by in silico methods for potential erk1/ interaction sites identified three potential d-domains in the intrinsically disordered region, 706–, 732–, and 676–. the previously described conserved tv-box is part of the d3-domain, and this d-domain is the only one conserved throughout nhe evolution. since erk and erk are 84 % identical by sequence and share many if not all functions, and erk is the more widely studied enzyme of the two, we focused in vitro studies on erk to discriminate between the roles of each d-domain, we investigated the interaction between the disordered tail of nhe ) and recombinantly produced human iaerk by nmr spectroscopy, which previously had provided insight into the transient structure and conserved regions of hnhe1cdt. first, we measured perturbations of chemical shifts and peak intensities of the hnhe1cdt wt arising from addition of iaerk to a 1: molar ratio using 15n,1h-hsqc spectra. signals from all residues of the d3-domain disappeared, and decreased intensities and chemical shift perturbations were observed in the c-terminal neighbouring residues, suggesting this domain engages in the interaction. additionally, two phe residues in the distal end of hnhe1cdt 809– as well as a phe residue within a potential substrate site, 778– were highly perturbed. importantly, although the latter resembles a substrate site, both are reminiscent of the canonical erk f-site motif, suggesting several interaction sites between hnhe1cdt and iaerk additional yet minor perturbations were observed in the linking regions between these sites, which were partially caused by slight ph variations. since some residues in d are unassigned due to their overlap in the nmr spectra, and perturbations were observed close to this site, we cannot exclude d to also contribute to the interaction. lastly, as escherichia coli expression of erk can lead to autophosphorylation of y, we assessed the level of erk autophosphorylation by native page, showing that more than 80 % of erk is non- and less than 20 % mono-phosphorylated. to ensure that this had no effect on the interaction with nhe1cdt we fully dephosphorylated erk with the tyr phosphatase heptp and re-analysed the interaction, which gave an identical binding profile, in accordance with the low activity of the erk mono-phosphorylated state. in conclusion, nhe1cdt interact with iaerk through multiple contact sites involving the d3-domain, as the dominating d-domain, and two f-sites.fig. inactive erk interacts preferably with the d3-domain and two f-sites of hnhe1cdt. a position of transient helices, tv- and lsd-boxes, predicted d-domains and f-sites, as well as erk phosphorylation sites in the hnhe1cdt. b combined chemical shift perturbations Δδ of wt hnhe1cdt by iaerk interaction. c 15n,1h-hsqc peak intensity ratios of hnhe1cdt wt in the presence/absence of iaerk d difference in relaxation rates Δr between hnhe1cdt and nhe1cdt:iaerk e–g 15n,1h-hsqc peak intensity ratios of e hnhe1cdt d3-axa, f hnhe1cdt f1-a, and g hnhe1cdt f2-aa in the presence/absence of iaerk h internally urea referenced secondary cα chemical shifts of wt hnhe1cdt identify the presence of several transient helices. ● indicate the position of prolines, □ unassigned residues, and blue box severe peak overlap we next investigated whether interfering with any of these contact sites would affect the interactions and first exploited the knowledge that mapk interaction is severely perturbed by mutations of ΦxΦ to axa in d-domains. hence, we constructed axa variants of all three d-domains alone and in combination, both in the full-length hnhe for cellular studies and in hnhe1cdt for in vitro studies. in d3-axa, chemical shift perturbations in the two f-sites upon erk addition were preserved in the interaction with iaerk whereas no perturbations were observed in the axa-mutated d3-domain, implying that this site is important for the interaction. no notable effects of d1- and d2-axa substitutions were observed, arguing against their involvement in the interactions. this data also indicated that the f-sites interacted with iaerk independently of the d-domain. to assess this further the f-sites were individually mutated by substituting ftp778– with atp778– and fp811– with aa811–. both f-site variants showed strongly decreased chemical shift perturbations at the mutation sites upon erk addition, leaving the other f-site and the d3-domain unaffected. this conclusively identified all three sites as erk interaction sites. further, substitutions at each site left the other sites unaffected, indicating that these regions of hnhe interact independently with iaerk nhe does not fold upon binding to erk but may be a flexible wrapper to further address how hnhe1cdt interacted with iaerk we analysed the complex by size exclusion chromatography and compared elution profiles with those of the individual proteins. since hnhe1cdt is an idr, it has a larger hydrodynamic radius than iaerk, and hnhe1cdt thus eluted first from the column. subtracting individual runs from that of the mixture revealed a broad peak with an elution volume smaller than that of iaerk yet larger than that of hnhe1cdt, suggesting that hnhe1cdt folds or wraps around iaerk circular dichroism spectroscopic analyses, as well as nmr chemical shift analyses, did not indicate folding upon binding formation of significant secondary structure, suggesting that hnhe1cdt forms a relatively extended structure around iaerk to substantiate this conclusion, we recorded 15n transverse relaxation rates of the unbound and the iaerk2-bound hnhe1cdt, and analysed their differences. since d residues broadened beyond detection in the complex, their r values could not be extracted. however, for residues interacting with iaerk a significant increase in r rates is expected compared to those of hnhe1cdt alone, due to the larger radius of gyration of the complex or due to chemical exchange between different states. indeed, residues from both f-sites had substantially larger r rates in the complex compared to hnhe1cdt alone, and many residues between these sites were also affected, although not to the same extent. this result supports the sec results and suggests a substantial interaction area between the two proteins. the loss of nmr signal upon binding imposes a challenge for obtaining affinity constants. although nmr peak intensities are only directly proportional to the populations when all states can be accounted for, they may provide apparent upper-limit affinities. from a titration of hnhe1cdt with iaerk global fitting of intensity changes using residues ltv684– of the d3-domain gave a kdapp of 16 ± 2 μm, while the other interaction sites had relatively lower affinities with apparent affinities, kdapp of 86 ± 26 μm and 69 ± 14 μm. attempts to substantiate this and obtain stoichiometries by isothermal titration calorimetry or microscale thermophoresis were unsuccessful, due to limited stability of concentrated erk as well as adhesion of erk to the capillary tubing. instead, to determine the stoichiometry of the interaction we used non-denaturing mass spectrometry, allowing the non-covalent complex to be captured at low micromolar concentration. deconvolution of the spectrum using in-house software revealed three main charge state envelopes, corresponding to hnhe1cdt, iaerk, and from the 1: complex. a minor population of iaerk dimers was also detected, most likely due to the presence of a hexahistidine-tag, as noted. these data unequivocally showed that hnhe1cdt and iaerk form a 1: complex, fully in-line with the perturbation of the relaxation times and the size exclusion data. collectively, these data indicated that nhe1cdt binds to erk with medium affinity, through at least three independent contact sites forming a 1: complex.fig. native mass spectrum and molecular model of the hnhe1cdt:iaerk complex. a non-denaturing mass spectrum of the hnhe1-erk complex, acquired from 200 mm ammonium acetate. deconvolution was performed using the unidec software and the individual charge series fits are displayed below, corresponding to nhe, erk, nhe1:erk complex, and minor contribution of erk dimer. b proposed model for the hnhe1-erk interactions. the hnhe1cdt interacts with erk at three sites, exploiting a d-domain and two f-sites. at the d-domain recognition site, k l and v of the hnhe1cdt d3-domain, as well as d d y and h of the iaerk d-domain recognition site are shown as sticks finally, we used the nmr chemical shifts and relaxation data together with known structures of iaerk complexes to model the hnhe1cdt-iaerk interaction. when modelled at the d-domain recognition site of iaerk each individual d-domain when bound to the d-domain recognition site allowed for either of the f-sites to reach the f-site recognition site. in each case the model predicted long id linkers between the binding sites, which were long enough to allow nhe to wrap around iaerk this is in accordance with the nmr data and supports their uncoupled behaviour. collectively, these data suggest that hnhe1cdt could interact with iaerk in a tripartite 1: interaction exploiting a d-domain and two f-sites. erk phosphorylates hnhe1cdt at six consensus sites in a distinct order and with different kinetics in silico analysis predicted six canonical erk substrate sites in hnhe1cdt, several of which were previously shown to be phosphorylated in vivo and in vitro. we therefore used active erk and time-resolved nmr spectroscopy to investigate whether hnhe1cdt could be phosphorylated by aerk in vitro, and how the individual d-domains and f-sites might contribute to this. addition of catalytic amounts of aerk to 15n-labelled hnhe1cdt resulted in changes in chemical shifts diagnostic of specific s/t-phosphorylation events. from nmr assignments of the fully phosphorylated state of hnhe we identified all six sites to be phosphorylated in a distinct temporal order and with different rate constants. phosphorylated residues s693p and t779p appeared first and simultaneously. after a lag phase, when s693p and t779p had essentially reached saturation, s785p, s723p, s726p, and s771p appeared in close succession. s771p was the last to appear and did not reach saturation in the time frame of the experiment. two-state behaviour was observed for s and t, whereas phosphorylation of s and s showed more complex behaviour with interlinked rates. this can be inferred from the observation of two intermediates, i.e. phosphorylation of the neighbour in the self-unphosphorylated state, and vice versa. to determine if s and t acted as priming sites for subsequent phosphorylations of the four other sites, these were mutated to alanines in two different variants and the phosphorylation kinetics repeated. the same order of phosphorylation was observed, which indicated that the relative order of phosphorylation depended solely on substrate specificity and/or recognition domain affinity, and that neither s or t acted as priming sites. in summary, these results demonstrate complex behaviour of hnhe1cdt phosphorylation by aerk with a defined order, and additional intermediate states. s and t were the first and most reactive aerk phosphorylation sites in hnhe1cdt, but did not act as priming sites, followed by s, then s simultaneously with s, followed by dually phosphorylated s and s and lastly s fig. active erk phosphorylates six p-sites in hnhe1cdt in a distinct order and with different kinetics. a 15n,1h-hsqc spectra of unphosphorylated and erk phosphorylated hnhe1cdt wt. addition of catalytic amounts of aerk to 15n-labelled hnhe1cdt resulted in diagnostic changes in chemical shifts evident of specific s/t-phosphorylation events. b zoom on s/t and sp/tp regions of a. red labels/arrows indicate phosphorylation-induced peak shifts, and blue arrows/labels peak shifts of neighbouring residues sensing phosphorylation. c time courses of hnhe1cdt phosphorylations and order of peak appearances. d phosphorylation time courses. zoom i–v on boxes in b. peaks of unphosphorylated and phosphorylated states simultaneously disappear/appear with time, in a distinct order. dashed lines encircle multiple peaks of the same residue reporting on distinct phosphorylation states. stars indicate position of intermediates. e relative positions of erk phosphorylation sites to d-domains and the f-sites in hnhe1cdt f changes of peak intensities with time reported on the phosphorylation rates at the individual sites. s and t were the first and most reactive aerk phosphorylation sites in hnhe1cdt, followed by s, then s simultaneously with s, followed by dually phosphorylated s and s and lastly s g kinetics of t phosphorylation shown by peak disappearance of d and v concomitant with peak appearance for v777* and t779p. apparent rate constants can be extracted by fitting the unphosphorylated disappearing peak or the appearing phosphorylated peak of either the phosphorylated residue or close neighbours, and should, for a two-state reaction, be the same irrespective of which peak is used for fitting. two-state behaviour was observed for s and t h–j phosphorylation of s and s showed more complex behaviour with interlinked rates. these residues are so close that a neighbouring phosphorylation event would influence their chemical shifts, leading to the observation of intermediates, i.e. phosphorylation of the neighbour in the self-unphosphorylated state, and vice versa. the observation of two intermediates i and i suggested parallel phosphorylation of s and s both peaks of the dually unphosphorylated state disappeared with faster rates than the final peaks from the dually phosphorylated state appeared. thus, the apparent phosphorylation rates of each site were highly dependent on the phosphorylation state of the neighbour, i.e. k and k as well as k and k are not identical. i appeared first with a rate similar to the fast decay of the unphosphorylated states, whereas i appeared concomitantly with the dually phosphorylated state but much slower and with low intensities. thus, although both orders of phosphorylation were observed, the main path was via intermediate i i.e. phosphorylation of s first, followed by phosphorylation of s k the weak peaks reporting on the unphosphorylated states of s and s initially gained intensity before starting to decrease due to phosphorylation. as peak intensities are strongly dependent on dynamics, this observed increase may result from altered dynamics caused by the nearby phosphorylation of t l apparent rate constants for the individual phosphorylation sites and the effect of d-domain and f-site mutations. docking site mutations do not change the order of phosphorylation events, yet modulate the individual rates in a distant dependent manner. based on single measurements and standard deviations from the exponential fits, the apparent rates are significantly different except for s wt compared to d3-axa, s wt compared to f2-aa, and s wt compared to d1d2- d-domains play differential roles in scaffolding and activation we subsequently asked whether the d-domains and f-sites affected the nhe phosphorylation pattern in vitro, as well as the scaffolding function in vivo. all sites were analysed in vitro using the axa mutations of the d-domains and alanine mutations of the f-sites, and were analysed together with wt hnhe using the same batch of aerk. for all variants, all six s/t-sites were phosphorylated in the same order as in the wt hnhe but with altered kinetics. analysing those data is not trivial. since s and s as well as s t and s are close in sequence, they sense the phosphorylation state of the neighbour, leading to intermediate states with altered dynamics and/or chemical environment. the t phosphorylation site overlaps with the f1-site, so the effect of f mutation may be either due to the loss of the interaction or due to the altered intrinsic affinity of the phosphosite. also, the slow phosphorylation sites s and s may be affected by altered kinetics of the fast sites due to simple intramolecular substrate competition.table apparent rate constants, k app for hnhe1cdt phosphorylation by aerk and effect of d-domain and f-site mutations k s723/s726a afitting of s and s individually would require fitting to biexponentials due to their extensive crosstalk, which in turn reduces fitting accuracy. therefore, only decrease of the s peak intensity was fitted, which reports on both s and s phosphorylation due to their crosstalk in detail, s phosphorylation was not affected by the d3-axa mutation, but was slowed down by the d1d2- and f1-a variants, and accelerated by the most distant f2-aa variant. similarly, s723/s phosphorylations were slowed down by all mutations except the most distant one, f2-aa that again led to accelerated rates. t phosphorylation was slowed down by all mutations, and s was strongly decreased by both f-site mutants. taken together, mutations of the d-domains and f-sites affected phosphorylation in a distance dependent manner. in three cases the rate of phosphorylation went up, suggesting that the presence of this site was inhibitory. in all three cases this occurred for the site furthest away from the mutation, reflecting competition between sites. this suggests that the d-domains and f-sites are not mandatory for phosphorylation, but rather exert regulatory roles, and that each site uses the most optimal erk interaction site to become phosphorylated. to determine whether the d-domain and f-site variants affected hnhe1-erk interaction in a cellular context we used immunofluorescence analysis, which revealed that hnhe localized predominantly to the plasma membrane region of ap- cells, although some intracellular hnhe labeling was also seen, fully consistent with previous reports and with the pla studies. stimulation with epidermal growth factor induced distinct plasma membrane ruffles, to which hnhe clearly localized, in congruence with its role in cell motility, and similar to the localization of nhe to prolactin-induced ruffles, which we recently demonstrated. erk1/ localized diffusely in the cytosol and nucleus, as well as in plasma membrane regions, where it partially co-localized with hnhe. in a cellular context we refrained from using the f1-variant, as the phenylalanine is part of a phosphorylation site, thus complicating the interpretations. similar to wt hnhe all hnhe variants localized predominantly to the plasma membrane region, suggesting that the mutations did not affect nhe membrane targeting. compared to cells expressing wt hnhe co-localization with erk1/ was unaltered both under basal and stimulated conditions in cells expressing hnhe1-d1d2-, suggesting that d and d are not important for in vivo scaffolding and in congruence with their role in phosphorylation. interestingly, whereas stimulation by egf tended to increase co-localization in cells expressing wt hnhe and d1d2- the opposite was true in cells expressing the d3-axa, d1d2d3- and f2-aa-variants. hence, in these variants, a loss of colocalization was seen, which suggests that scaffolding as well as the dynamics of the hnhe1-erk interaction are dependent on both the d3-domain and the f2-site. these data fully support the in vitro nmr data and highlight both the d3-domain and f2-site as important for scaffolding.fig. nhe co-localizes with erk shown by immunofluorescence. a immunofluorescence images of ap- cells treated or not for 15 min with egf. merged images were zoomed to highlight the co-localization of erk1/ and nhe. all other variants are shown in additional file 6: figure s data are representative of three independent biological replicates. b representative line scans across membrane areas of images as in a. line scans were performed using olympus image analysis software. the figure shows the pixel intensities at each wavelength over the line shown, in the absence and presence of egf as shown. data are examples based on analysis of at least cells in three to four independent replicates per condition. c summary of line scan analysis data for all variants. the figure shows the percentage of cells with nhe1-erk1/ co-localization in both membranes, based on the experiments illustrated in b. data are shown as mean percentage with sem error bars, based on analysis of at least cells in three to five independent replicates per condition. §§ and §§§, p <  and p < , respectively, relative to ap1 + egf; * p <  relative to own control in absence of egf. two-way anova, Šídák’s multiple comparisons test nhe regulates erk phosphorylation status in a cellular context to address whether nhe regulates erk activity, we next assessed erk1/ activation by determining relative t202/y -t185/y phosphorylation, corresponding to erk1/ activation. untransfected ap- cells, or ap- cells stably expressing wt hnhe were exposed to egf to induce erk1/ activity. as seen, despite the known low level of egf receptor expression in chinese hamster ovary -derived cells such as ap- cells, stimulation of cells expressing wt hnhe with egf evoked a modest but significant increase in erk1/ phosphorylation at these sites, i.e. erk1/ activation. importantly, the presence of wt hnhe was necessary for detectable activation of erk1/ by egf. this suggests that hnhe is important for erk1/ activation in this context. to assess whether the interaction-site mutations would affect nhe1-regulated erk activity, we exposed untransfected ap- cells or ap- cells expressing wt and variant hnhe1s, to egf and determined erk1/ activity. compared to cells expressing wt hnhe cells expressing hnhe1-d3-axa, hnhe1-d1d2d3- or hnhe1-f2-aa variants exhibited a significant decrease in relative egf-induced erk1/ phosphorylation. these data show that hnhe is important for egf-induced erk1/ activation in a cellular context, in a manner dependent on its d3-domain and f2-site. these data further support that this at least in part reflects a scaffolding role of hnhe involving the three independent low affinity sites. notably, the effects of the mutations on erk1/2-nhe co-localization and erk1/ activity are comparable if not identical, and mirrors the interaction monitored by nmr spectroscopy.fig. nhe regulates the phosphorylation status of cellular erk1/ erk1/ phosphorylation in untransfected ap- cells or ap- cells expressing wt and variant hnhe as indicated. cells were stimulated or not with human recombinant egf for 15 min. a representative immunoblots of p-erk1/ -t185/y phosphorylation) and total erk1/ under the conditions shown. the arrows indicate erk and erk, respectively. b summary data from three to eight independent biological replicates per condition. blots were scanned, and band intensities quantified using un-scan-it graph digitizer software, as described in the “methods”. quantified data are shown as means with sem error bars, normalized to those in untransfected, unstimulated ap- cells. *, **, and ***, p < , p < , and p < , respectively, compared to untransfected control; #, ##, and ###, p < , p < , and p < , respectively, compared to the same cells in absence of egf. two-way anova, holm-Šídák’s multiple comparisons test discussion nhe activity after various stimuli is regulated by erk1/ and the nhe c-terminal tail is directly phosphorylated by erk in vitro and in vivo. vice versa, nhe can regulate erk1/ activity, yet molecular details and mechanistic understanding of their interaction have been lacking. in conjunction with yeast two-hybrid studies suggesting interaction of nhe with mapks, these studies led us to hypothesize that nhe and erk engage in direct physical interaction. supporting this hypothesis, we report here that erk and nhe interact directly in vivo as well as in vitro, and that erk phosphorylates multiple sites in hnhe1cdt. based on nmr analyses, in conjunction with various combinations of d-domain and f-site mutations, we suggest that hnhe1cdt scaffolds iaerk and that they interact in a non-cooperative modular manner that involves a d-domain and two f-sites. nmr titrations revealed the affinity of the d3-domain to be in the low micromolar range, in agreement with known d-domain affinities. d-domain and f-site mutations did not prevent erk2-mediated hnhe phosphorylation in vitro, but altered its kinetics. in vivo, hnhe and erk1/ co-localized at the plasma membrane in a manner sensitive to erk1/ stimulation, and mutations in the hnhe d3-domain and f-site altered erk1/ activity. thus, a central conclusion of this work is that nhe and erk directly interact and engage in physical and functional reciprocal interactions. this provides a novel molecular framework for understanding previous reports of both nhe1-mediated scaffolding and regulation of erk1/ and erk1/2-mediated phosphorylation of nhe. the organization of idps as “flexible wrappers” has previously been suggested as a general scaffolding mechanism, involved in bipartite interactions, for example of ste with the yeast mapk fus and of p21cip with cyclin-dependent kinases. upon identification of the f-site, a bipartite modular, non-cooperative recognition system was originally suggested for kinases with the d-domain and f-site acting independently or in combination. the tripartite interaction between hnhe1cdt and iaerk uncovered here extends this concept. for hnhe the multi-site interaction may function in a manner analogous, but not identical, to a fuzzy complex, where several binding sites are at play concomitantly, yet with none of the interactions seemingly affecting each other. the “hot potato hypothesis” was originally suggested by perham in to describe the handover of substrates and intermediates in multi-enzyme complexes. indeed, the non-cooperative, multi-site scaffold interaction between hnhe and erk may function similarly to holding a hot potato; the sites do not cooperate to increase the affinity, but will “shuffle” dynamically with sites being sometimes off, sometimes on, and hence we term this type of scaffold interaction a “shuffle complex”.fig. scaffolding of erk by the intrinsically disordered nhe1cdt via a “shuffle complex”. suggested topology of the hnhe1cdt-iaerk complex indicating the tripartite binding mode between hnhe1cdt and human iaerk a–b as nhe functions as a dimer in the cell, one or both nhe1s in a dimer can shuffle an erk1/ c alternatively, one nhe binds erk1/ whereas the other nhe in the dimer can scaffold other members of the signalling complex. alternating conformations are indicated encompassing the individual sites and combinations nhe is, to the best of our knowledge, the first membrane protein described to scaffold members of the mapk pathway, spanning all four levels of the mapk hierarchy. many soluble scaffold proteins acting together with, for example, g protein-coupled receptors and growth factor receptors, have been described, but detailed interaction data have not provided insight into how scaffolding and regulation are coupled. in the cell, meks, phosphatases, and substrates all compete for the d-domain recognition site on erk, and it is currently not known how nhe interacts with the other mapk members, including mek, and assembles a signalling complex. as nhe acts as a dimer in vivo, we propose that upon release of the d3-domain from one nhe monomer due to competition with mek, the shuffle complex organization keeps erk in place via scaffolding by d-domains of the other nhe subunit in the nhe dimer, or by the f-sites. furthermore, in a potential cellular complex, the remaining d-domains as well as f-sites will be available for further scaffolding of other members of the mapk hierarchy, as suggested by yeast two-hybrid screens, potentially mek, although this remains to be explored. nhe is the first example of an id substrate of erk for which detailed interaction data now exist, and to the best of our knowledge, no other idp or idr has to date been experimentally linked to erk phosphorylation or scaffolding. it has been noted that the d-domain of the tyrosine-phosphatase ptp-sl resides in a region with high disorder propensity. disorder predictions of the nuclear pore protein tpr show its erk2-interacting f-site to reside in an idr, similar to the nucleoporin fg-repeat regions and to hnhe1cdt. thus, it appears that the f-site recruitment site interaction may be frequently exploited by idps. however, it remains to be seen whether the multi-site shuffle interaction is a novel canonical idp/idr-erk interaction mode or if it is a unique scaffolding function specific to nhe all six putative erk phosphorylation sites of hnhe1cdt were phosphorylated by aerk in vitro. previous in vivo phosphoproteomics have mapped phosphorylation at five of these sites, yet without identification of the responsible kinases, and with no information on the sequence of individual phosphorylation events. further, some of these sites, i.e. s t and s were previously identified upon in vitro phosphorylation of nhe by erk. no non-canonical phosphorylation was detected in the present study, in contrast with previous reports identifying s s and s as erk-dependent nhe phosphorylation sites. while it is possible that additional complexity may be introduced in the in vivo setting, our data underscore the major advantage of nmr for direct identification of phosphorylation sites. physiological roles have been proposed for all six phosphorylations, although their interplay and dynamics have never previously been assessed. s and s were reported to be phosphorylated by p mapk in murine pro-b-cells, and phosphorylation of s was suggested to mediate apoptosis-induced alkalinization by nhe. based on studies of nhe mutants expressed in nhe1-deficient cho cells, s was reported to mediate erk-dependent nhe activation, and later, s t and s but not s s or s which were assigned roles in erk-dependent nhe phosphorylation after sustained acidosis in renal cells. in amphiuma erythrocytes, phosphorylation of residues corresponding to s and s were detected by ms, where s was constitutively phosphorylated. the precise downstream effects of these phosphorylations are not currently known, but they are likely to both impact nhe structural dynamics and hence activity, and to contribute to the scaffolding role of nhe in regulation of erk, hence fine-tuning cellular erk signaling. timing of signalling events is crucial for many cellular functions, and phosphorylation events that are interdependent or distributive with very different rate constants are possible ways of controlling signal duration and strength. indeed, it has been suggested that such temporally ordered phosphorylations serve as platforms for signal integration. our findings provide evidence for a distinct temporal order of erk phosphorylation of hnhe1cdt with the occurrence of specific intermediates. these intermediates could function as tightly regulated docking sites or thresholds that convert graded signals to switch-like responses. such dynamics in erk signalling have been observed to affect the half-life of an erk downstream effector, the transcription factor c-fos. timed phosphorylation events in hnhe1cdt may therefore similarly partake in control of the erk signal duration. the hnhe1cdt has other confirmed phosphorylation sites than those demonstrated in the present study, and many more putative ones, several of which are close to the erk interaction sites, for example s phosphorylated by rsk. these sites may mediate interactions with other binding partners, introducing additional layers of complexities, for example of pathway crosstalk. the close proximity of the primary s and t phosphorylation sites to the d3-domain and f1-site, respectively, suggests regulatory role for the interaction of hnhe1cdt with, and phosphorylation by, erk analogously, the first phosphorylation events in hnhe1cdt may change the binding mode and/or dynamics with erk in a way that regulates the phosphorylation of succeeding sites, although we showed that they do not act as priming sites. the effects of hnhe variants on the phosphorylation kinetics support the hypothesis that all sites are at play within the shuffle complex, where the sites closest to the phosphorylation site are exploited for interaction with erk the current data does not allow us to evaluate whether all six sites are phosphorylated during nhe activation. some sites, and most likely the slowest ones observed here, may only react under certain physiological conditions. we hypothesize that such conditional phosphorylations could be important for a rheostatic regulation of both erk and nhe consequently, the results of this work open a series of new questions, both regarding the generality of shuffle complexes in scaffolding by idps, but also concerning the functional roles and spatial and temporal interconnectivity of the six identified phosphorylation sites in hnhe  in this work we have demonstrated that the intrinsically disordered region of hnhe acts as a membrane scaffold engaging erk in a multi-site shuffle complex. we show that the interaction is recapitulated in vivo, and that co-regulation of hnhe and erk manifests in distinct in vivo effects on erk1/ activity and in vitro effects on hnhe phosphorylation. our work provides a molecular mechanism for the important scaffolding function of nhe and characterizes a direct interaction between the intrinsically disordered hnhe1cdt and erk leading to hnhe1cdt phosphorylation and regulation of erk1/ activity. 
extracellular signal-regulated kinase is an s/t kinase with more than known substrates, and with critical roles in regulation of cell growth and differentiation and currently no membrane proteins have been linked to erk scaffolding. time-resolved nmr spectroscopy revealed that erk phosphorylated the disordered tail of hnhe at six sites in vitro, in a distinct temporal order, with the phosphorylation rates at the individual sites being modulated by the docking sites in a distant dependent manner. using nuclear magnetic resonance spectroscopy and immunofluorescence analysis we demonstrate that erk scaffolding by hnhe occurs by one of three d-domains and by two non-canonical f-sites located in the disordered intracellular tail of hnhe mutation of which reduced cellular hnhe1-erk1/ co-localization, as well as reduced cellular erk1/ activation. this work characterizes a new type of scaffolding complex, which we term a “shuffle complex”, between the disordered hnhe1-tail and erk and provides a molecular mechanism for the important erk scaffolding function of the membrane protein hnhe which regulates the phosphorylation of both hnhe and erk 
 extracellular signal-regulated kinase is an s/t kinase with more than known substrates, and with critical roles in regulation of cell growth and differentiation and currently no membrane proteins have been linked to erk scaffolding. methods and  here, we identify the human na+/h+ exchanger as a membrane scaffold protein for erk and show direct hnhe1-erk1/ interaction in cellular contexts. using nuclear magnetic resonance spectroscopy and immunofluorescence analysis we demonstrate that erk scaffolding by hnhe occurs by one of three d-domains and by two non-canonical f-sites located in the disordered intracellular tail of hnhe mutation of which reduced cellular hnhe1-erk1/ co-localization, as well as reduced cellular erk1/ activation. time-resolved nmr spectroscopy revealed that erk phosphorylated the disordered tail of hnhe at six sites in vitro, in a distinct temporal order, with the phosphorylation rates at the individual sites being modulated by the docking sites in a distant dependent manner.  this work characterizes a new type of scaffolding complex, which we term a “shuffle complex”, between the disordered hnhe1-tail and erk and provides a molecular mechanism for the important erk scaffolding function of the membrane protein hnhe which regulates the phosphorylation of both hnhe and erk electronic supplementary material the online version of this article contains supplementary material, which is available to authorized users. keywords nhe1intrinsically disordered proteinphosphorylationmapkshuffle complexnmrscaffoldhttp://dx.doi.org/ /100008394natur og univers, det frie forskningsråd12-12694212-128803; 4181-00344pedersen stine f. kragelund birthe b. http://dx.doi.org/http://dx.doi.org/ /100008398villum fonden http://dx.doi.org/http://dx.doi.org/ /501100004191novo nordisk issue-copyright-statement© the author 2016 