BACKGROUND
dysregulated signalling is a prominent feature in cellular transformation and tumorigenesis.
the proto-oncogene casitas b-lineage lymphoma , encodes an e <dig> ubiquitin ligase that downregulates ptk-directed cell signaling through ubiquitination, thereby targeting these kinases for lysosomal or proteasomal degradation  <cit> .
cbl is a member of the cbl family of proteins, so characterized based on a highly conserved n-terminal region that contains the structural components required for ubiquitin ligase activity.
in simpler eukaryotic organisms, such as caenorhabditis elegans and dictyostelium discoideum, only one cbl protein is present, but in mammals there are three, including cbl, cbl-b and cbl-c.
the conserved n-terminus of cbl family proteins contains a substrate tyrosine kinase-binding domain , a linker helix region  and a ring domain .
the tkbd confers specificity to cbl’s ligase activity based on the selective recruitment of phosphorylated substrates containing an xpyxxp, dpyr or raxnqpy motif  <cit> .
the ring domain mediates the transfer of ubiquitin  from an e <dig> ub-conjugating enzyme to the substrate  <cit> .
within the lhr is a conserved tyrosine  that is crucial for regulating ligase activity.
phosphorylation of this tyrosine enhances ligase activity and is essential for ubiquitination of receptor ptks .
in addition to the highly conserved n-terminus, cbl and cbl-b also have extensive c-termini that confer adaptor-like functions to these proteins based on the ability to mediate multiple protein-protein interactions.
these include a proline rich region that mediates interactions with sh <dig> domain-containing proteins and a tyrosine rich region that, upon phosphorylation, becomes a binding motif for other sh <dig> domain-containing proteins  <cit> .
cbl and cbl-b terminate with an ubiquitin-associated domain, which is crucial for homo- and heterodimerization of these two cbl proteins  <cit> .fig.
<dig> model depicting linker helix -mediated regulation of cbl ligase.
a domains of cbl’s n-terminal ubiquitin ligase region.
a blue rectangle and navy wavy line correspondingly depict the tkbd and its substrate-binding site.
the lhr and its components are indicated with ll <dig> coloured purple, ll <dig> green and lh yellow.
a yellow hexagon depicts the lh tyr.
the ring is coloured orange with the two loops representing the e2-binding site.
b model of unphosphorylated cbl coloured as in .
when tyr <dig> is unphosphorylated, the lh is clamped to the tkbd and movement of the ring restricted to the tkbd face opposing the substrate-binding site.
rotation about ll <dig> allows cbl to fluctuate between an autoinhibited state where the e2-binding site is occluded  and a catalytically competent state where e <dig> is able to bind .
c model of phosphorylated cbl coloured as in .
a brown hexagon and circle represent the phosphorylated lh tyr.
when tyr <dig> is phosphorylated, the lh clamps into the ring domain and ll <dig> rotates 180°, juxtaposing the ring domain and substrate-binding face of the tkbd.
the tyr <dig> binding site on the tkbd cannot accommodate a phosphate moiety, thereby eliminating the lh-tkbd interaction.
whether the ring domain is flexible in solution is unknown.
d model depicting possible states of cbl tyr  <dig> mutants coloured as in .
the lh tyr <dig> mutant is shown as an x in a red square.
cbl y371x mutants are predicted to favour different states depending on the chemical nature of the substitution.
less disruptive mutants are expected to maintain the unphosphorylated equilibrium , whereas more disruptive mutants are expected to perturb the lh-tkbd interaction, leaving the ring in an open, catalytically competent conformation with greater access to space surrounding the tkbd
cbl abnormalities are associated with a number of human cancers.
high expression of cbl is commonly observed in several human breast cancer cell lines as well as primary breast and prostate cancer tissues  <cit> .
in addition, cbl mutations are observed in myelodysplastic syndromes-myeloproliferative neoplasms  and non-small cell lung cancers  <cit> .
cbl is suggested to have different roles in pathological processes depending on its function as a ligase or adaptor.
breast and prostate cancer cell line oncogenic characteristics are reduced when cbl is knocked down, and, in breast cancer cells, independent of its ligase activity, cbl mediates suppression of transcriptional activity induced by the tumour suppressor transforming growth factor-β <dig>  <cit> .
in contrast, more in-depth studies of select mds/mpn mutants suggest that these mutants potentiate transformation because they fail to ubiquitinate and thereby downregulate ptk signalling  <cit> .
the highest frequency of missense mutations in mds/mpn clinical samples occurs at tyr371; identified mutations include cbl y371s, y371h, y371c, and y371d.
cbl y371s and y371h have been investigated extensively in independent studies  <cit> .
cbl y371s promotes colony formation in soft-agar assays and induces tumour formation when injected into nude mice.
likewise, cbl y371h promotes cytokine-independent growth in cells but only when endogenous cbl is knocked down.
both display defects in their ability to ubiquitinate the ptk egfr.
for tyr <dig> mutations in the lhr, although defective ptk ubiquitination contributes to aberrant cell signalling, it is not sufficient to potentiate transformation.
cbl y371f shows reduced egfr ubiquitination in vitro and in cells  <cit> , but it is significantly less efficient in promoting colony formation in soft agar assays and does not induce tumour formation when injected into mice  <cit> .
a number of factors contribute to how phosphorylation of tyr <dig> regulates cbl’s ligase activity.
in the unphosphorylated state, cbl’s ring domain rotates between an open, catalytically competent conformation and a closed conformation   <cit> .
in the closed conformation, the e2-binding site on the ring domain is occluded and cbl is autoinhibited.
although the ring can bind e <dig> thioesterified with ub  in the unphosphorylated state, the ring domain is restricted to the tkbd face opposite the substrate-binding site by interactions between the lhr and tkbd.
the lhr comprises a long loop  followed by an α-helix  and then a second shorter loop .
tyr <dig> is on the lh, and, in the unphosphorylated state, tyr <dig> and several other residues from the lh, including tyr <dig>  bind to the tkbd, limiting movement between the ring and tkbd to rotation about ll <dig>  phosphorylation of tyr <dig> eliminates this lhr-tkbd interaction – modelling suggests the tyr <dig> binding pocket on the tkbd is too small to accommodate a phosphate group.
instead, ptyr371-lhr forms a new set of interactions with the ring domain that stabilizes e2 ~ ub during ub transfer  <cit> .
movement within ll <dig> is more restricted by this new set of interactions but a conformational change in ll <dig> juxtaposes the ring domain with the substrate-binding face of the tkbd .
in solution, whether ll <dig> is flexible and adopts multiple conformations or prefers the crystallographically observed conformation where the ring is juxtaposed with the substrate binding face of the tkbd is unclear.
we postulate that y <dig> mutations might have different oncogenic potentials due to flexibility differences in cbl’s linker region mediated by the lhr-tkbd interaction .
we predict that, while a large aromatic residue like phe cannot duplicate the tyr371-tkbd interactions, neither will it perturb those interactions; hence, cbl y371f can adopt and readily maintain conformations observed in the unphosphorylated state.
on the other hand, mutations, such as y371s, introduce a polar residue into a hydrophobic pocket and are likely to perturb or occlude the lh-tkbd interaction, thereby unlocking the lhr from the tkbd and leaving cbl in an intermediate activity state where the e2 ~ ub binding site on the ring domain is exposed but lacking the ptyr371-lhr e2 ~ ub stabilizing component .
to investigate the influence of mutations and phosphorylation on cbl’s lhr flexibility and ring accessibility in solution, we performed small-angle x-ray scattering  analysis and surface plasmon resonance  assays on ptyr371-cbl, unphosphorylated cbl and a selection of cbl variants.
we then performed focus formation assays to explore the relationship between this flexibility and oncogenic potential.
RESULTS
cbl’s lhr-tkbd interactions affect ring binding affinity for e2 ~ ub
previous studies suggest cbl y <dig> mutants have the potential to perturb or abrogate lhr-e <dig> interactions essential for ligase activity  <cit> .
to investigate this possibility, we used spr to measure binding of the substrate e <dig> ubch5b conjugated to ub  to variants of the n-terminal fragment of cbl comprising the tkbd, linker region and ring domain , as performed previously  <cit> .
we tested the unphosphorylated n-cbl variants containing the tyr <dig> mutations identified in mds/mpn clinical samples as well as n-cbl y371a, which behaves similarly to the mds/mpn tyr <dig> mutants  <cit> , and n-cbl y371f based on its reduced transformation potential in cells and mice.
in addition, we tested cbl y371e, which has previously been shown to stimulate cbl activity in vitro  <cit> .
n-cbl y368f was also included in our assays because this mutation is required to generate homogeneously phosphorylated ptyr371-n-cbl; previous studies demonstrate this mutation does not affect the structure or ligase activity in vitro or in cells  <cit> .
we also included n-cbl m222e, which disrupts the closed conformation without perturbing the tkdb-lhr interaction  <cit> , so that we could investigate changes induced by rotation about ll <dig>  we predicted that, if the ring domain were unfolded, no binding of ubch5b–ub to n-cbl y <dig> variants is expected, whereas, if these variants simply weaken the lhr-tkbd interaction, cbl y <dig> mutants will adopt the closed conformation less frequently and thus bind ubch5b–ub more tightly than wild-type n-cbl.
none of the n-cbl y <dig> mutants is expected to bind ubch5b–ub as tightly as ptyr371-n-cbl because ptyr <dig> eliminates the closed, autoinhibited conformation and forms additional interactions with ubch5b ~ ub.
all of the n-cbl variants in our spr assay bind ubch5b–ub with similar or higher affinity than wild-type n-cbl  but more weakly than ptyr371-n-cbl , suggesting mutations in the lhr do not affect the competency of the ring domain to bind ubch5b–ub.
other mutations in the lhr also affect lhr-tkbd interactions as evidenced by the higher binding affinity of n-cbl y368f  for ubch5b–ub than wild-type n-cbl.table  <dig> dissociation constants  for interactions between cbl variants and ubch5b–ub
k
standard errors of the mean are indicated
the spr data demonstrate that the chemical characteristics of the side chain of the mutated amino acid in the n-cbl y <dig> variants influence binding affinity for ubch5b–ub: the greater potential the mutation has to perturb the tyr <dig> binding pocket on the tkbd, the higher the binding affinity for ubch5b–ub.
the highest binding affinities are observed when tyr is substituted with an amino acid that has a shorter, non-aromatic and polar or charged side chain like n-cbl y371s , y371e , and y371d ; the binding affinities of these mutants are comparable to n-cbl m222e , which disrupts the ring-tkbd interaction in the closed, autoinhibited conformation.
in contrast, when tyr <dig> is substituted with phe, the ubch5b–ub binding affinity is comparable to wild-type  and the lhr-tkbd interaction is unperturbed, as demonstrated by the crystallographic structure of n-cbl y371f .
for the remaining mds/mpn mutants, polar and aromatic or short and hydrophobic amino acids are substituted for tyr <dig> .
these are expected to weaken the interactions within the tyr <dig> binding pocket but not as effectively as the group of mutants with short and polar or charged side chains.
thus, the ubch5b–ub binding affinity of these mutants is expected to fall between wild-type and the more disruptive mutants and this is indeed what we observe in our spr assays: n-cbl y371h , y371a  and y371c .
characterization of wild-type and ptyr <dig> cbl by saxs
initially, we sought to investigate how tyr <dig> phosphorylation affects the conformation of cbl in solution by performing saxs analysis on ptyr371-n-cbl and wild-type n-cbl.
the monomeric state of these n-cbl variants was confirmed by the saxs-derived overall parameters  and gel filtration chromatography .
the deduced molecular masses  ranged from  <dig> to 48 kda and are comparable with a predicted monomer mass of ~45 kda for n-cbl.fig.
<dig> saxs analysis of wild-type  and ptyr371-n-cbl.
a
top: wt experimental scattering data  versus theoretical scattering curve from crystal structure  before  and after normal modes refinement .
bottom: ptyr371-n-cbl experimental scattering data  versus theoretical scattering from the eom  <dig>  ensemble .
b
p distribution of wt  and ptyr371-n-cbl .
c normalized kratky plot of wt and ptyr371-n-cbl coloured as in .
d eom  <dig>  distributions of the random pool  and the ensembles of wt and ptyr371-n-cbl coloured as in
r
r
d
max
v
porod
v
dammif
mmporod
mmdammif
rg radius of gyration, d
max maximum particle distances, v volume, mm molecular mass
the saxs-derived radius of gyration  of wild-type n-cbl variant is ~ <dig>  nm whereas ptyr371-n-cbl is ~ <dig>  nm, and the calculated maximum particle distances  are ~ <dig>  and ~ <dig>  nm, respectively.
these differences indicate significant conformational changes in n-cbl upon phosphorylation of tyr <dig>  the presence of an extended tail in the p function of ptyr371-n-cbl  suggests rearrangements of the ring domain that make the particle more elongated as compared to the native one.
in addition, the normalized kratky plots  for wild-type and ptyr371-n-cbl demonstrate significant differences.
the wild-type reveals the peak at s rg ≈ √ <dig>  as expected for globular particles  <cit> , whereas for ptyr371-n-cbl the peak position is significantly shifted suggesting the presence of structural disorder.
the theoretical scattering computed from the native, unphosphorylated crystal structure  gives an overall reasonable fit to the experimental data but also displays some systematic deviations .
these deviations point to possible differences between solution and crystalline states of the native protein.
the crystal structure was therefore refined using an iterative normal mode analysis  <cit> .
this procedure yielded a model which was similar to the original structure  and provided a significant improvement of the fit from χ =  <dig>  to  <dig> .
ab initio shape reconstruction further confirms the globular state of unphosphorylated cbl .fig.
<dig> comparison of crystal structure of n-cbl  and the model derived from iterative normal mode analysis.
the unphosphorylated, catalytically competent n-cbl from the crystal structure  is coloured cyan and is superposed with the normal mode analysis-refined model.
the tkbd is shown in blue, the ring domain in orange and the lhr in yellow as a surface model
given the expected flexibility of ptyr371-n-cbl, structural modelling was performed using an ensemble optimization method   <cit> .
here, ensembles of models with variable conformations are selected from a pool of randomly generated models such that the scattering from the ensemble fits the experimental data, and the distributions of the overall parameters  in the selected pool are compared to the original pool.
as a negative control, eom was first used on the data from n-cbl and the selected ensembles showed predominantly compact conformations .
in contrast, the selected ensembles for ptyr371-n-cbl displayed broader distributions of predominantly extended conformations, which were on average more extended than the random pool .
these data indicate that ptyr371-n-cbl is extended and flexible in solution, suggesting that phosphorylation of the lh allows the ring domain to adopt multiple conformations in solution and access multiple surfaces of the tkbd .fig.
<dig> cartoon ensemble representation of ptyr371-n-cbl.
the tkbd domain is coloured blue.
the predicted ring domain conformations are shown in several colours as indicated in the table.
the missing loops connecting the ring and tkbd domains are coloured according to the corresponding ring domains but presented as transparent surfaces
characterization of n-cbl mutants by saxs
to study how lhr-tkbd interactions influence the flexibility of ll <dig> in solution, we performed saxs on a concentration series of our n-cbl tyr <dig> mutants, n-cbl y368f and n-cbl m222e.
the monomeric state of all n-cbl variants was confirmed by the saxs-derived overall parameters  and gel filtration chromatography  with the exception of n-cbl y371c, where strong concentration-dependent effects were observed; for this reason, n-cbl y371c was omitted from further saxs analysis.
for the remaining n-cbl variants, the deduced mms ranged from  <dig> to 48 kda pointing to a monomeric state in solution.
comparison of the saxs-derived parameters as well as quantification of the flexibility  estimated by using eom revealed a link between the conformation of n-cbl in solution and the extent of perturbation of the tyr371-tkbd interaction similar to the trend observed in our spr data .
our n-cbl y <dig> variants were classified into four categories, depending on the extent of lhr-tkbd perturbation observed based on our spr findings: complete , strongly-perturbing , moderately perturbing , and non-perturbing .
ptyr371-n-cbl, which abolishes the lhr-tkbd interaction, appears the most elongated, whereas unphosphorylated, wild-type n-cbl appears more compact.
compared to wild-type n-cbl, the strongly perturbing mutants, n-cbl y371s, y371e and y371d, are partially elongated  but not as much as ptyr371-n-cbl.
conformational rearrangements of these mutants are also confirmed by the corresponding rflex values of approximately 84 %, 90 % and 88 % compared to approximately 60 % for wild-type n-cbl.
the moderately and non-perturbing mutants have rg and dmax values more similar to the wild-type n-cbl, although with different degrees of flexibility .
notably, n-cbl y371f appears slightly more compact and less flexible than wild-type n-cbl, with an rg of  <dig>  nm, a dmax of  <dig>  nm, and an rflex of approximately 56 %.
together with spr, these data demonstrate that, in solution, ring domain accessibility is connected to n-cbl conformational rearrangements induced by mutation or modification of tyr <dig> in the lhr.table  <dig> average ensemble outcome r
g and d
max from eom  <dig>  together with the χ value and the quantification of flexibility  for each n-cbl variant
r
d
r
fig.
<dig> eom  <dig>  analysis of n-cbl tyr <dig> mutants, n-cbl y368f and n-cbl m222e.
mutants were clustered according to their ability to perturb the lhr-tkbd interaction based on saxs parameters shown in table  <dig>  a strongly perturbing mutants.
b moderately perturbing mutants.
c weakly or non-perturbing mutants
the tyr371-tkbd interaction appears to play a critical role in mediating how elongated n-cbl is in solution.
although n-cbl y368f and m222e have higher binding affinities for ubch5b–ub than wild-type, at saxs resolution both have characteristics comparable to wild-type, suggesting they are similarly compact in solution.
although n-cbl m222e has a comparable binding affinity to the strongly-perturbing n-cbl y <dig> mutants, in solution, it appears more compact, as both rg and dmax are similar to wild-type n-cbl and flexibility is relatively limited .
n-cbl y368f has a higher binding affinity for ubch5b–ub than wild-type, but at the level of resolution provided by saxs its characteristics and compactness are similar to those of the wild-type protein, suggesting similar behaviour in solution.
transformation potential of cbl y <dig> mutants in cells
to test the transforming potential of cbl y <dig> mutants in cells, we performed focus formation assays using 3t <dig> cells stably transfected with n-terminally flag-tagged cbl variants.
relative protein expression levels of each variant were assessed by immunoblotting .
foci were visualized with sulforhodamine b staining and counted manually with a dna safe imager .
afterwards, the sulforhodamine b stain was extracted and the a <dig> measured to compare relative cell densities  <cit> .
foci counts and relative cell densities were analyzed by one-way anova followed by dunnett’s test with wild-type cbl as the control.
cbl70z was included among the variants tested as a positive transforming control  <cit> .
other variants tested included the same set used in our biochemical assays except in a full-length context.fig.
<dig> transformation potential of cbl variants in focus formation assays.
a immunoblot of lysates from 3t <dig> fibroblasts infected with flag-tagged cbl variants using α-flag antibody  and α-actin  antibody as a loading control.
b sulforhodamine b-stained 3t <dig> fibroblasts infected with indicated cbl variants.
c mean number of foci formed by cbl variant-infected 3t <dig> fibroblasts shown in a bar graph .
no foci were present in 3t <dig> cells infected with wild-type cbl, cbl m222e or cbl y368f.
double asterisks  denote significant differences  between indicated cbl variant and wild-type using anova followed by dunnett’s multiple comparisons test.
error bars indicate standard deviation.
d as in  but for a <dig> of extracted sulforhodamine b from cbl-infected 3t <dig> fibroblasts
foci were observed in cells transfected with cbl y <dig> mutants and cbl70z, but not in wild-type, cbl y368f- or cbl m222e-transfected cells .
when analyzed by one-way anova followed by dunnett’s test, significant differences  in number of foci and cell densities were observed between cells infected with wild-type cbl and all the y <dig> mutants except cbl y371f .
significantly more foci were present in cbl70z, cbl y371e and the mds/mpn cbl y <dig> mutant set and the relative cell density was significantly greater compared to wild-type cbl; however, there is no correlation between mutations that promote moderate or strong lhr-tkbd perturbations and transformation potential.
based on the ability to form foci, all the cbl variants except cbl y368f and m222e have the ability to potentiate transformation in cells.
based on statistical analyses of the number of foci and relative cell density, cbl70z, cbl y371e and the mds/mpn cbl y <dig> mutant set have significantly higher numbers of foci than wild-type cbl.
for cbl y371f, neither the number of foci nor the relative cell density are significantly different than in cells infected with wild-type cbl; these results suggest this mutant may potentiate transformation but not to the same extent as the other cbl y <dig> mutants or cbl70z.
discussion
in this work, we employed saxs and spr to characterize how modifications and mutations in cbl affect the conformation and accessibility of the ring domain in solution and subsequently investigate the relationship between these characteristics and the ability of cbl variants to potentiate transformation in cells.
our results show that a mutation  within the ring-tkbd interface regulates accessibility of the ring domain only, whereas mutation or modification of tyr <dig> within the lhr mediates the lhr-tkbd interaction, thereby regulating ring domain accessibility as well as the space sampled by the ring domain relative to the tkbd.
the chemical nature of the substitution or modification controls the lhr-tkbd interaction and shifts the equilibrium between the native conformation where the ring is restricted to the face of the tkbd opposing the substrate-binding site and open conformations where the ring domain can access other surfaces of the tkbd and potentially other regions of cbl.
none of the tyr <dig> mutants can adopt the ptyr <dig> conformation that is critical for activation of e2 ~ ub and rtk ubiquitination, but all of the tyr <dig> mutants perturb the lhr-tkbd conformation observed in the native state except cbl y371f.
likewise, all of the tyr <dig> mutants except cbl y371f clearly potentiate transformation in our focus formation assays, whereas cbl y371f forms foci but statistical analyses of the number of foci formed and relative cell densities suggest that this mutant has less transformation potential than the others.
previous studies have shown that cbl y371f is not transforming in soft agar colony formation assays nor does it promote tumour formation in nude mice  <cit> .
these data indicate that the flexibility of the lhr features in the cbl tyr <dig> mutant transformation mechanism.
met <dig> is a key residue in maintaining the ring-tkbd interaction in native cbl.
a met to glu mutation was used to perturb this interaction and investigate the importance of the ring-tkbd interaction on cbl’s conformation in solution.
our previous work and spr studies show that mutation of met <dig> in the tkbd to glu increases cbl’s e2–ub binding affinity by approximately 8-fold and enhances cbl’s in vitro autoubiquitination rate  <cit> .
though cbl m222e has a higher binding affinity for ubch5b–ub and is more active than wild-type cbl, saxs analysis demonstrates that cbl m222e has parameters  comparable to wild-type cbl, suggesting both adopt a compact conformation.
in cells, cbl m222e does not enhance egfr ubiquitination  nor does it potentiate transformation in our focus formation assays.
the lack of effect of this mutant in these assays suggests structural regulation of cbl’s ligase activity requires more than a simple rotation that exposes the e2-binding site on the ring domain.
previous work has shown that phosphorylation of the conserved lhr tyr in the cbl family enhances activity in vitro and is essential for receptor ptk ubiquitination in cells, where cbl’s e <dig> ligase activity features prominently in egfr downregulation via lysosomal degradation  <cit> .
the rate and pattern of cbl-mediated egfr ubiquitination determines whether egfr is directed to the lysosome or recycled to the membrane.
x-ray crystallography and nmr studies of cbl and cbl-b have shown that this phosphorylation event eliminates ring-mediated autoinhibition, optimally positions ub for transfer and juxtaposes the ring domain with the tkbd substrate-binding site  <cit> .
notably, in the crystal structures of cbl and cbl-b bound to zap <dig> peptide and e <dig> or e2 ~ ub, the conformations of ring domain relative to the tkbd are identical.
here, we show that, in solution, when tyr <dig> is phosphorylated, the rg and dmax increase and eom analysis demonstrates the linker region becomes flexible, allowing the ring domain to sample the space surrounding the tkbd rather than maintain the crystallographically observed conformation.
this is consistent with other e <dig> ligases, where translational and rotational movement of the ring domain are required for ub transfer to select substrate lysine sites  as well as polyub chain formation  <cit> .
cbl binds egfr directly through its tkbd and indirectly through growth factor receptor binding 2-mediated interactions in the region c-terminal to the ring domain  <cit> .
cbl-mediated ubiquitination of egfr is observed for both binding sites, highlighting a potential essential role for ring flexibility in cbl substrate ubiquitination.
our saxs and spr data show that mutations within the lhr perturb the native lhr-tkbd conformation and that the extent of perturbation is dependent on the chemical nature of the amino acid substitution.
in the native state, trp <dig>  ala <dig>  val <dig>  and met <dig> form a hydrophobic pocket around the aromatic ring of tyr <dig> and the side chain of ser <dig> forms a hydrogen bond with the hydroxyl group of tyr <dig>  the more disruptive the mutation is to the hydrophobic environment, the greater the perturbation in the lhr-tkbd interaction.
when tyr <dig> is mutated to an amino acid with a charged side chain like asp or glu or a small, polar side chain like ser, the ubch5b–ub binding affinity is approximately 8-fold enhanced compared to native cbl and the rg and dmax increase to values comparable to ptyr371-cbl.
additionally, saxs analysis reveals these tyr <dig> mutants are the most flexible compared to native cbl.
mutation of tyr <dig> to smaller hydrophobic amino acids like cys or ala only moderately perturbs the lhr-tkbd interaction as evidenced by an approximately 2-fold enhancement in the ubch5b–ub binding affinity and the moderate increase in rg, dmax and rflex compared to native cbl.
likewise, when tyr <dig> is mutated to his, only moderate to weak perturbation of the lhr-tkbd interaction occurs.
there is an approximately 2-fold enhancement in the ubch5b–ub binding affinity and saxs analysis shows evidence of more flexibility  compared to native cbl.
when tyr <dig> is mutated to phe, the hydrophobic interactions are not disturbed and the native state is maintained: the ubch5b–ub binding affinity, rg, dmax and rflex values are comparable to native cbl.
in addition, previous in vitro autoubiquitination assays show native cbl and cbl y371f have comparable catalytic efficiencies  <cit> .
thus, in solution, it appears as though cbl y371f mimics the behaviour of native cbl.
mutation of tyr <dig> is not the only site at which the lhr-tkbd interaction can be perturbed.
our previous work and current data show that mutation of tyr <dig> to phe also disrupts the lhr-tkdb interaction  <cit> .
cbl y368f binds ubch5b–ub approximately 2-fold more tightly than native cbl and has an approximately 2-fold enhancement in catalytic efficiency for in vitro autoubiquitination.
in addition, this mutant displays a slight increase in all saxs-based parameters  compared to native cbl.
however, in contrast to the cbl tyr <dig> mutants, cbl y368f does not promote focus formation in our assays nor does it compromise egfr ubiquitination in cells  <cit>  and previous work has also shown this mutant does not form colonies in soft agar assays nor promote tumour growth in nude mice  <cit> .
though it is clear that tyr368-tkbd interactions also play a role in the conformations cbl adopts in solution, the significance of this interaction in cbl’s ligase activity in cells is unclear.
other studies have shown that cbl Δy <dig> is defective in egfr ubiquitination and has the ability to potentiate transformation in cells and in mice  <cit> .
in addition, cbl y368c has also been identified in mds/mpn clinical samples, and, like cbl y371c and y371s, has been shown to potentiate transformation in cells and in mice; however, whether ptk ubiquitination is compromised remains unknown  <cit> .
as observed for tyr <dig>  the chemical nature of the mutation at tyr <dig> might factor into regulation of cbl’s ligase activity as well as transformation potential.
while the ability to ubiquitinate ptks is essential for cbl-mediated downregulation of ptk signalling, previous work has clearly shown that it is not the only prerequisite for cbl-dysfunction driven transformation.
thien et al.
<cit>  propose that the lhr-tkbd stability of cbl mutants contributes to their ability to potentiate transformation based on the analysis of a number of linker region and ring mutants: cbl Δy <dig>  Δy <dig> and y371a promote transformation but not cbl y368f or y371f.
cbl y368f is slightly more flexible than native cbl but retains the ability to ubiquitinate receptor ptks, whereas cbl y371f cannot ubiquitinate receptor ptks but is able to maintain the native conformation in solution.
thus, it seems that both characteristics contribute to the transformation potential of cbl mutants.
cbl functions as both an ubiquitin ligase and adaptor in receptor ptk-mediated cell signalling.
although enzymatic activity is slower in the absence of tyr <dig> phosphorylation, cbl is still functional  <cit> .
it may be that when the lhr-tkbd interaction is perturbed, cbl ubiquitinates substrates that bind to regions other than the canonical tkbd-binding site or the more flexible mutants might ubiquitinate “dead end” lysines on substrates such that biologically viable ubiquitination chains are not formed.
alternatively, perturbed lhr-tkbd interactions might disrupt oligomerization or interactions with other proteins or lead to the non-sequential recruitment of substrates to exposed surfaces that interfere with controlled signalling.
CONCLUSIONS
we have shown that lhr-tkbd interactions regulate cbl’s ligase activity and conformations in solution.
mutations or modifications within the lhr prevent cbl from maintaining a closed conformation where the ring domain is restricted to a surface opposite the tkbd-substrate binding site; instead, depending on the nature of the alteration, cbl becomes flexible and the ring domain can access multiple surfaces of the tkbd.
transformation only occurs when mutations within the lhr  perturb the native state and  fail to ubiquitinate ptks.

 dysregulated signalling is a prominent feature in cellular transformation and tumorigenesis. the proto-oncogene casitas b-lineage lymphoma, encodes an e ubiquitin ligase that downregulates ptk-directed cell signaling through ubiquitination, thereby targeting these kinases for lysosomal or proteasomal degradation. cbl is a member of the cbl family of proteins, so characterized based on a highly conserved n-terminal region that contains the structural components required for ubiquitin ligase activity. in simpler eukaryotic organisms, such as caenorhabditis elegans and dictyostelium discoideum, only one cbl protein is present, but in mammals there are three, including cbl, cbl-b and cbl-c. the conserved n-terminus of cbl family proteins contains a substrate tyrosine kinase-binding domain, a linker helix region and a ring domain. the tkbd confers specificity to cbl’s ligase activity based on the selective recruitment of phosphorylated substrates containing an xpyxxp, dpyr or raxnqpy motif. the ring domain mediates the transfer of ubiquitin from an e ub-conjugating enzyme to the substrate. within the lhr is a conserved tyrosine that is crucial for regulating ligase activity. phosphorylation of this tyrosine enhances ligase activity and is essential for ubiquitination of receptor ptks. in addition to the highly conserved n-terminus, cbl and cbl-b also have extensive c-termini that confer adaptor-like functions to these proteins based on the ability to mediate multiple protein-protein interactions. these include a proline rich region that mediates interactions with sh domain-containing proteins and a tyrosine rich region that, upon phosphorylation, becomes a binding motif for other sh domain-containing proteins. cbl and cbl-b terminate with an ubiquitin-associated domain, which is crucial for homo- and heterodimerization of these two cbl proteins.fig. model depicting linker helix -mediated regulation of cbl ligase. a domains of cbl’s n-terminal ubiquitin ligase region. a blue rectangle and navy wavy line correspondingly depict the tkbd and its substrate-binding site. the lhr and its components are indicated with ll coloured purple, ll green and lh yellow. a yellow hexagon depicts the lh tyr. the ring is coloured orange with the two loops representing the e2-binding site. b model of unphosphorylated cbl coloured as in. when tyr is unphosphorylated, the lh is clamped to the tkbd and movement of the ring restricted to the tkbd face opposing the substrate-binding site. rotation about ll allows cbl to fluctuate between an autoinhibited state where the e2-binding site is occluded and a catalytically competent state where e is able to bind. c model of phosphorylated cbl coloured as in. a brown hexagon and circle represent the phosphorylated lh tyr. when tyr is phosphorylated, the lh clamps into the ring domain and ll rotates 180°, juxtaposing the ring domain and substrate-binding face of the tkbd. the tyr binding site on the tkbd cannot accommodate a phosphate moiety, thereby eliminating the lh-tkbd interaction. whether the ring domain is flexible in solution is unknown. d model depicting possible states of cbl tyr mutants coloured as in. the lh tyr mutant is shown as an x in a red square. cbl y371x mutants are predicted to favour different states depending on the chemical nature of the substitution. less disruptive mutants are expected to maintain the unphosphorylated equilibrium, whereas more disruptive mutants are expected to perturb the lh-tkbd interaction, leaving the ring in an open, catalytically competent conformation with greater access to space surrounding the tkbd cbl abnormalities are associated with a number of human cancers. high expression of cbl is commonly observed in several human breast cancer cell lines as well as primary breast and prostate cancer tissues. in addition, cbl mutations are observed in myelodysplastic syndromes-myeloproliferative neoplasms and non-small cell lung cancers. cbl is suggested to have different roles in pathological processes depending on its function as a ligase or adaptor. breast and prostate cancer cell line oncogenic characteristics are reduced when cbl is knocked down, and, in breast cancer cells, independent of its ligase activity, cbl mediates suppression of transcriptional activity induced by the tumour suppressor transforming growth factor-β. in contrast, more in-depth studies of select mds/mpn mutants suggest that these mutants potentiate transformation because they fail to ubiquitinate and thereby downregulate ptk signalling. the highest frequency of missense mutations in mds/mpn clinical samples occurs at tyr371; identified mutations include cbl y371s, y371h, y371c, and y371d. cbl y371s and y371h have been investigated extensively in independent studies. cbl y371s promotes colony formation in soft-agar assays and induces tumour formation when injected into nude mice. likewise, cbl y371h promotes cytokine-independent growth in cells but only when endogenous cbl is knocked down. both display defects in their ability to ubiquitinate the ptk egfr. for tyr mutations in the lhr, although defective ptk ubiquitination contributes to aberrant cell signalling, it is not sufficient to potentiate transformation. cbl y371f shows reduced egfr ubiquitination in vitro and in cells, but it is significantly less efficient in promoting colony formation in soft agar assays and does not induce tumour formation when injected into mice. a number of factors contribute to how phosphorylation of tyr regulates cbl’s ligase activity. in the unphosphorylated state, cbl’s ring domain rotates between an open, catalytically competent conformation and a closed conformation. in the closed conformation, the e2-binding site on the ring domain is occluded and cbl is autoinhibited. although the ring can bind e thioesterified with ub in the unphosphorylated state, the ring domain is restricted to the tkbd face opposite the substrate-binding site by interactions between the lhr and tkbd. the lhr comprises a long loop followed by an α-helix and then a second shorter loop. tyr is on the lh, and, in the unphosphorylated state, tyr and several other residues from the lh, including tyr bind to the tkbd, limiting movement between the ring and tkbd to rotation about ll phosphorylation of tyr eliminates this lhr-tkbd interaction – modelling suggests the tyr binding pocket on the tkbd is too small to accommodate a phosphate group. instead, ptyr371-lhr forms a new set of interactions with the ring domain that stabilizes e2 ~ ub during ub transfer. movement within ll is more restricted by this new set of interactions but a conformational change in ll juxtaposes the ring domain with the substrate-binding face of the tkbd. in solution, whether ll is flexible and adopts multiple conformations or prefers the crystallographically observed conformation where the ring is juxtaposed with the substrate binding face of the tkbd is unclear. we postulate that y mutations might have different oncogenic potentials due to flexibility differences in cbl’s linker region mediated by the lhr-tkbd interaction. we predict that, while a large aromatic residue like phe cannot duplicate the tyr371-tkbd interactions, neither will it perturb those interactions; hence, cbl y371f can adopt and readily maintain conformations observed in the unphosphorylated state. on the other hand, mutations, such as y371s, introduce a polar residue into a hydrophobic pocket and are likely to perturb or occlude the lh-tkbd interaction, thereby unlocking the lhr from the tkbd and leaving cbl in an intermediate activity state where the e2 ~ ub binding site on the ring domain is exposed but lacking the ptyr371-lhr e2 ~ ub stabilizing component. to investigate the influence of mutations and phosphorylation on cbl’s lhr flexibility and ring accessibility in solution, we performed small-angle x-ray scattering analysis and surface plasmon resonance assays on ptyr371-cbl, unphosphorylated cbl and a selection of cbl variants. we then performed focus formation assays to explore the relationship between this flexibility and oncogenic potential.  cbl’s lhr-tkbd interactions affect ring binding affinity for e2 ~ ub previous studies suggest cbl y mutants have the potential to perturb or abrogate lhr-e interactions essential for ligase activity. to investigate this possibility, we used spr to measure binding of the substrate e ubch5b conjugated to ub to variants of the n-terminal fragment of cbl comprising the tkbd, linker region and ring domain, as performed previously. we tested the unphosphorylated n-cbl variants containing the tyr mutations identified in mds/mpn clinical samples as well as n-cbl y371a, which behaves similarly to the mds/mpn tyr mutants, and n-cbl y371f based on its reduced transformation potential in cells and mice. in addition, we tested cbl y371e, which has previously been shown to stimulate cbl activity in vitro. n-cbl y368f was also included in our assays because this mutation is required to generate homogeneously phosphorylated ptyr371-n-cbl; previous studies demonstrate this mutation does not affect the structure or ligase activity in vitro or in cells. we also included n-cbl m222e, which disrupts the closed conformation without perturbing the tkdb-lhr interaction, so that we could investigate changes induced by rotation about ll we predicted that, if the ring domain were unfolded, no binding of ubch5b–ub to n-cbl y variants is expected, whereas, if these variants simply weaken the lhr-tkbd interaction, cbl y mutants will adopt the closed conformation less frequently and thus bind ubch5b–ub more tightly than wild-type n-cbl. none of the n-cbl y mutants is expected to bind ubch5b–ub as tightly as ptyr371-n-cbl because ptyr eliminates the closed, autoinhibited conformation and forms additional interactions with ubch5b ~ ub. all of the n-cbl variants in our spr assay bind ubch5b–ub with similar or higher affinity than wild-type n-cbl but more weakly than ptyr371-n-cbl, suggesting mutations in the lhr do not affect the competency of the ring domain to bind ubch5b–ub. other mutations in the lhr also affect lhr-tkbd interactions as evidenced by the higher binding affinity of n-cbl y368f for ubch5b–ub than wild-type n-cbl.table dissociation constants for interactions between cbl variants and ubch5b–ub k standard errors of the mean are indicated the spr data demonstrate that the chemical characteristics of the side chain of the mutated amino acid in the n-cbl y variants influence binding affinity for ubch5b–ub: the greater potential the mutation has to perturb the tyr binding pocket on the tkbd, the higher the binding affinity for ubch5b–ub. the highest binding affinities are observed when tyr is substituted with an amino acid that has a shorter, non-aromatic and polar or charged side chain like n-cbl y371s, y371e, and y371d; the binding affinities of these mutants are comparable to n-cbl m222e, which disrupts the ring-tkbd interaction in the closed, autoinhibited conformation. in contrast, when tyr is substituted with phe, the ubch5b–ub binding affinity is comparable to wild-type and the lhr-tkbd interaction is unperturbed, as demonstrated by the crystallographic structure of n-cbl y371f. for the remaining mds/mpn mutants, polar and aromatic or short and hydrophobic amino acids are substituted for tyr. these are expected to weaken the interactions within the tyr binding pocket but not as effectively as the group of mutants with short and polar or charged side chains. thus, the ubch5b–ub binding affinity of these mutants is expected to fall between wild-type and the more disruptive mutants and this is indeed what we observe in our spr assays: n-cbl y371h, y371a and y371c. characterization of wild-type and ptyr cbl by saxs initially, we sought to investigate how tyr phosphorylation affects the conformation of cbl in solution by performing saxs analysis on ptyr371-n-cbl and wild-type n-cbl. the monomeric state of these n-cbl variants was confirmed by the saxs-derived overall parameters and gel filtration chromatography. the deduced molecular masses ranged from to 48 kda and are comparable with a predicted monomer mass of ~45 kda for n-cbl.fig. saxs analysis of wild-type and ptyr371-n-cbl. a top: wt experimental scattering data versus theoretical scattering curve from crystal structure before and after normal modes refinement. bottom: ptyr371-n-cbl experimental scattering data versus theoretical scattering from the eom ensemble. b p distribution of wt and ptyr371-n-cbl. c normalized kratky plot of wt and ptyr371-n-cbl coloured as in. d eom distributions of the random pool and the ensembles of wt and ptyr371-n-cbl coloured as in r r d max v porod v dammif mmporod mmdammif rg radius of gyration, d max maximum particle distances, v volume, mm molecular mass the saxs-derived radius of gyration of wild-type n-cbl variant is ~  nm whereas ptyr371-n-cbl is ~  nm, and the calculated maximum particle distances are ~ and ~  nm, respectively. these differences indicate significant conformational changes in n-cbl upon phosphorylation of tyr the presence of an extended tail in the p function of ptyr371-n-cbl suggests rearrangements of the ring domain that make the particle more elongated as compared to the native one. in addition, the normalized kratky plots for wild-type and ptyr371-n-cbl demonstrate significant differences. the wild-type reveals the peak at s rg ≈ √ as expected for globular particles, whereas for ptyr371-n-cbl the peak position is significantly shifted suggesting the presence of structural disorder. the theoretical scattering computed from the native, unphosphorylated crystal structure gives an overall reasonable fit to the experimental data but also displays some systematic deviations. these deviations point to possible differences between solution and crystalline states of the native protein. the crystal structure was therefore refined using an iterative normal mode analysis. this procedure yielded a model which was similar to the original structure and provided a significant improvement of the fit from χ =  to. ab initio shape reconstruction further confirms the globular state of unphosphorylated cbl.fig. comparison of crystal structure of n-cbl and the model derived from iterative normal mode analysis. the unphosphorylated, catalytically competent n-cbl from the crystal structure is coloured cyan and is superposed with the normal mode analysis-refined model. the tkbd is shown in blue, the ring domain in orange and the lhr in yellow as a surface model given the expected flexibility of ptyr371-n-cbl, structural modelling was performed using an ensemble optimization method. here, ensembles of models with variable conformations are selected from a pool of randomly generated models such that the scattering from the ensemble fits the experimental data, and the distributions of the overall parameters in the selected pool are compared to the original pool. as a negative control, eom was first used on the data from n-cbl and the selected ensembles showed predominantly compact conformations. in contrast, the selected ensembles for ptyr371-n-cbl displayed broader distributions of predominantly extended conformations, which were on average more extended than the random pool. these data indicate that ptyr371-n-cbl is extended and flexible in solution, suggesting that phosphorylation of the lh allows the ring domain to adopt multiple conformations in solution and access multiple surfaces of the tkbd.fig. cartoon ensemble representation of ptyr371-n-cbl. the tkbd domain is coloured blue. the predicted ring domain conformations are shown in several colours as indicated in the table. the missing loops connecting the ring and tkbd domains are coloured according to the corresponding ring domains but presented as transparent surfaces characterization of n-cbl mutants by saxs to study how lhr-tkbd interactions influence the flexibility of ll in solution, we performed saxs on a concentration series of our n-cbl tyr mutants, n-cbl y368f and n-cbl m222e. the monomeric state of all n-cbl variants was confirmed by the saxs-derived overall parameters and gel filtration chromatography with the exception of n-cbl y371c, where strong concentration-dependent effects were observed; for this reason, n-cbl y371c was omitted from further saxs analysis. for the remaining n-cbl variants, the deduced mms ranged from to 48 kda pointing to a monomeric state in solution. comparison of the saxs-derived parameters as well as quantification of the flexibility estimated by using eom revealed a link between the conformation of n-cbl in solution and the extent of perturbation of the tyr371-tkbd interaction similar to the trend observed in our spr data. our n-cbl y variants were classified into four categories, depending on the extent of lhr-tkbd perturbation observed based on our spr findings: complete, strongly-perturbing, moderately perturbing, and non-perturbing. ptyr371-n-cbl, which abolishes the lhr-tkbd interaction, appears the most elongated, whereas unphosphorylated, wild-type n-cbl appears more compact. compared to wild-type n-cbl, the strongly perturbing mutants, n-cbl y371s, y371e and y371d, are partially elongated but not as much as ptyr371-n-cbl. conformational rearrangements of these mutants are also confirmed by the corresponding rflex values of approximately 84 %, 90 % and 88 % compared to approximately 60 % for wild-type n-cbl. the moderately and non-perturbing mutants have rg and dmax values more similar to the wild-type n-cbl, although with different degrees of flexibility. notably, n-cbl y371f appears slightly more compact and less flexible than wild-type n-cbl, with an rg of  nm, a dmax of  nm, and an rflex of approximately 56 %. together with spr, these data demonstrate that, in solution, ring domain accessibility is connected to n-cbl conformational rearrangements induced by mutation or modification of tyr in the lhr.table average ensemble outcome r g and d max from eom together with the χ value and the quantification of flexibility for each n-cbl variant r d r fig. eom analysis of n-cbl tyr mutants, n-cbl y368f and n-cbl m222e. mutants were clustered according to their ability to perturb the lhr-tkbd interaction based on saxs parameters shown in table  a strongly perturbing mutants. b moderately perturbing mutants. c weakly or non-perturbing mutants the tyr371-tkbd interaction appears to play a critical role in mediating how elongated n-cbl is in solution. although n-cbl y368f and m222e have higher binding affinities for ubch5b–ub than wild-type, at saxs resolution both have characteristics comparable to wild-type, suggesting they are similarly compact in solution. although n-cbl m222e has a comparable binding affinity to the strongly-perturbing n-cbl y mutants, in solution, it appears more compact, as both rg and dmax are similar to wild-type n-cbl and flexibility is relatively limited. n-cbl y368f has a higher binding affinity for ubch5b–ub than wild-type, but at the level of resolution provided by saxs its characteristics and compactness are similar to those of the wild-type protein, suggesting similar behaviour in solution. transformation potential of cbl y mutants in cells to test the transforming potential of cbl y mutants in cells, we performed focus formation assays using 3t cells stably transfected with n-terminally flag-tagged cbl variants. relative protein expression levels of each variant were assessed by immunoblotting. foci were visualized with sulforhodamine b staining and counted manually with a dna safe imager. afterwards, the sulforhodamine b stain was extracted and the a measured to compare relative cell densities. foci counts and relative cell densities were analyzed by one-way anova followed by dunnett’s test with wild-type cbl as the control. cbl70z was included among the variants tested as a positive transforming control. other variants tested included the same set used in our biochemical assays except in a full-length context.fig. transformation potential of cbl variants in focus formation assays. a immunoblot of lysates from 3t fibroblasts infected with flag-tagged cbl variants using α-flag antibody and α-actin antibody as a loading control. b sulforhodamine b-stained 3t fibroblasts infected with indicated cbl variants. c mean number of foci formed by cbl variant-infected 3t fibroblasts shown in a bar graph. no foci were present in 3t cells infected with wild-type cbl, cbl m222e or cbl y368f. double asterisks denote significant differences between indicated cbl variant and wild-type using anova followed by dunnett’s multiple comparisons test. error bars indicate standard deviation. d as in but for a of extracted sulforhodamine b from cbl-infected 3t fibroblasts foci were observed in cells transfected with cbl y mutants and cbl70z, but not in wild-type, cbl y368f- or cbl m222e-transfected cells. when analyzed by one-way anova followed by dunnett’s test, significant differences in number of foci and cell densities were observed between cells infected with wild-type cbl and all the y mutants except cbl y371f. significantly more foci were present in cbl70z, cbl y371e and the mds/mpn cbl y mutant set and the relative cell density was significantly greater compared to wild-type cbl; however, there is no correlation between mutations that promote moderate or strong lhr-tkbd perturbations and transformation potential. based on the ability to form foci, all the cbl variants except cbl y368f and m222e have the ability to potentiate transformation in cells. based on statistical analyses of the number of foci and relative cell density, cbl70z, cbl y371e and the mds/mpn cbl y mutant set have significantly higher numbers of foci than wild-type cbl. for cbl y371f, neither the number of foci nor the relative cell density are significantly different than in cells infected with wild-type cbl; these results suggest this mutant may potentiate transformation but not to the same extent as the other cbl y mutants or cbl70z. discussion in this work, we employed saxs and spr to characterize how modifications and mutations in cbl affect the conformation and accessibility of the ring domain in solution and subsequently investigate the relationship between these characteristics and the ability of cbl variants to potentiate transformation in cells. our results show that a mutation within the ring-tkbd interface regulates accessibility of the ring domain only, whereas mutation or modification of tyr within the lhr mediates the lhr-tkbd interaction, thereby regulating ring domain accessibility as well as the space sampled by the ring domain relative to the tkbd. the chemical nature of the substitution or modification controls the lhr-tkbd interaction and shifts the equilibrium between the native conformation where the ring is restricted to the face of the tkbd opposing the substrate-binding site and open conformations where the ring domain can access other surfaces of the tkbd and potentially other regions of cbl. none of the tyr mutants can adopt the ptyr conformation that is critical for activation of e2 ~ ub and rtk ubiquitination, but all of the tyr mutants perturb the lhr-tkbd conformation observed in the native state except cbl y371f. likewise, all of the tyr mutants except cbl y371f clearly potentiate transformation in our focus formation assays, whereas cbl y371f forms foci but statistical analyses of the number of foci formed and relative cell densities suggest that this mutant has less transformation potential than the others. previous studies have shown that cbl y371f is not transforming in soft agar colony formation assays nor does it promote tumour formation in nude mice. these data indicate that the flexibility of the lhr features in the cbl tyr mutant transformation mechanism. met is a key residue in maintaining the ring-tkbd interaction in native cbl. a met to glu mutation was used to perturb this interaction and investigate the importance of the ring-tkbd interaction on cbl’s conformation in solution. our previous work and spr studies show that mutation of met in the tkbd to glu increases cbl’s e2–ub binding affinity by approximately 8-fold and enhances cbl’s in vitro autoubiquitination rate. though cbl m222e has a higher binding affinity for ubch5b–ub and is more active than wild-type cbl, saxs analysis demonstrates that cbl m222e has parameters comparable to wild-type cbl, suggesting both adopt a compact conformation. in cells, cbl m222e does not enhance egfr ubiquitination nor does it potentiate transformation in our focus formation assays. the lack of effect of this mutant in these assays suggests structural regulation of cbl’s ligase activity requires more than a simple rotation that exposes the e2-binding site on the ring domain. previous work has shown that phosphorylation of the conserved lhr tyr in the cbl family enhances activity in vitro and is essential for receptor ptk ubiquitination in cells, where cbl’s e ligase activity features prominently in egfr downregulation via lysosomal degradation. the rate and pattern of cbl-mediated egfr ubiquitination determines whether egfr is directed to the lysosome or recycled to the membrane. x-ray crystallography and nmr studies of cbl and cbl-b have shown that this phosphorylation event eliminates ring-mediated autoinhibition, optimally positions ub for transfer and juxtaposes the ring domain with the tkbd substrate-binding site. notably, in the crystal structures of cbl and cbl-b bound to zap peptide and e or e2 ~ ub, the conformations of ring domain relative to the tkbd are identical. here, we show that, in solution, when tyr is phosphorylated, the rg and dmax increase and eom analysis demonstrates the linker region becomes flexible, allowing the ring domain to sample the space surrounding the tkbd rather than maintain the crystallographically observed conformation. this is consistent with other e ligases, where translational and rotational movement of the ring domain are required for ub transfer to select substrate lysine sites as well as polyub chain formation. cbl binds egfr directly through its tkbd and indirectly through growth factor receptor binding 2-mediated interactions in the region c-terminal to the ring domain. cbl-mediated ubiquitination of egfr is observed for both binding sites, highlighting a potential essential role for ring flexibility in cbl substrate ubiquitination. our saxs and spr data show that mutations within the lhr perturb the native lhr-tkbd conformation and that the extent of perturbation is dependent on the chemical nature of the amino acid substitution. in the native state, trp ala val and met form a hydrophobic pocket around the aromatic ring of tyr and the side chain of ser forms a hydrogen bond with the hydroxyl group of tyr the more disruptive the mutation is to the hydrophobic environment, the greater the perturbation in the lhr-tkbd interaction. when tyr is mutated to an amino acid with a charged side chain like asp or glu or a small, polar side chain like ser, the ubch5b–ub binding affinity is approximately 8-fold enhanced compared to native cbl and the rg and dmax increase to values comparable to ptyr371-cbl. additionally, saxs analysis reveals these tyr mutants are the most flexible compared to native cbl. mutation of tyr to smaller hydrophobic amino acids like cys or ala only moderately perturbs the lhr-tkbd interaction as evidenced by an approximately 2-fold enhancement in the ubch5b–ub binding affinity and the moderate increase in rg, dmax and rflex compared to native cbl. likewise, when tyr is mutated to his, only moderate to weak perturbation of the lhr-tkbd interaction occurs. there is an approximately 2-fold enhancement in the ubch5b–ub binding affinity and saxs analysis shows evidence of more flexibility compared to native cbl. when tyr is mutated to phe, the hydrophobic interactions are not disturbed and the native state is maintained: the ubch5b–ub binding affinity, rg, dmax and rflex values are comparable to native cbl. in addition, previous in vitro autoubiquitination assays show native cbl and cbl y371f have comparable catalytic efficiencies. thus, in solution, it appears as though cbl y371f mimics the behaviour of native cbl. mutation of tyr is not the only site at which the lhr-tkbd interaction can be perturbed. our previous work and current data show that mutation of tyr to phe also disrupts the lhr-tkdb interaction. cbl y368f binds ubch5b–ub approximately 2-fold more tightly than native cbl and has an approximately 2-fold enhancement in catalytic efficiency for in vitro autoubiquitination. in addition, this mutant displays a slight increase in all saxs-based parameters compared to native cbl. however, in contrast to the cbl tyr mutants, cbl y368f does not promote focus formation in our assays nor does it compromise egfr ubiquitination in cells and previous work has also shown this mutant does not form colonies in soft agar assays nor promote tumour growth in nude mice. though it is clear that tyr368-tkbd interactions also play a role in the conformations cbl adopts in solution, the significance of this interaction in cbl’s ligase activity in cells is unclear. other studies have shown that cbl Δy is defective in egfr ubiquitination and has the ability to potentiate transformation in cells and in mice. in addition, cbl y368c has also been identified in mds/mpn clinical samples, and, like cbl y371c and y371s, has been shown to potentiate transformation in cells and in mice; however, whether ptk ubiquitination is compromised remains unknown. as observed for tyr the chemical nature of the mutation at tyr might factor into regulation of cbl’s ligase activity as well as transformation potential. while the ability to ubiquitinate ptks is essential for cbl-mediated downregulation of ptk signalling, previous work has clearly shown that it is not the only prerequisite for cbl-dysfunction driven transformation. thien et al. propose that the lhr-tkbd stability of cbl mutants contributes to their ability to potentiate transformation based on the analysis of a number of linker region and ring mutants: cbl Δy Δy and y371a promote transformation but not cbl y368f or y371f. cbl y368f is slightly more flexible than native cbl but retains the ability to ubiquitinate receptor ptks, whereas cbl y371f cannot ubiquitinate receptor ptks but is able to maintain the native conformation in solution. thus, it seems that both characteristics contribute to the transformation potential of cbl mutants. cbl functions as both an ubiquitin ligase and adaptor in receptor ptk-mediated cell signalling. although enzymatic activity is slower in the absence of tyr phosphorylation, cbl is still functional. it may be that when the lhr-tkbd interaction is perturbed, cbl ubiquitinates substrates that bind to regions other than the canonical tkbd-binding site or the more flexible mutants might ubiquitinate “dead end” lysines on substrates such that biologically viable ubiquitination chains are not formed. alternatively, perturbed lhr-tkbd interactions might disrupt oligomerization or interactions with other proteins or lead to the non-sequential recruitment of substrates to exposed surfaces that interfere with controlled signalling.  we have shown that lhr-tkbd interactions regulate cbl’s ligase activity and conformations in solution. mutations or modifications within the lhr prevent cbl from maintaining a closed conformation where the ring domain is restricted to a surface opposite the tkbd-substrate binding site; instead, depending on the nature of the alteration, cbl becomes flexible and the ring domain can access multiple surfaces of the tkbd. transformation only occurs when mutations within the lhr perturb the native state and fail to ubiquitinate ptks. 
previous studies have revealed that select point mutations in tyr can potentiate transformation in cells and mice but not all possible mutations do so. to explore how tyr mutations affect cbl’s properties, we used surface plasmon resonance to measure cbl mutant binding affinities for e conjugated with ubiquitin, small angle x-ray scattering studies to investigate cbl mutant conformation in solution and focus formation assays to assay cbl mutant transformation potential in cells. lhr flexibility, ring domain accessibility and transformation potential are associated with the extent of lhr-substrate-binding domain perturbation affected by the chemical nature of the mutation. ubiquitincblmyeloproliferative neoplasmssaxstransformation potentialhttp://dx.doi.org/ /501100000289cancer research ukhttp://dx.doi.org/ /501100000781european research council647849huang danny t. issue-copyright-statement© the author 2016 more disruptive mutants like cbl y371d or y371s are more extended and the ring domain is more accessible, whereas cbl y371f mimics native cbl in solution. c-cbl’s lhr mutations are only oncogenic when they disrupt the native state and fail to ubiquitinate ptks. here, we investigate how tyr mutations affect cbl’s conformation in solution and how this relates to cbl’s ability to potentiate transformation in cells. correspondingly, the only tyr mutants that potentiate transformation in cells are those that perturb the lhr-substrate-binding domain interaction. to trigger oncogenic potential, cbl tyr mutants must perturb the lhr-substrate-binding domain interaction and eliminate ptk ubiquitination. phosphorylation of a conserved residue on the linker helix region between the substrate-binding and ring domains is required to ubiquitinate ptks, thereby flagging them for degradation.
 casitas b-lineage lymphoma is a ring ubiquitin ligase that negatively regulates protein tyrosine kinase signalling. phosphorylation of a conserved residue on the linker helix region between the substrate-binding and ring domains is required to ubiquitinate ptks, thereby flagging them for degradation. this conserved tyr is a mutational hotspot in myeloproliferative neoplasms. previous studies have revealed that select point mutations in tyr can potentiate transformation in cells and mice but not all possible mutations do so. to trigger oncogenic potential, cbl tyr mutants must perturb the lhr-substrate-binding domain interaction and eliminate ptk ubiquitination. although structures of native and ptyr371-cbl are available, they do not reveal how tyr mutations affect cbl’s conformation. here, we investigate how tyr mutations affect cbl’s conformation in solution and how this relates to cbl’s ability to potentiate transformation in cells.  to explore how tyr mutations affect cbl’s properties, we used surface plasmon resonance to measure cbl mutant binding affinities for e conjugated with ubiquitin, small angle x-ray scattering studies to investigate cbl mutant conformation in solution and focus formation assays to assay cbl mutant transformation potential in cells. cbl tyr mutants enhance e2–ub binding and cause cbl to adopt extended conformations in solution. lhr flexibility, ring domain accessibility and transformation potential are associated with the extent of lhr-substrate-binding domain perturbation affected by the chemical nature of the mutation. more disruptive mutants like cbl y371d or y371s are more extended and the ring domain is more accessible, whereas cbl y371f mimics native cbl in solution. correspondingly, the only tyr mutants that potentiate transformation in cells are those that perturb the lhr-substrate-binding domain interaction.  c-cbl’s lhr mutations are only oncogenic when they disrupt the native state and fail to ubiquitinate ptks. these findings provide new insights into how lhr mutations deregulate c-cbl. electronic supplementary material the online version of this article contains supplementary material, which is available to authorized users. keywords ubiquitincblmyeloproliferative neoplasmssaxstransformation potentialhttp://dx.doi.org/ /501100000289cancer research ukhttp://dx.doi.org/ /501100000781european research council647849huang danny t. issue-copyright-statement© the author 2016 