Biochem. J. (2012) 447, 1–16 (Printed in Great Britain)
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doi:10.1042/BJ20120769
REVIEW ARTICLE
Teaching an old dogma new tricks: twenty years of Shc adaptor signalling
Melanie K. B. WILLS and Nina JONES1
Department of Molecular and Cellular Biology, University of Guelph, Guelph, ON, Canada, N1G 2W1
Shc (Src homology and collagen homology) proteins are
considered prototypical signalling adaptors in mammalian cells.
Consisting of four unique members, ShcA, B, C and D, and
multiple splice isoforms, the family is represented in nearly
every cell type in the body, where it engages in an array
of fundamental processes to transduce environmental stimuli.
Two decades of investigation have begun to illuminate the
mechanisms of the flagship ShcA protein, whereas much remains
to be learned about the newest discovery, ShcD. It is clear,
however, that the distinctive modular architecture of Shc proteins,
their promiscuous phosphotyrosine-based interactions with a
multitude of membrane receptors, involvement in central cascades
including MAPK (mitogen-activated protein kinase) and Akt, and
unconventional contributions to oxidative stress and apoptosis all
require intricate regulation, and underlie diverse physiological
function. From early cardiovascular development and neuronal
differentiation to lifespan determination and tumorigenesis, Shc
adaptors have proven to be more ubiquitous, versatile and dynamic
than their structures alone suggest.
INTRODUCTION
PLCγ (phospholipase Cγ ) [6]. Indeed, the Shc (Src homology
and collagen) protein was one of the first characterized cellular
adaptors, and remains a prototype for this class of molecule [7],
that functions to recruit and juxtapose several sequential members
of a pathway [5]. Broadly, adaptors may serve to amplify or
convert signals, or organize substrates in a cascade, and are defined
by the unique complement of modules and amino acid motifs that
equip them to participate in the transduction process [8].
In the ensuing twenty years since Shc was first identified,
three additional related proteins have been identified in mammals,
prompting the common designations ShcA, B, C and D, according
to their order of discovery. The family is best characterized by its
participation in the major pathways of growth factor signalling,
especially the Ras/MAPK (mitogen-activated protein kinase)
and PI3K (phosphatidylinositol-3 kinase)/Akt cascades proximal
to various stimuli conveyed through RTKs (receptor tyrosine
kinases), integrins and cytosolic kinases, as well as cytokine,
antigen and G-protein-coupled receptors [7]. Additionally,
the 66 kDa isoform of ShcA performs unconventional roles
in the cellular stress response [9]. All of the homologues
share a common architecture defined by an N-terminal PTB
(phosphotyrosine-binding) domain and a C-terminal SH2 domain,
both of which can independently co-ordinate phosphorylated
tyrosine residues of active receptors. The domains are connected
by a central CH (collagen homology) 1 region containing
The cell’s capacity to recognize and respond to diverse stimuli is
contingent upon two fundamental phenomena: inducible protein
phosphorylation and molecular recognition events that enable
transduction components to discern transient changes in residues
and motifs. Indeed, the past three decades have witnessed both
the birth and evolution of the signal transduction discipline,
founded on observations that tyrosine phosphorylation could
mediate oncogenic transformation of cells [1], and propelled by
the subsequent discovery of a conserved pTyr (phosphotyrosine)
recognition region, henceforth termed an SH2 (Src homology
2) domain, that was evident in several cytosolic kinases
and responsible for orchestrating the cellular consequences of
phosphorylation [2,3]. The concept that protein interactions are
mediated by distinct, highly discerning, independently folded
domains has since proven to be a unifying paradigm in cell
signalling regulation [3,4]. Possession of one or more of these
modules confers specific abilities to the parent protein irrespective
of its other functions, thereby endowing the cell with a method of
recycling and recombining successful signalling strategies [5].
Shortly after postulation of the domain theory, a novel protein
came to light that was defined by an SH2 domain and the absence
of any apparent catalytic function, thereby distinguishing it from
the majority of known SH2 proteins of the day, such as Src and
Key words: adaptor protein, antigen receptor, cell regulation,
EGFR (epidermal growth factor receptor), oxidative stress,
phosphotyrosine signalling, phosphotyrosine-binding domain
(PTB domain), receptor tyrosine kinase, signal transduction, Src
homology and collagen homology (Shc), Src homology 2 domain
(SH2 domain).
Abbreviations used: ALK, anaplastic lymphoma kinase; AngII, angiotensin II; AP2, adaptor-related protein 2; BCR, B-cell receptor; CB, cytochrome c binding; CH, collagen homology; CNS, central nervous system; Crk, v-crk sarcoma virus CT10 oncogene homologue; cyt c, cytochrome c ; E11.5, embryonic
day 11.5; ECM, extracellular matrix; EGF, epidermal growth factor; EGFR, EGF receptor; ERK, extracellular-signal-regulated kinase; ES, embryonic stem;
GBM, glioblastoma; GDNF, glial-cell-line-derived neurotrophic factor; Grb2, growth factor receptor-bound protein 2; Gab1, Grb2-associated binding
protein 1; IL-3, interleukin 3; IQGAP1, IQ motif-containing GTPase-activating protein 1; KI, knockin; KO, knockout; LTP, long-term potentiation; Lyn, v-yes-1
Yamaguchi sarcoma viral related oncogene homologue; MAPK, mitogen-activated protein kinase; MEF, mouse embryonic fibroblast; PCD, programmed
cell death; PEST, Pro-Glu-Ser-Thr; PI3K, phosphoinositide 3-kinase; Pin1, peptidylprolyl cis–trans isomerase, NIMA-interacting 1; PKC, protein kinase
C; PLCγ, phospholipase Cγ; PNS, peripheral nervous system; PP2A, protein phosphatase 2A; PTB, phosphotyrosine-binding; PTEN, phosphatase and
tensin homologue deleted on chromosome 10; PTP, protein tyrosine phosphatase; pTyr, phosphotyrosine; PyV MT, polyoma virus middle T; RMS, rostral
migratory stream; ROS, reactive oxygen species; RTK, receptor tyrosine kinase; SFK, Src family kinase; SH2, Src homology 2; SH3, Src homology 3; Shc,
Src homology and collagen homology; SOS, Son of sevenless; SVZ, subventricular zone; TCR, T-cell receptor; Trk, tropomyosin-related kinase; ZAP-70,
ζ-chain (TCR)-associated protein kinase 70 kDa.
1
To whom correspondence should be addressed (email [email protected]).
c The Authors Journal compilation c 2012 Biochemical Society
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M. K. B. Wills and N. Jones
consensus tyrosine residues that are subject to phosphorylation
upon engagement of the adaptor with an RTK, and subsequently
serve as recognition motifs for proximal signalling effectors
[10]. Thus, by relocating to receptors in response to stimuli
and recruiting downstream transduction components, Shc proteins
provide a molecular platform for the development of the signalling
complex. As the past decades have revealed though, the diversity
of cellular processes involving these adaptors places Shc at a
signalling crossroads, where its influence may extend beyond
individual cascades to encompass pathway integration.
DISCOVERY AND CHARACTERIZATION
Identification of the first Shc adaptor arose from efforts to
elucidate novel SH2 domain-containing proteins, in attempt
to better define the mechanisms of tyrosine kinase signalling.
Using a DNA probe designed against the SH2 domain of the
c-fes tyrosine kinase, Pelicci et al. [6] screened human cDNA
libraries for complementary sequences. Their search yielded a
homologue sharing considerable identity with the SH2 domains
of other proteins, most notably the cytosolic kinase Src (60 %).
Further inspection of the gene and its products revealed three
protein isoforms, designated p66, p52 and p46 for their respective
molecular masses in kDa (Figure 1). Structurally, the proteins
are identical through the PTB–CH1–SH2 core, and are solely
distinguished by length of their N-termini. p46 and p52 are
generated from different start codons within the same transcript,
whereas p66 is a product of alternative splicing that endows it with
a unique N-terminal 110-amino-acid extension [11]. Functionally,
Shc proteins gained early notoriety as molecular mediators of
pTyr-based signalling events [12].
Targeted gene perturbation studies in mice have since revealed
striking functional differences between the ShcA isoforms.
Although concurrent disruption of p66, p52 and p46 results in
embryonic lethality by E11.5 (embryonic day 11.5) [13], animals
lacking p66 alone experience a 30 % increase in lifespan, owing
to the transcript’s involvement in cellular oxidative stress and
rendering p66 ShcA one of a small subset of human genes that
has been implicated in the aging process [9].
Although p46 and p52 are broadly expressed across various
tissues and developmental time points, p66 is notably absent
from select cell types, including those of haematopoietic lineage
[6,14]. It has also become apparent that all of the ShcA
isoforms are markedly down-regulated in quiescent cells of the
mature CNS (central nervous system) [15]. Accordingly, several
independent groups identified two novel homologues in the human
and mouse brain that were believed to assume pTyr adaptor
responsibilities in the absence of ShcA [16–18]. Although size
estimate discrepancies exist in the literature, ShcB (Sck/Sli/Shc2)
is commonly reported as a single 68 kDa protein [19–21],
whereas ShcC (Rai/N-Shc/Shc3) is often cited as two isoforms
of 64 kDa and 52 kDa [21–25]. In contrast with the embryoniclethal ShcA-null animals, single and compound ShcB/C-knockout
mice are viable, although subject to reductions in specific neuron
populations [19].
The paradigm of Shc signalling within and beyond the nervous
system stands to be challenged again, by the recent discovery
of another mammalian homologue, ShcD [Shc4/RaLP (Rai-like
protein)]. This adaptor was revealed by two separate groups as
a result of BLAST queries of the mouse and human genomes
[26,27]. Protein and RNA profiling in the mouse has identified
ShcD in an array of embryonic and adult tissues, and has shown
that it is differentially regulated throughout development [28]. Its
expression pattern is distinct from that of the other Shc proteins,
c The Authors Journal compilation c 2012 Biochemical Society
and includes notable representation in the CNS, muscle, epithelia
and bone precursors [28]. Although the contributions of this
protein to organismal development and maintenance have yet to
be determined, incentive now exists to address this gap in adaptor
knowledge.
Shc protein architecture
As discussed above, ShcA was first identified as an SH2-domaincontaining protein, thereby establishing its role in pTyr signalling.
However, the subsequent discovery and characterization of an
additional novel domain capable of co-ordinating phosphorylated
tyrosine residues [29,30] revealed this N-terminal PTB domain to
be the dominant point of contact between ShcA and many of its
ligands [7]. In its unbound state prior to contacting a target ligand,
the PTB domain is structurally disordered; only upon complex
formation does the fully folded structure emerge [31]. Analyses
of peptide-bound domains demonstrate that, despite differences in
gross architecture, the core pTyr-binding pockets of the Shc PTB
and SH2 modules share a related conformation and amino acid
composition, characterized by non-contiguous Arg-Arg-Lys-Ser
residues [32]. Nevertheless, the domains are selective for different
amino acid motifs in their compatible ligands, with SH2 target
specificity defined by residues C-terminal to the pTyr residue, and
having the sequence pTyr--Xaa-Ile/Leu/Met ( representing a
hydrophobic moiety) [33], whereas the PTB domain recognizes
the phosphorylated residue in an N-terminal context given by
Asn-Pro-Xaa-pTyr, with isoleucine or a similar hydrophobic
amino acid at the − 5 position relative to the pTyr [34]. Notable
exceptions to tyrosine specificity of the domain include phosphoindependent interactions with cytoskeletal regulator IQGAP1 (IQ
motif-containing GTPase-activating protein 1) [35], as well as
the phosphatases PTP (protein-tyrosine phosphatase)-PEST (ProGlu-Ser-Thr) [36], PP2A (protein phosphatase 2A) [37] and PTPε
[38].
Intriguingly, the Shc PTB domain also resembles phospholipidbinding PH (pleckstrin homology) domains. Using residues
distinct from the pTyr-binding pocket, it selectively co-ordinates
PtdIns(4,5)P2 and related components of the membrane bilayer,
albeit with a lower affinity than the tyrosine interaction
[32,39]. Although PtdIns(4,5)P2 does not affect the PTB–
pTyr association [40], pTyr engagement with the domain does
decrease its phospholipid affinity [32], suggesting that Shc could
be thermodynamically driven from the membrane to activated
receptors, but not vice versa. Indeed, lipid interaction is considered
instrumental in preemptively targeting ShcA to the membrane and
positioning it in proximity to certain receptors, including IL-3
(interleukin 3) [39]. Of note, some of the residues responsible
for phospholipid recognition are absent from ShcD, despite their
conservation in the other Shc homologues [27]. The consequence
of this deletion on subcellular localization and signalling response of the adaptor have not directly been addressed, although
ShcD is considered a physiologically relevant substrate of several
RTKs [26,27].
In all of the vertebrate Shc proteins, the PTB and SH2 domains
are connected by a CH1 region rich in glycine and proline
residues [11,22], that can contribute to Pro-Xaa-Xaa-Pro motifs
that bind SH3 (Src homology 3) domains of cellular kinases such
as Src, Fyn and Lyn (v-yes-1 Yamaguchi sarcoma viral related
oncogene homologue) [7]. The best-characterized features of the
region, however, are conserved tyrosine residues in positions
239/240 and 317 of p52 ShcA that become phosphorylated
upon engagement of Shc proteins with active kinases, and
represent canonical pTyr-Xaa-Asn-Xaa recognition motifs for
Shc family adaptor protein signalling
Figure 1
3
Shc protein architecture
Mammalian homologues share a common modular structure and conserved CH1 region tyrosine (Y) residues (green). Three novel tyrosine phosphorylation sites are found within the CH1 region
of ShcC and D (peach), whereas a serine (S) residue in the CH2 region of p66 ShcA (yellow) is required for the oxidative stress response. It has likewise been detected in ShcD, although it is not
known whether the residue undergoes phosphorylation. Cysteine residues (C, blue) involved in oligomerization, and a cyt c-binding motif and catalytic core (CB, purple) are also found in the CH2
region of select Shc proteins. The adaptin binding motif (A, teal) is conserved in all of the homologues except for ShcD. The percentage amino acid identity relative to ShcA is given above the PTB
and SH2 domains of ShcB, C and D.
the Ras/MAPK signalling adaptor, Grb2 (growth factor receptorbound protein 2) [10]. Additionally, ShcC and ShcD contain novel
tyrosine phosphorylation sites that can co-ordinate the Crk (v-crk
sarcoma virus CT10 oncogene homologue) and Grb2 adaptors
respectively (Figure 1, peach residues) [26,41]. Near the Cterminal end of the CH1 region lies a binding motif for the adaptin
proteins involved in clathrin-mediated internalization [42]. This
Arg-Asp-Leu-Phe-Asp-Met-Lys-Pro-Phe-Glu sequence [42] is
notably absent from ShcD, yet conserved in ShcA, B and C
(Figure 1, teal residues) [26,27].
Unique to the longer isoforms of the four Shc proteins is an
N-terminal CH2 region, which is likewise enriched in glycine
and proline residues [11]. This attribute is particularly salient in
ShcD, which contains several SH3-domain-recognition sequences
of undetermined utility [26]. The CH2 region has been most
intensively scrutinized in p66 ShcA, as it constitutes the structural
epicentre of the adaptor’s oxidative stress response. As described
below, at least three key sequences contribute to this function: a
Ser36 -Pro37 motif that undergoes phosphorylation [9], Cys59 that
mediates tetramerization [43], and a CB [cyt c (cytochrome c)binding] region that doubles as a catalytic core to generate ROS
(reactive oxygen species) [44]. Although present in p52 ShcA,
the CB region has been deemed non-functional [44]. Intriguingly,
ShcD shares the Cys59 oligomerization residue [43], and may also
possess the p66 Ser-Pro motif, as revealed by sequence alignment
[26,27].
Overall, the CH2–PTB–CH1–SH2 orientation uniquely
distinguishes Shc adaptors, and although PTB and SH2 domains
are individually common in signalling proteins, they are rarely
found within the same molecule [22]. Amino acid sequence
comparisons reveal that ShcA and D share the highest identity
(Figure 1). Across the family, the PTB and SH2 domains are most
conserved, whereas the CH1 and CH2 regions are more divergent
[26]. Striking similarity in exon organization between human
ShcA, B and C argues that they arose from a common ancestral
gene, as does the existence of apparent orthologues in Drosophila
and Fugu rubripes, and putative loci in Caenorhabditis elegans
[22]. Interestingly, the nematode lacks both pTyr consensus sites
of the CH1 region, whereas the fruitfly contains a Tyr239/240
sequence that becomes phosphorylated upon stimulation [22,45].
This motif, and Tyr317 , are found in all of the vertebrate Shc
adaptors, thus implying that expanded pTyr signalling capacity is
associated with increased organismal complexity [22].
Regulation
Fundamentally, the ability of Shc proteins to engage in controlled
signal propagation in a given cell is contingent on their expression,
localization, target recognition, and acquisition and removal of
phosphate moieties, all of which must proceed in an orchestrated
manner. Although efforts have focused on elucidating the details
of ShcA dynamics, ShcB, C and D remain relatively unexplored
from a regulatory perspective.
As previously noted, the isoforms of ShcA are differentially
expressed [6]. Analysis of the locus has revealed two promoters of
the TATA-/Inr + class that independently regulate transcription
of p46/p52 and p66, the latter of which can be epigenetically
repressed through cytosine methylation and histone deacetylation
[14]. Consistent with the role of this isoform in mediating ROSresponsive apoptosis, patients suffering ESRD (end-stage renal
disease) experience considerably reduced CpG methylation in the
p66 promoter [46].
c The Authors Journal compilation c 2012 Biochemical Society
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M. K. B. Wills and N. Jones
Homologue- and isoform-specific differences also extend to
subcellular localization of the proteins. In neurons, for example,
ShcB is distributed throughout the soma and processes, whereas
ShcC is limited to the cell body [47]. Elsewhere, ShcA is
predominantly a cytoplasmic protein that can concentrate in
cellular niches. An N-terminal targeting sequence in p46 that
is masked in the longer isoforms uniquely allows this adaptor
to accumulate in the mitochondrial matrix [48]. Conversely, a
fraction of p66 can relocate to the mitochondrial intermembrane
space in response to appropriate apoptotic stimuli [44,49,50].
Some estimates suggest that roughly 5 % of p52 ShcA is already
membrane-associated in unstimulated cells [39]; however, it
is generally observed in the cytosol and rough endoplasmic
reticulum, from which it translocates to the periphery upon growth
factor stimulation [51,52].
Subsequently, rapid phosphorylation of Shc proteins upon
binding active RTKs is a vital step in the transduction process
that primes the adaptor to interact with downstream partners.
Recent evidence suggests, however, that the implications of
Shc post-translational modification extend beyond generating
recognition motifs for effectors. George et al. [40] report
that although the isolated SH2 domain of p52 ShcA binds
representative target ligands in solution, it loses this ability
in the context of the full-length Shc protein. Strikingly,
phosphorylation of tyrosine residues in the CH1 region not
only restores SH2-mediated binding, but also enhances the
affinity of the interaction 50-fold, implying that the Shc protein
employs a phospho-dependent switch that inducibly controls
SH2 activation. Conversely, molecular dynamics simulations
and in silico binding free energy estimates predict that
the conformational change accompanying phosphorylation of
Tyr317 decreases the affinity of Shc for representative ligands.
This single modification was found to enhance structural
rigidity of the adaptor and decouple the concerted movement
of the PTB and SH2 domains [53,54], thereby disrupting
the interaction of both domains with their cognate motifs on the
EGF (epidermal growth factor) RTK [54]. Considering that Shc
engagement with a stimulated receptor potentiates the signal and
also protects the RTK from phosphatases, this structural shift
is postulated to control detachment of the adaptor following
its activation, thereby facilitating eventual termination of the
transduction process [54]. However, the model does not account
for additional phosphorylation of Tyr239/240 , which usually occurs
concomitantly with Tyr317 . Whether this further alters molecular
conformation remains to be seen.
Under these circumstances, biochemical and molecular
simulation data appear to predict contradictory consequences
of CH1 phosphorylation, although this may be the result
of differences in experimental approaches. The biochemical
perspective focused on the influence of phosphorylation in
modifying the binding behaviour of the SH2 domain towards
receptor motifs with which it failed to interact in its full-length
inactive form. Unlike the in silico analysis, it did not consider the
consequences of Shc phosphorylation on interactions for which
the SH2 domain is a physiologically relevant principal point of
contact. Thus it is possible that the models describe independent
concurrent phenomena, or that the regulatory mechanisms are
distinct and exclusive to certain subsets of receptor–adaptor
interactions. In the former scenario, Shc phosphorylation by
its partner RTK could conceivably destabilize the primary
PTB-mediated interaction, while simultaneously priming a
secondary association between the SH2 domain and another
ligand. Indeed the utility of the double domain structure of
Shc and its contributions to signalling cross-talk are presently
undercharacterized. Beyond their direct implications for Shc
c The Authors Journal compilation c 2012 Biochemical Society
signalling, these discoveries also challenge the assumption that
independently folded domains act as fully autonomous modules
within signalling proteins. Instead, they paint a more dynamic
picture of contextual adaptability and regulation, hinting at subtle
mechanisms by which transducers could integrate information.
The findings likewise emphasize the requirement to regulate
Shc phosphorylation, and indeed studies have revealed a small
repertoire of phosphatase enzymes capable of this task. The
first to be identified was the ubiquitous PTP-PEST, which
interacts constitutively with the PTB domains of p52 and p66
ShcA via an unconventional Asn-Pro-Leu-His recognition motif
in its C-terminus [36,55]. The association is further enhanced
by treatment with a DAG (diacylglycerol) second messenger
analogue, which induces PKC (protein kinase C)-dependent
serine phosphorylation of ShcA p52 at residue 29 (Ser138 in p66)
and thereby provides an additional binding sequence for PTPPEST [55,56]. Importantly, this association has been found to
reduce Shc tyrosine phosphorylation and Shc-mediated MAPK
activation following insulin signalling [56], and during antigen
receptor-stimulated lymphocyte activation [57].
Tumour suppressor PTEN (phosphatase and tensin homologue
deleted at chromosome 10), PP2A and PTPε are likewise
capable of dephosphorylating Shc, impeding its interaction with
Grb2 and suppressing subsequent MAPK activation [37,38,58].
Notably a role has been established for PTEN in negatively
regulating Shc-mediated cell migration [59,60], thus implicating
the phosphatase–adaptor interaction in the metastatic mechanism.
Like PTP-PEST, PP2A and PTPε stably associate with the ShcA
PTB domain under basal conditions; however, both phosphatases
demonstrate distinct context-dependent regulation of ShcA. In
the case of PP2A, growth factor stimulation and consequent
phosphorylation of Shc Tyr317 inhibit the phosphatase and cause
it to dissociate from the adaptor [37]. Within 30 min of signal
initiation, the interaction is restored.
Conversely, the capacities of the three ShcA isoforms
to act as PTPε substrates depend on the kinase used initially to
phosphorylate the adaptor, and whether it confers a protective
effect due to complex formation. Proximal to the cytosolic kinase
Src, Shc is subject to dephosphorylation by PTPε, whereas it is
resistant to the phosphatase when downstream of RTKs including
Neu and EGFR (EGF receptor) [38]. This is postulated to reflect
the difference in affinities between the kinases and adaptor, and
competitive PTB domain engagement by protective RTKs that
probably precludes phosphatase binding.
Although the finer details of Shc regulation are still subject
to investigation, it is evident that numerous mechanisms
integrate these adaptors into cellular networks that require
inducible, reversible and highly specific interactions to maintain
homoeostasis.
MOLECULAR MECHANISMS
The canonical role of Shc adaptors is to mediate signal
transduction events at the membrane, by providing a molecular
scaffold for the recruitment and assembly of signalling
components. The details of these exchanges continue to be
revealed, at increasingly fine levels of resolution. Additionally, the
adaptors are now appreciated to engage in a variety of homologueand isoform-specific activities.
RTK signalling
Growth factors comprise a spectrum of secreted compounds that
mediate growth, survival and differentiation of responsive cells
by selectively activating compatible transmembrane RTKs [61].
Shc family adaptor protein signalling
Although numerous RTKs exist, EGFR is considered a prototype
for this class of transducer and ShcA, its archetypal adaptor
in mediating Ras/MAPK activation (Figure 2, A). Accordingly,
we will discuss the EGFR–Shc interaction as a model of RTK
signalling, with the caveat that only the general phenomenon can
be extrapolated to other receptors. Nevertheless, the relevance of
this system transcends vertebrate signalling, as the EGFR and
Shc homologues DER (Drosophila relative of EGFR) and dShc
(Drosophila Shc) are found to associate in Drosophila [45].
Transduction begins when a ligand binds to the extracellular
region of the RTK, stimulating receptor dimerization and
autophosphorylation of conserved tyrosine residues on its
cytoplasmic tail that represent recognition motifs for downstream
targets. Uniquely, three such residues in EGFR seem to coordinate ShcA. Numbered according to classic nomenclature,
pTyr992 is a minor SH2-domain-binding site, whereas pTyr1148
dominantly engages the PTB domain and pTyr1173 is considered
a secondary motif that facilitates interaction with both the PTB
and SH2 domains of ShcA [62–64]. Although the sites initially
appeared redundant [65], subsequent analyses have found a
more robust signalling output when the PTB domain is involved
[64], and raise the possibility that optimal MAPK activation
may be achieved through co-operation of both domains [66].
Paradoxically, a dissenting line of evidence contends that Shc
is responsive to EGF stimulation even after compound ablation
of all of the aforementioned sites [67,68]; however, we offer an
alternative explanation below.
Recent quantitative proteomic analyses of early signalling
events have revealed that phosphorylation of EGFR Tyr1148
and Tyr1173 occurs within 5 s of ligand engagement, followed
immediately by recruitment and phosphorylation of Shc [69].
Accordingly, EGF stimulation appears to have two distinct effects
on the intrinsic kinase of the RTK; in addition to increasing
the general catalytic activity of the enzyme, EGF also alters
substrate specificity and promotes preferential phosphorylation
of Shc Tyr317 relative to EGFR autophosphorylation sites [70].
Considering that the activation of another proximal EGFR partner,
PLCγ , lags behind that of Shc [69], the possibility thus exists
that a temporal component partly defines the consequences of
transduction. Evidence further suggests that ShcA can mobilize
directly from the cytoplasm to the EGFR without prior association
with membrane phospholipids, although this is not true of all
receptors. Interaction between ShcA and IL-3, for example, can
be abrogated by mutating the three key residues in the PTB domain
that constitute a phospholipid-binding pocket [39].
p66, p52 and p46 ShcA are all subject to phosphorylation
[52] on two consensus binding motifs for the Grb2 adaptor,
corresponding to Tyr239/240 and Tyr317 in the CH1 region of human
p52 [10], as detected both in vitro [6,52] and in vivo in the
mouse and rat [15,71]. Tyr239/240 [10] and Tyr317 [72] have each
been proposed as the dominant Grb2-binding site that recruits
the adaptor into the developing transduction complex. According
to the latter scenario, Tyr317 mediates MAPK activation, whereas
Tyr239/240 stimulates c-myc expression by unknown mechanisms
[72]. Notably though, the EGFR variant used in these experiments
lacked the Shc-binding sites, so the outcome may not represent
canonical EGFR signalling. Regardless of the motif(s) of origin,
activation of the MAPK cascade, as detailed in Figure 2 (A), is
the best-characterized outcome of signalling through p52 and p46
ShcA [7,11].
In contrast with these isoforms, however, p66 fails to augment
MAPK phosphorylation upon ligand stimulation, despite binding
both EGFR and Grb2 (Figure 2, C) [11,52,73,74]. The response
is particularly perplexing considering that p66 shares identical
PTB, CH1 and SH2 structures with the shorter p52/46 isoforms.
5
Nevertheless, a model has emerged whereby the adaptor is
recruited to active EGFR, phosphorylated first on tyrosine
residues by the RTK, and then on serine/threonine residues of
the CH2 region in a MEK [MAPK/ERK (extracellular-signalregulated kinase) kinase]-dependent manner [73]. Although
the pTyr motifs bind and recruit Grb2, serine/threonine
phosphorylation destabilizes the p66–EGFR interaction, thereby
decoupling the fledgling signalling complex from Ras, and
sequestering Grb2–SOS (Son of sevenless) away from the
membrane and the other ShcA isoforms, which are not subject
to phospho-serine/threonine regulation [73] (Figure 2). The
consequences of this behaviour include accelerated inactivation of
ERK [73] and suppressed transcription from the c-fos promoter,
which is specifically attributed to the CH2 region [11].
Despite the focus on co-ordinating Grb2, phospho-independent
interactions have also surfaced that diversify the downstream
consequences of Shc signalling. Proximal to the EGF family
receptors, ShcA and the scaffold IQGAP1 have been identified in
membrane ruffles, where they associate via the Shc PTB domain
[35]. As ShcA can have a role in lamellipodia formation, the
interaction is postulated to connect RTK activation to cytoskeletal
reorganization through Shc [35].
A major component of the EGFR response, beyond signalling,
involves internalizing and sorting the receptor, destining it for
recycling or degradation (Figure 2, E). Within 5 [75] to 20 [52]
min of stimulation, activated EGFR and ShcA are found to colocalize in intracellular vesicles identified as early endosomes by
their immunoreactivity against EEA1 (early endosome antigen 1)
[76]. Specific tracking of phosphorylated Shc-recognition motifs
on EGFR suggest that the receptor retains its signalling capacity
through the initial internalization process [76]. Conversely, only
a limited subset of LAMP-1 (lysosomal-associated membrane
protein 1)-positive late endosomes or lysosomes contain Tyr1173 phosphorylated EGFR, and ShcA is accordingly absent from
these compartments [76]. In addition to accompanying EGFR
through part of its endocytic journey, ShcA has structural
and functional precedence to participate in the internalization
process. As discussed above, a motif in the CH1 region binds
the α- and β-adaptin components of the AP2 (adaptor-related
protein 2) complex [42], which is involved in clathrin-coated pit
formation leading to endocytosis [75]. The interaction between
p52 ShcA and α-adaptin is markedly induced by EGF stimulation,
thereby enhancing the amount of AP2 juxtaposed with EGFR.
Moreover, ligand internalization is perturbed by disabling the
adaptin-binding motif of ShcA [75]. Although the AP2 complex
can interact directly with the EGFR in a phospho-independent
manner, it appears that Shc inducibly enhances recruitment of
its components in response to signalling, therefore helping to
facilitate the internalization process upon receptor activation [75].
By comparison, little is known about the associations between
EGFR and ShcB, C and D, beyond their potential to interact
[18,26,77]. Inaugural studies reported that ShcB and ShcC recruit
Grb2 to the activated receptor [78], and that the isolated ShcC
SH2 domain can act as a dominant negative to repress EGFR
signalling through ERK and Elk [77]. For ShcC, however, the
phenomenon has been inconsistent and hard to recapitulate. Fulllength endogenous ShcC is not phosphorylated following EGF
stimulation of a neuroblastoma cell line, neither does it affect
MAPK activation when overexpressed [21]. Indeed, this adaptor
appears to interact with a more limited repertoire of RTKs, and its
reduced affinity for Grb2 renders it a poor transducer of the SOS–
Ras cascade [21,24]. In contrast, ShcD has been found to augment
the Ras/MAPK response proximal to receptors including EGFR
[27], possibly as a result of its additional unique Grb2-recognition
motif [26].
c The Authors Journal compilation c 2012 Biochemical Society
6
Figure 2
M. K. B. Wills and N. Jones
Molecular mechanisms of Shc proteins
A: ShcA (p52) is best characterized as an adaptor protein proximal to RTKs such as EGFR. Extracellular ligand engagement causes the receptor to dimerize and autophosphorylate multiple tyrosine
residues in its cytoplasmic region, which serve as recognition motifs for the Shc PTB and/or SH2 domains. The intrinsic EGFR kinase phosphorylates tyrosine residues of the Shc CH1 region, to
which the Grb2 adaptor can bind and initiate the Ras/MAPK cascade. B: Shc can also recruit the cytosolic kinase Src to the RTK; conversely, active Src can phosphorylate Shc. C: like p52, p66
also interacts with EGFR and Grb2; however, it exerts an inhibitory effect on the MAPK pathway. D: non-catalytic receptors including integrins use indirect methods to recruit Shc. Upon binding to
the ECM, select β subunits are subject to phosphorylation by Src family kinases to generate an Shc-binding site. Alternatively, the transmembrane domain of certain α subunits interacts with the
membrane-spanning adaptor caveolin-1. This recruits and activates the Src family kinase Fyn, which phosphorylates Shc and promotes its interaction with Grb2. Regardless of the origin of stimuli,
the canonical MAPK pathway proceeds with the relocalization of Grb2 and its constitutive binding partner, the guanine-nucleotide-exchange factor SOS, to the developing transduction complex in
proximity to the farnesylated membrane-associated Ras GTPase. SOS facilitates the exchange of GDP for GTP to activate Ras, which in turn activates the first of the cytosolic kinases in the cascade
that terminates with the MAPK ERK. The pathway can promote diverse cellular responses including migration, invasion, proliferation, differentiation and survival. E: through its adaptin-binding motif,
Shc also has a role in facilitating receptor endocytosis that occurs concurrently with signaling. F: in contrast, ShcC is predominantly involved in survival signaling through PI3K/Akt, proximal to the
GDNF receptor Ret. As with EGFR, ligand stimulation causes Shc to relocate to the phosphorylated receptor. Both ShcC, and its constitutive binding partner, Gab1, become phosphorylated upon
binding the RTK, prompting an interaction between Gab1 and the p85 regulatory subunit of PI3K. The catalytic p110 subunit of PI3K can then phosphorylate membrane phospholipids that recruit
the Akt kinase, which is activated by PDK (phosphoinositide-dependent kinase) 1 and PDK2. Akt itself then phosphorylates several downstream targets that suppress apoptosis. G: at focal adhesion
contacts with the ECM, p66 ShcA senses and responds to mechanical tension, initiating detachment-mediated apoptosis (anoikis) in the absence of sufficient cytoskeletal integrity. H: upon oxidative
challenge, p66 ShcA is phosphorylated at Ser36 by PKC, and targeted to the mitochondrion via Pin1-mediated isomerization. In the intermembrane space (IM), p66 oxidizes cyt c of the electron
transport chain, and reduces O2 to H2 O2 to increase mitochondrial permeability and promote apoptosis. M, matrix.
Overall, the ShcA cascade is thought to be a ‘redundant
but dominant’ path to MAPK activation from the EGFR [79].
Redundancy arises from the principal role of ShcA in recruiting
Grb2, which can also bind directly to EGFR [62], and dominance
is owed to the cell’s predicted preference for this longer path over
the more streamlined one [79]. Insights gleaned from MEFs
(mouse embryonic fibroblasts) harbouring a genetic deletion
of all of the ShcA isoforms suggest that although the EGFR–
MAPK pathway is functional in the absence of ShcA, the
adaptor is required to sensitize cells to low concentrations of
growth factor [13]. In a different experimental system, the
reduced proliferative potential of senescing hepatocytes has
been attributed to diminished ERK activation, despite consistent
levels of EGFR and Shc proteins and sustained phosphorylation
capacity across time. Instead, the change in ERK responsiveness is
postulated as a consequence of age-induced destabilization of the
EGFR–Shc complex [80]. Thus it appears that ShcA plays a key
role in fine-tuning the MAPK signalling output from the EGFR.
A second major cellular hub involving Shc adaptors,
particularly ShcA and ShcC, is the PI3K/Akt pathway [21,24,81],
c The Authors Journal compilation c 2012 Biochemical Society
although it currently lacks the level of characterization afforded to
Shc–MAPK. In particular, the consequences of ShcC proximal
to the GDNF (glial-cell-line-derived neurotrophic factor) RTK
Ret have received attention. In neuronal and thyroid carcinoma
cells, ShcC facilitates pro-survival signalling by complexing with
the adaptor Gab1 (Grb2-associated binding protein 1), which can
recruit the p85 subunit of PI3K to the receptor upon stimulation,
thereby activating this cascade as portrayed in Figure 2 (F)
[21,24]. By contrast, ShcC appears to have little effect on PI3K
signalling in neural progenitor cells [82], whereas it represses
Akt phosphorylation in lymphocytes [83], thereby emphasizing
the highly contextual nature of Shc-mediated transduction.
Cytosolic kinase signalling
From the infancy of Shc characterization, the role of cytosolic
enzymes in regulating these adaptors has been sought. The SFKs
(Src family kinases) constitute a collection of proto-oncogenic
non-RTKs implicated in a variety of cellular phenomena [84].
Shc family adaptor protein signalling
The founding member of the family, v-Src, was initially
identified as the transforming factor in Rous sarcoma virus,
and later revealed to have a cellular counterpart, c-Src, that
could likewise contribute to malignancy when deregulated [84].
SFKs are also important proximal components in RTK-mediated
signalling events [85]. In cells transformed by v-Src, all of
the isoforms of ShcA are found to be highly phosphorylated
[86]. Interestingly v-Src phosphorylates ShcA at both Tyr239/240
and Tyr317 , whereas c-Src targets only Tyr239/240 and depends on
engagement of the Shc PTB domain with phosphatidylinositols
[87–89]. Activation of these sites by SFKs likely primes Shc to
engage downstream effectors in an analogous fashion to receptormediated signalling (Figure 2, B). Additionally, SFK regulation
of Shc proximal to RTK stimulation may constitute a parallel
pathway of Shc involvement in growth factor transduction.
To that end, the aforementioned studies of receptor–adaptor
dynamics that demonstrate ligand-induced ShcA phosphorylation
in the absence of Shc-binding sites in the EGFR cytoplasmic
tail have been perplexing [67,68]. However, the deletions and
targeted mutations employed in these investigations failed to
account for EGFR Tyr974 and Tyr845 , sites of Src binding and
phosphorylation respectively [90,91]. Furthermore, the EGFR–
Src interaction is ligand dependent and known to enhance Shc
phosphorylation [91]. We therefore propose that EGF-mediated
Shc signalling that occurs independently of a direct interaction
between EGFR and the adaptor is in fact orchestrated by an SFK,
which could localize to the receptor and subsequently activate
ShcA.
Conversely, ShcA overexpression has been found to enhance the
ligand-dependent association between EGFR and c-Src, as well as
up-regulate Src enzyme activity [52]. Both p66 and p52 directly
bind and activate c-Src even in the absence of RTKs, via a noncanonical Shc interface [92]. The consequence of the association
appears to involve STAT (signal transducer and activator of
transcription) phosphorylation and nuclear translocation, as well
as induction of proteins including p21, WAF1 and Cip1 [92].
In addition to reciprocal regulatory involvement with SFKs,
mounting evidence suggests that Shc is also subject to serine
phosphorylation by PKC. Treatment of cells with a potent PKC
activator fails to induce tyrosine phosphorylation of Shc, and
instead produces a unique pattern of serine phosphorylation that
can be prevented by pretreatment with a PKC inhibitor, and is
not recapitulated by administering growth factors such as FGF-1
(fibroblast growth factor 1) [56,93]. Intriguingly, this stimulation
induces a pTyr-independent association between Shc and Grb2
that is disrupted by the serine/threonine-specific PP1 (protein
phosphatase 1) [93]. However, the utility of this interaction is
unclear, as subsequent analyses in Shc-null MEFs revealed that
PKC-mediated activation of Ras/ERK proceeds independently of
Shc [56]. Intriguingly though, one of the novel phosphorylation
sites corresponds to Ser29 in p52, which is involved in phosphatase
regulation of ShcA, as described above [56].
Signalling through non-catalytic receptors
Shc adaptors are also involved in transducing signals downstream
of transmembrane proteins that lack intrinsic tyrosine kinase
activity, such as integrins and antigen receptors of T- and
B-lymphocytes. Often the signals converge on the common
Ras/MAPK pathway already discussed, although mounting
evidence also highlights the existence of novel Shc functions.
Regardless of their origin, non-catalytic receptors require
accessory kinases to initiate pTyr-mediated recruitment of signalling components. This is exemplified by the TCR (T-cell receptor),
7
a multimeric complex involved in recognition of processed
antigen. Upon engagement of the epitope-binding dimer, the
associated CD3 signal transduction subunits are phosphorylated
by the SFK Lck, on conserved ITAMs (immune-receptor tyrosinebased activation motifs). This is followed by recruitment of a
second kinase, ZAP-70 [ζ -chain (TCR)-associated protein kinase
70 kDa] [94]. As reviewed by Finetti et al. [95], the SH2 domain
of p52 ShcA binds phospho-CD3, whereas ZAP-70 engages the
PTB domain and can also phosphorylate the adaptor, in addition
to Lck. In the case of p52, this can activate Ras/MAPK and
Rac pathways, potentially influencing T-cell activation, survival
and cytoskeletal dynamics. Similar mechanisms exist to couple
p52 to the BCR (B-cell receptor). Conversely, both p66 ShcA
and ShcC are considered negative regulators of TCR and BCR
signalling, reducing the receptors’ sensitivity to antigen and
increasing levels of apoptosis [83,95]. Accordingly, loss of these
antagonists triggers autoimmune phenotypes characterized by
hyperactive cells and self-reactive antibodies [95].
In non-haematopoietic cells proteinaceous ECM (extracellular
matrix) represents both a substratum for attachment as well as
a source of information and stimuli for the cell. Accordingly,
non-catalytic heterodimeric integrin receptors provide vital
mechanical and chemical links between the ECM of the
surroundings and the cytoskeletal and signalling networks of
the cell interior, thereby influencing processes such as migration,
proliferation and apoptosis [96]. A subset of integrin receptors are
capable of transducing signals through the ShcA adaptor protein,
including those for laminin (α6β4) [97,98], collagen (α2β1) [99],
collagen/laminin (α1β1), fibronectin (α5β1) [100] as well as
the promiscuous receptor that can engage vitronectin (αvβ3)
[99,100]. Several mechanisms have been proposed to connect
Shc adaptors to active integrins and transduce ‘outside-in’ signals
(Figure 2, D). In one strategy, intracellular kinases phosphorylate
integrins directly to generate consensus Shc-recognition motifs.
This is the case with the laminin-binding α6β4 integrin, which
sustains phosphorylation on its β4 subunit upon ligation and
subsequently recruits Shc, via its PTB and SH2 domains, to
signal through the MAPK pathway [97,98]. β3 Integrin is
also inducibly phosphorylated and becomes a direct target for
Shc [101].
However, not all β subunits are subject to phosphorylation. In
an indirect binding model, Shc recruitment occurs independently
of the integrin cytoplasmic tail and any tyrosine residue motifs
located therein. Instead, ligand engagement by select integrins
stimulates the transmembrane region of the α subunit to
interact with the membrane-spanning adaptor caveolin-1, which
constitutively binds the SFK Fyn [100,102]. Alternatively, Yes
(v-yes-1 Yamaguchi sarcoma viral oncogene homologue), Lck,
or Lyn may substitute for Fyn [103]. Activation of the kinase
and its subsequent SH3-domain-mediated association with ShcA
facilitate Shc phosphorylation of Tyr317 , recruitment of Grb2
and initiation of the Ras/MAPK cascade that can lead to cell
proliferation [100,102].
Owing to their promiscuity, Shc proteins can integrate simultaneous stimuli from multiple sources, such as growth factors
and ECM, to tailor the cellular response. In one experimental
system, ShcA was found to promote integrin-mediated haptotaxis
in the absence of growth factors, by a PTB-domain-dependent
mechanism. Upon the addition of EGF, migration proceeded
independently of Shc, whereas proliferation required the Shc
SH2 domain [99]. Postnatal angiogenesis also appears to require
the integrative powers of ShcA to assimilate signals from the
VEGF (vascular endothelial growth factor) and integrin receptors,
invoking a cell survival response upon engagement with a
compatible ECM substrate and growth factor [104].
c The Authors Journal compilation c 2012 Biochemical Society
8
M. K. B. Wills and N. Jones
Recent developments also implicate p66 ShcA in anoikis, the
apoptotic programme initiated when adherent cells detach from
the matrix (Figure 2, G). Intriguingly though, this function is
distinct from the role of p66 in oxidative-stress-induced apoptosis,
and instead relies on recruitment of the adaptor to focal adhesions,
the integrin-based mechanosensory structures that anchor the cell
to the ECM [105]. Here, p66 is speculated to infer the attachment
status of the cell based upon tension in cytoskeletal stress fibres
radiating from the foci. A loss of mechanical integrity triggers p66
to activate the GTPase RhoA, which initiates anoikis. Although
the precise mechanism remains unclear, it appears to require the
intact p66 PTB domain, and involve release of Ras-mediated
repression of RhoA (Figure 2, G) [105,106]. In attached cells,
or those lacking p66, the pro-apoptotic behaviours of RhoA are
inhibited.
have reduced fertility and intolerance to cold and starvation, owing
to deficits in adipogenesis and glucose homoeostasis [113].
The collective p66 body of work briefly highlighted in the
present review underscores the complexity of cellular regulatory
systems and the diverse and dynamic functionality of ShcA.
PHYSIOLOGICAL CONTRIBUTIONS
The functional importance and non-redundancy of Shc proteins
are best revealed by their diverse and unique contributions
to animal development and maintenance. From embryonic
lethality to prolonged lifespan, the consequences of Shc genetic
perturbations are vast and far-reaching. In the past decade,
emphasis has shifted from establishing the gross phenotypic
changes in the mouse that arise from reverse-genetic analysis
of Shc, to dissecting the specific intracellular requirements of
signalling events that underlie physiology.
Oxidative stress and apoptosis
Arguably the most divergent of the Shc protein functions is
its role in oxidative stress and PCD (programmed cell death).
Although the topic has been comprehensively reviewed [107–
112], the oxidoreductase activity of p66 ShcA remains a pivotal
juxtaposition to canonical Shc signalling and an intensive area of
investigation. The first indication of the adaptor’s unprecedented
function arose from a landmark study that selectively removed
the p66 isoform of ShcA in mice, leaving p52 and p46 intact
[9]. Surprisingly, the genetic ablation extended rodent lifespan
by 30 %, and conferred resistance to oxidative stress at both
the organismal and cellular level. Ser36 in the CH2 region was
identified as a critical component of the p66-mediated apoptotic
response to oxidative challenge, as phosphorylation of this residue
is required to trigger cell death upon exposure to UV or H2 O2 [9].
Models have since emerged that portray p66 ShcA as both a
sensor and a proponent of ROS, and position it in an intricate
network of cell fate decisions (Figure 2, H). It appears that the
initial oxidative stimulus can be conveyed to ShcA via PKCβ,
which phosphorylates Ser36 of p66 [49]. Phospho-Ser36 -Pro37 then
becomes a consensus recognition site for the prolyl isomerase
Pin1 (peptidylprolyl cis–trans isomerase, NIMA-interacting 1),
which induces cis–trans isomerization around this bond and
targets p66 for mitochondrial uptake [49]. A further level of
regulation may be achieved by context-dependent oligomerization
of p66. The isolated CH1 region seems to exist as a noncovalent dimer that is incapable of eliciting apoptosis. However,
under oxidizing conditions when redox scavengers are saturated,
the dimer tetramerizes via disulfide bridging of the conserved
Cys59 , and primes the protein to initiate PCD [43]. Strikingly,
the translocated activated p66 is situated in the mitochondrial
intermembrane space, from which it can interact with cyt c of
the electron transport chain and engage in redox exchanges that
provoke apoptosis [44]. Contained within the CB motif of the
CH1 region are three critical residues, Glu132 , Glu133 and Trp134 ,
that constitute the redox centre of p66. This core facilitates the
binding and oxidation of cyt c, and serves to transfer electrons to
molecular oxygen to generate H2 O2 . In turn, this ROS increases
inner mitochondrial membrane permeability causing the organelle
to swell, rupture and release pro-apoptotic factors into the cytosol
[44].
Not surprisingly, p66 has been implicated in a number of
disorders with metabolic and inflammatory characteristics that
can be prevented by selective ablation of the protein in murine
models. As revealed recently, however, p66 deletion is only
advantageous under select, controlled circumstances. In a natural
wild environment, p66-null mice fail to thrive and are found to
c The Authors Journal compilation c 2012 Biochemical Society
Cardiovascular system
Considering the broad expression of ShcA in the adult, the
discovery that it contributes most potently to heart and circulatory
system morphogenesis at the early stages of mouse development
came as a surprise. Global disruption of all three ShcA isoforms in
a murine model causes defects in heart structure and function and
reduces vascular complexity, thereby suggesting problems with
angiogenic remodelling. ShcA KO (knockout) embryos succumb
to gross cardiovascular dysfunction by E11.5 [13].
Analyses of cultured fibroblasts recovered from mutant
embryos have revealed a distinct role for ShcA in facilitating
ERK2 activation following stimulation with low concentrations
of growth factor. In the absence of the adaptor, larger
stimuli are required to elicit a comparable MAPK response.
Additionally, the ShcA-null MEFs demonstrate reduced ERK
phosphorylation and altered cytoskeletal organization proximal
to integrin engagement with ECM. Accordingly, ShcA-deficient
embryos experience considerable and specific reduction in active
MAPK, corresponding with the areas involved in cardiovascular
development that normally express ShcA [13].
Intriguingly, a similar phenotype arises from selective
incapacitation of the ShcA PTB domain alone. Genetic KI
(knockin) embryos expressing ShcA with a non-functional PTB
domain die at approximately E11.5 and exhibit cardiac deficits
reminiscent of full ShcA KO [114]. This is not true of KI
embryos with mutations to the SH2 domain or all of the CH1
region pTyr motifs (Tyr3Phe mutation) that abrogate interactions
with up- or down-stream targets respectively. Instead these
animals can survive to birth, but demonstrate impaired motor coordination due to abnormal development of muscle spindles and
the monosynaptic stretch reflex [114]. Thus it appears that midgestational heart morphogenesis requires ShcA with a functional
PTB domain, but occurs independently of the CH1 region tyrosine
residues [114], which have long been considered the primary
effector regions of the adaptor.
Further support for this hypothesis has come from studies of the
adaptor in postnatal heart function. Cardiomyocyte-specific ShcA
KO produces dilated cardiomyopathy similar to cardiomyocyterestricted expression of the same PTB mutation described above
[115]. Notably, although conditional removal of the CH1 region
pTyr motifs in cardiomyocytes yields phenotypically normal
offspring, these animals rapidly succumb to experimentally
induced heart failure, thus implying that the Grb2 consensus sites
contribute little to basal cardiac function, but are required to elicit
an appropriate response to biomechanical stress [115].
Shc family adaptor protein signalling
Indeed the involvement of CH1 phosphotyrosine residues in
cardiovascular homoeostasis appears to be highly contextual.
In culture, cardiac fibroblasts exposed to thrombin demonstrate
increased p46/52 phosphorylation on Tyr239/240 and Tyr317 ,
concomitant with enhanced Erk activation, whereas the same
treatment of cardiomyocytes fails to augment ShcA tyrosine
phosphorylation [116].
The cardiac implications of ShcA further depend on the isoform
in question. Expression of p66 has been shown to decrease
during postnatal maturation of myocytes, and is re-induced upon
exposure to hypertrophic agonists; the adaptor also undergoes
different patterns of phosphorylation relative to p46/52 [116].
Consistent with its role in oxidative stress and apoptosis, p66 has
a deleterious role in the heart following chronic exposure to Ang
II (angiotensin II), an instigating factor in cardiac senescence.
Selective deletion of the p66 isoform confers resistance to Ang
II-mediated hypertrophy and apoptosis of cardiomyocytes and
endothelial cells in mice [117].
Overall, compelling evidence suggests that various tissues
of the heart differentially utilize unique combinations of
the signalling modalities of distinct ShcA isoforms, under the
evolving demands of morphogenesis, maintenance, aging
and stress, thereby underscoring the complex and dynamic
contributions of this adaptor to cardiac physiology.
Nervous system
Genetic models and in vitro analyses have likewise fostered
a vision of the dynamic interplay between ShcA, B and C
proteins over the course of nervous system development and into
adulthood. Contrary to its generalized expression elsewhere in
the body, ShcA is highly restricted and tightly regulated in the
brain. During gestation and beyond, its expression is confined to
areas of active neuroblast proliferation, whereas post-mitotic cells
show marked reduction of the protein coincident with cellular
differentiation [15]. Accordingly, it has been reported that the
initial process of neural induction from ES (embryonic stem) cells
is characterized by up-regulation of p66 ShcA [118]. In support of
this observation, experimentally induced overexpression of p66 in
cultured ES cells exposed to neuralizing conditions accelerates
the loss of pluripotency and acquisition of neural markers, such
as nestin, as occurs very early in the commitment process [118].
The contributions of ShcA proteins to neurogenesis from this point
forward have been analysed by tissue-specific deletion or mutation
in mice. McFarland et al. [119] reported that animals experienced
considerable microencephaly when ShcA was excised under
control of the nestin promoter, thereby implicating ShcA in
survival mechanisms of this precursor population. Strikingly,
a similar phenotype results from conditional expression of
a phosphorylation-defective dominant negative adaptor (Shc
Tyr3Phe) that lacks the central CH1 pTyr residues that serve as
Grb2 consensus sequences. Its detrimental consequences have
been attributed to neural progenitor apoptosis. Although the brain
masses of nestin-induced Shc Tyr3Phe mice were reduced to 50 %
of control values for the lifetime of the animals, the period of
enhanced apoptosis that gave rise to microencephaly was limited
to a narrow developmental window [119].
In contrast to the CH1-region-independent contributions of
ShcA to cardiovascular signalling [114,115], conserved pTyr
motifs appear to be critical mediators of nervous system
development [119]. However, differences in the molecular
strategies and genetic backgrounds of experimental animals used
in these studies preclude direct comparison of the severity of the
Tyr3Phe mutation in the heart and nervous system. Additional off-
9
target effects of the dominant–negative protein under a non-native
promoter also cannot be ruled out.
Although ShcA expression is initially high and progressively
decreases with developmental stage [15], ShcC is absent early in
embryogenesis and is strongly up-regulated prior to birth [19], at
which point it is detected in quiescent neurons [120]. Expression
continues to escalate postnatally for 6–8 weeks in mice [19].
Indeed ShcA and ShcC display a pattern of reciprocal expression
in pre- and post-mitotic neuronal cells that has been postulated as
a molecular commitment switch, wherein ShcA down-regulation
and ShcC induction demarcate the transition between cellular
proliferation and differentiation [120]. This orchestration is
consistent with the findings that ShcA is required to maintain
neuronal precursors [119], whereas ShcC prevents apoptosis
and promotes neuronal maturation, as gauged by morphological
changes when the adaptor is overexpressed in primary neuronal
cultures [120]. The survival influence of ShcC depends on its
SH2 domain, and involves sustained phosphorylation of Akt and
inhibition of the pro-apoptotic protein Bad (BCL2-associated
agonist of cell death), both of which are consequences of the PI3K
cascade. Meanwhile, the adaptor’s contribution to differentiation
is linked to prolonged Erk1/2 activation [120]. Both PI3K
pathway augmentation and delayed persistent MAPK signalling
are facilitated by ShcC proximal to the GDNF receptor Ret
[21]. Although a consensus has yet to be reached regarding the
biochemical changes that drive and accompany the shift in Shc
expression, these findings suggest that the molecular basis of the
transition may lie in the divergent signalling properties of the two
adaptors.
However, despite the appeal of the ShcA/ShcC developmental
switch model, initial data from the genetic ShcC KO mouse
did not appear to support a fundamental role for this adaptor
in establishing and maintaining post-mitotic neurons. Curiously,
ShcC KO animals are viable and display no overt morphological
abnormalities [19]. The possibility that ShcB, which likewise
demonstrates a modest increase in CNS expression near the end
of gestation, performs compensatory functions in the absence of
ShcC was also tested. Double KO mice carrying global compound
ShcB and ShcC ablation are viable and neuroanatomically
normal in the CNS, though they exhibit reduced numbers of
sympathetic neurons in the superior cervical ganglia that cannot
be recapitulated by singular ablation of ShcB or ShcC [19].
Loss occurs late in development, consistent with the hypothesis
that ShcB/C support post-mitotic neurons. Both the ShcB and
ShcB/ShcC KOs also lose discrete populations of sensory neurons
from the dorsal root ganglia, although the animals retain sensory
function. Evidently, the KO phenotypes are less severe than
anticipated under a model that implicates ShcC as a critical
factor in post-mitotic neuron survival. Sakai et al. [19] surmized
that ShcB and C are not exclusive mediators of RTK signalling
in neurons, but instead participate in only a subset of cellular
responses. To that end, ShcD has recently been detected in postmitotic embryonic neurons and it associates with several neuronal
RTKs including TrkB (Tropomyosin-related kinase B) and MuSK
(muscle, skeletal, receptor tyrosine kinase) [26,121], although its
role in this context has yet to be investigated [28]. It remains to
be seen whether this uncharacterized protein will shed more light
on the role of the Shc adaptor switch in neurogenesis.
Comprehensive Shc protein expression profiles have similarly
been generated for the adult CNS and PNS (peripheral
nervous system), complementing burgeoning functional studies
to establish the roles of these adaptors after development. At
maturity ShcA is present in the PNS [78], but is restricted to the
olfactory epithelium [15] and SVZ (subventricular zone) of the
brain [47], both of which are involved in adult neurogenesis.
c The Authors Journal compilation c 2012 Biochemical Society
10
M. K. B. Wills and N. Jones
Accordingly, mice generated using McFarland’s strategy to
conditionally remove ShcA from nestin-expressing cells [119]
exhibit smaller more disorganized SVZ niches postnatally
[122]. The size reduction has been attributed to decreased cell
proliferation, whereas tissue architecture is altered on structural
and physiological levels housing many cells of indeterminate
origin [122]. This phenotype mirrors the role of ShcA in
development, bolstering the hypothesis that the adaptor is central
to the maintenance and self-renewal properties of neural stem and
precursor cells.
ShcB and ShcC have been widely detected in neurons of
the adult CNS, where they display distinct profiles within the
substructures of the brain and spinal cord. Both proteins appear to
be excluded from glia [47,120] with the exception of the human
gastrointestinal tract, where ShcC is evident in enteric glial cells,
but absent from the surrounding neurons [25].
Consistent with their opposing roles in nervous system
development, ShcA and ShcC are contextually regulated in motor
neurons. Although ShcC predominates under homoeostasis,
injury shifts the balance in favour of ShcA and suggests a related
role for this protein in regeneration [123].
Intriguingly, further characterization of the ShcC KO mice at
maturity has revealed more subtle phenotypes that emphasize the
utility of this adaptor. ShcC KO animals are more susceptible
to ischaemic damage and reperfusion injury following strokelike cerebral artery occlusion, presenting larger infarct volumes,
increased cellular apoptosis via the intrinsic mitochondrial
pathway, more dramatic cognitive impairment and elevated
incidence of mortality [23]. Indeed cortical neurons isolated from
ShcC-null animals show an increased sensitivity to hypoxic and
oxidative stressors, coincident with decreased PI3K activity
and Akt phosphorylation, prompting the authors to surmize that
ShcC normally exerts neuroprotective effects in the wake of stress
[23].
Moreover, a novel function in hippocampus-dependent learning
and memory has been ascribed to the adaptor. ShcC-null animals
demonstrate superior spatial and nonspatial learning as a result
of enhanced hippocampal LTP (long-term potentiation) [124].
Increased synaptic plasticity in the absence of ShcC coincides
with elevated tyrosine phosphorylation of the postsynaptic
NMDA (N-methyl-D-aspartate) receptor, which represents a
physiological binding partner of ShcC and a key mediator of
LTP [124].
Despite earlier reports to the contrary, ShcC has also recently
been identified in neurogenic regions of the adult rodent brain,
including the SVZ and RMS (rostral migratory stream). Evidence
from the ShcC KO mice implicates this adaptor in facilitating
migration of neural progenitors from the SVZ along the RMS to
their final destination in the olfactory bulb, through its influence
on metalloprotease expression and Wnt/β-catenin signalling [82].
DISEASE: CANCERS AND BEYOND
It remains to be seen whether the cardiovascular and neurological
deficits apparent in Shc KO models are paralleled in naturally
occurring human conditions. Yet, with such broad involvement
in fundamental signalling pathways, metabolism, morphogenesis
and physiology, Shc proteins are increasingly implicated in a
spectrum of disorders that arise from cellular disregulation.
Although ectopic or aberrant stimulation can provoke undesired
cell proliferation or invasion, withdrawal of other tissues from
adequate trophic support can lead to degenerative conditions. In
the case of Huntington’s disease, a novel link has recently been
reported between p46/52 ShcA and the TrkB RTK. In vitro, the
c The Authors Journal compilation c 2012 Biochemical Society
expanded mutant huntingtin protein reduces ShcA expression,
effectively repressing Ras/ERK activation proximal to TrkB
and its ligand, BDNF (brain-derived neurotrophic factor), and
thereby rendering cells more sensitive to oxidative damage [125].
Conversely, patients with Alzheimer’s disease show enhanced
glial expression of ShcA and elevated ERK activation [126]. It has
been shown that amyloid precursor protein, a neuropathological
disease precursor, is cleaved to produce a membrane-bound Cterminal fragment that can become tyrosine-phosphorylated and
interact with Shc and Grb2, thereby implicating the components
in potentially detrimental signalling cascades [126,127].
Cancer investigations, however, have generated the most
extensive insights into Shc protein contributions to pathology.
From the time of its discovery, ShcA has been considered a
proto-oncogene in the light of its capacity to transform cells [6],
whereas ShcD was first characterized as a mediator of melanoma
metastasis [27]. Collectively, members of the Shc family have
been associated prognostically and/or mechanistically with
a variety of cancers including prostate, breast, thyroid and
neuroblastoma, discussed below, in addition to malignancies of
the brain [82], kidney [60] and colon [128].
Hormone-sensitive cancers
The steroid hormones of the body influence development of
neoplasms in certain tissues, including the prostate and breast.
Although ShcA has been found to engage in various tumorigenic
mechanisms, the prognosis depends largely on the tissue and Shc
isoform in question, as they can have opposite effects in the two
types of cancer [129].
The study of prostate cancer has focused on p66 ShcA
as a driver of proliferation. This isoform in particular is upregulated in primary prostate tumours and is induced upon
steroid hormone treatment of cultured cells [130]. Experimental
p66 overexpression or knockdown can respectively increase or
decrease prostate cancer cell proliferation [131], although the
findings were initially hard to reconcile due to the protein’s role
in oxidative apoptosis and lack of involvement in mitogenic
signalling. An alternative explanation proposes that steroid
hormones act to increase p66 expression and translocation into
mitochondria, where both the redox centre of the protein and
the resulting ROS it generates upon interaction with cyt c are
required to promote proliferation [129,132,133]. Although the
participation of ROS in mitogenic signalling is well documented
[134], prostate studies provide the first evidence implicating the
oxidoreductase activity of p66 Shc in an oncogenic phenotype
[132]. However, the circumstances under which p66 ShcA
stimulates cell death compared with proliferation are not yet
clear, and its contributions to cellular dynamics are evidently
more context-dependent than previously assumed.
Indeed breast cancer presents an opposing scenario in which
p66 expression implies a favourable patient outcome, particularly
in the context of the ratio of Tyr317 -phosphorylated ShcA to the
p66 isoform (pTyrShc/p66Shc) [135]. Evidently, elevated levels
of phosphorylated ShcA relative to the total amount of the p66
isoform are indicative of aggressive neoplasms and increased risk
of relapse. Clinical predictions arising from the pTyrShc/p66Shc
ratio are consistent with established prognostic indicators [135],
and can likewise forecast disease recurrence following select
pharmaceutical treatments [136].
To address the molecular contributions of Shc proteins to the
events of mammary tumorigenesis, several transgenic mouse
lines have been generated. In mammary epithelial cells, the
PyV MT (polyoma virus middle T) oncogene rapidly induces
Shc family adaptor protein signalling
metastatic tumours that develop in a manner reminiscent
of human breast cancer progression [137]. Mutation of the
residue responsible for cellular transformation and Shc binding
(Tyr250Phe PyV MT) has been found to reduce the oncogenic
severity of the phenotype compared with that of PyV MT
animals, thus implying that the Shc-recognition motif is important
for promoting tumour development [138]. An analogous study
was performed to investigate the tumorigenic contributions of
the Shc-binding site on the EGFR homologue Neu (ErbB-2;
HER-2), a negative prognostic indicator that is up-regulated
in 20–30 % of human breast cancers [139]. In the absence of
five key autophosphorylation sites, the RTK loses its ability
to transform cells. However, reconstituting the individual Shc
site (Tyr1227 ) restores transformation, enhances signalling through
MAPK and PI3K pathways, and promotes rapid development
of multifocal tumours [139,140]. Although intriguing, the
results of these transgenic studies should be interpreted with
caution, as recognition sequence mutations likely affect more
than one candidate binding partner. Indeed, all of the Shc family
PTB domains recognize Asn-Pro-Xaa-pTyr motifs and could
hypothetically co-ordinate with the PyV MT and Neu targets to
mediate a response, thereby obfuscating the contributions of the
individual adaptors.
The most compelling evidence of ShcA participation in breast
tumorigenesis comes from mouse studies in which this locus
was exclusively disrupted. In support of aforementioned findings,
mammary-tissue-specific ablation of ShcA prevented Neumediated tumour development [141]. Moreover, the contribution
of the three ShcA CH1 phosphorylation sites to PyV MT-mediated
oncogenesis was assessed using the ShcA Tyr3Phe KI strategy
described above. Expression of this signalling-compromised
ShcA substantially delayed tumour onset, reduced metastasis and
revealed site-specific phenotypic proclivities. Although mutation
of the equivalent Tyr317 site resulted in elevated tumour cell
apoptosis, attenuated angiogenic signalling was observed in
Tyr239/240Phe tumours, suggesting non-redundant roles for these
ShcA sites in breast cancer progression [141].
Cancers of the nervous and neuroendocrine origin
Aberrant signalling through the GDNF receptor Ret is common
to medullary and papillary thyroid carcinomas. It has been
demonstrated that Tyr1062 co-ordinates both the PTB and
SH2 domains of ShcA, and is important in Ret-mediated
transformation [142]. Accordingly, tyrosine-to-phenylalanine
amino acid mutations of Ret Tyr1062 and Shc Tyr317 independently
attenuate Ret-induced focus formation in vitro. ShcA Tyr317Phe
was specifically found to reduce ERK1/2 activation, thereby
suggesting the importance of the Shc adaptor and this CH1
tyrosine residue in facilitating the transformed phenotype [142].
Another hallmark of Ret-induced oncogenesis is cellular dedifferentiation, or the reduced expression of thyroid-specific
proteins. This is likewise thought to occur through the Shc–
Ras–MAPK pathway initiated at Ret Tyr1062 , since removal
of this site prevents down-regulation of thyroid markers
normally observed in the presence of the oncogenic receptor
[143]. As noted previously, however, mutations rendered
to the receptor do not provide definitive evidence of Shc
involvement.
Notably, ShcC is also a physiological substrate of Ret
that interacts through its PTB domain with Tyr1062 of the
receptor [21]. Its association with the Ret oncoprotein activates
the PI3K/Akt pathway, and promotes survival in thyroid
carcinoma cell lines when overexpressed [24]. The discovery
that ShcC is markedly up-regulated in human papillary thyroid
11
cancers further suggests the relevance of this interaction [24],
whereas observations that ShcA and C co-ordinate the same
Ret receptor residue yet potentiate different signals raise
the question of how their dynamics are maintained during
tumorigenesis.
Perhaps nowhere is this question of Shc competition better
illustrated than in the study of neuroblastoma, the most common
and fatal childhood tumour that develops from sympathetic
nervous system precursor cells and often presents paraspinally
in the abdomen or chest [144]. Intriguingly, it is capable of
spontaneous regression in patients under 1 year of age and
accumulating evidence implicates Shc proteins in cellular fate
decisions. In addition to classical prognostic markers, ShcC
overexpression has been found to predict poor clinical outcome
[145,146]. mRNA profiling of ShcA, B and C in patient tumours
revealed ubiquitous ShcA expression across all samples, although
the levels were more robust in lower-grade tumours, whereas
ShcB was minimal and mostly limited to earlier-stage cancers.
In contrast, ShcC expression was low in early stages of the
disease, and markedly elevated in advanced tumours, rendering it
a strong indicator of neuroblastoma progression [145]. This trend
in ShcA/ShcC gene expression was independently confirmed by
examining the respective proteins [146].
From the perspective of oncogenic mechanism, cultured
neuroblastoma cell lines have been used to investigate the
molecular determinants of the disease. Phosphorylated ShcC
is evident in a number of lines, particularly those in which
it associates with the constitutively active ALK (anaplastic
lymphoma kinase) and becomes a substrate for this enzyme [147].
Notably ShcA has also been detected in most of the neuroblastoma
cell lines tested, although its phosphorylation pattern is reciprocal
to that of ShcC [147]. Analysis of neuroblastoma patient tissue
likewise revealed that more advanced tumours were associated
with elevated ShcC phosphorylation [147]. The molecular
relevance of ShcC post-translational modification was further
investigated in cell lines expressing high levels of ALK, by
introducing a ShcC mutant devoid of conserved CH1 region
tyrosine residues. This was found to reduce activation of ERK1/2
and Akt, while impairing cellular motility, differentiation and
survival [148]. Most compelling, however, are the consequences
of abrogating Shc expression entirely. Although RNAi (RNA
interference)-mediated knockdown of ShcA impedes cellular
growth rate, ShcC attenuation reduces in vivo tumorigenicity
and promotes the appearance of neurite extensions and markers
of cellular differentiation [146]. Strikingly, neuronal maturation
observed in the absence of ShcC has been attributed to ShcA
through its sustained activation of ERK1/2. ShcA phosphorylation
is accordingly reduced in the presence of ShcC and restored
in its absence [146]. This supports a hypothesis that the Shc
adaptors have competing roles in neuroblastoma, and implies that
ShcC may participate in aggressive phenotypes by maintaining
cells in an undifferentiated state. The molecular processes by
which it accomplishes this and impedes ShcA have not yet been
elucidated. Paradoxically, this model directly opposes the putative
mechanism of neuronal maturation during development, in which
ShcC up-regulation is regarded as the switch from proliferation
to differentiation.
It is nevertheless consistent with recent findings that ShcC
is active in neurogenic regions of the adult brain where it
promotes cell migration. Accordingly, ShcC is also believed to
endow cancer stem cells of the high-grade CNS tumour, GBM
(glioblastoma), with their infiltrative capacity [82]. The adaptor is
consistently overrepresented in the stem/progenitor subpopulation
of GBM cells, regardless of its relative level in the tumour as a
whole. ShcC silencing reduces the invasive properties and overall
c The Authors Journal compilation c 2012 Biochemical Society
12
M. K. B. Wills and N. Jones
tumorigenicity of GBM-initiating cells. Intriguingly, however, the
migratory influence of ShcC in GBM does not involve the Wnt/βcatenin pathway as it does in normal neuroblasts. Instead, the
targets of ShcC appear more variable in the context of GBM
[82]. Collectively, these discoveries emphasize the importance of
temporal and environmental factors in defining the outcome of
Shc signalling. They likewise illustrate that cellular diversity in
heterogeneous tumours can be a crucial determinant of disease
progression.
CONCLUSIONS AND FUTURE PERSPECTIVES
In the 20 years since the first Shc protein was discovered,
great strides have been made in determining the physiological
roles, binding partners, molecular mechanisms, regulatory
processes and oncogenic contributions of this adaptor family.
Shc proteins are now appreciated to form vital links between
the interstitial environment and the informatic, architectural and
kinesthetic components of the cell, to integrate competing stimuli,
and, in the specific case of p66 ShcA, to respond to stress and
harness the redox potential of the mitochondrion to elicit
apoptosis. These advances have likewise expanded the conceptual
perspective of adaptor proteins in general. Although the recent
discovery of ShcD offers more opportunity for this type of basic
characterization, it appears overall that the reductionist chapter of
the signalling discipline may be drawing to a close while larger
more intractable questions remain to be addressed.
Competition or co-operation
One residual issue is the regulation of Shc protein dynamics when
the adaptors are co-expressed. Owing to the considerable identity
observed across the SH2 and PTB domains of this protein family,
distinct members can bind the same ligands using equivalent
domains that recognize the same target motifs [149]. Yet there are
well-established functional non-redundancies between different
Shc adaptors and even between isoforms of the same designate.
As noted by Nakamura et al. [41], ShcA and ShcC have equal
affinity for the TrkA receptor, although the signalling output from
each is distinct. These proteins co-express during development
and in neuroblastoma tumours; however, they mediate entirely
contradictory outcomes in the two scenarios. In a similar vein,
the three ShcA isoforms bind Grb2 upon EGF stimulation, but
only p66 fails to increase MAPK activation [11]. Considering the
unique opposing roles of the Shc proteins raises the question of
how their interplay is determined in the cell and whether their
relative levels are important for fine-tuning signals.
Shc promiscuity
Given that Shc adaptors complex with a multitude of membrane
receptors [7], they appear to represent a first point of signal
convergence in the cell, wherein diverse stimuli activate unique
receptors, which in turn recruit the common Shc protein.
Obermeier et al. [150] postulated that the differences in receptor–
transducer affinities could help to retain the molecular identity of
distinct cascades. They found, however, that Shc exhibited similar
binding characteristics with the Trk and EGF RTKs, despite their
opposing roles in differentiation and proliferation respectively. It
is not clear how the signal identity is retained in this context,
although the concern is not unique to Shc signalling. Indeed,
the ability of the ubiquitous MAPK pathway itself to transmit
diverse, yet precise, signals has long baffled cell biologists. As
noted by Brown and Sacks [151], the mechanism by which
c The Authors Journal compilation c 2012 Biochemical Society
a particular stimulus provokes a targeted response through the
MAPK pathway remains elusive, although contributing factors
are beginning to surface.
Molecular design and function
Shc proteins are unique among adaptors in possessing both a PTB
and an SH2 domain for pTyr recognition [22]. The significance
of this architecture is somewhat mysterious, although it suggests
the potential for signal integration or bifurcation. Consistent with
this prediction, ShcA-mediated migration and proliferation are
observed to be mutually exclusive, active under different circumstances and require differential engagement of the domains [99].
Likewise the phenotypes exhibited by mice with ShcA mutant
alleles depend on which region of the molecule is interrupted,
as well as the nature of the tissue itself [114]. Although these
findings support the functional relevance of the PTB and SH2
domains, they do not provide evidence for their concomitant use.
Moreover, isoforms of ShcA, B, C and D differ in the extent
of their N-terminal CH2 regions, but, with the exception of p66
ShcA, the importance of this sequence is unknown.
These observations inspire an even more elemental question
in the discipline: that of the overall relevance of Shc proteins to
signalling cascades. Other adaptors, such as Nck and Crk, possess
SH2 and SH3 domains that essentially convert a pTyr signal into
a proline interaction [152], thereby engaging effectors that do
not intrinsically contain phospho-specific domains. In contrast,
Shc responds to, and perpetuates, pTyr signalling by binding a
phospho motif of an activated receptor and recruiting Grb2 to its
own CH1 pTyr residues. Luzi et al. [22] questioned the necessity
of the Shc adaptor function, noting that Grb2 can localize directly
to active RTKs without assistance. Although adaptor proteins can
reinforce signals by expanding the repertoire of binding sites for
downstream components [8], this remains a contentious issue
for Shc as its two Grb2-recognition motifs appear to mediate
distinct physiological outcomes [72,141], thereby precluding a
simple additive model of amplification.
Indeed, Lai and Pawson’s [13] discovery that ShcA sensitizes
cells to low growth factor levels is among the most salient
arguments that Shc adaptors constitute signal modifying
components, rather than fundamental transduction architecture.
As mentioned above, the MAPK specificity conundrum is
concerned with the potentiation of unique signals through
common cascades. The duration of ERK activation has proven
important in determining outcome, as illustrated by the fate
decisions occurring in PC12 phaeochromocytoma cells [151].
Despite relying on the common MAPK pathway, EGF and nerve
growth factor responses in these cells are vastly different, inducing
proliferation and differentiation respectively. This effect has been
attributed to temporal regulation of the MAPK cascade, wherein
proliferation is a consequence of transient pathway activation,
whereas differentiation is achieved only upon sustained ERK
signalling [151]. These findings raise the intriguing possibility
that Shc proteins may in fact exist as signal amplitude or frequency
modulators to help direct the nature of the cell response.
The emerging paradigm in the signalling discipline appears to
shift emphasis from the details of molecular specificity governing
interactions, to the ‘informatic’ specificity by which the meaning
behind each signal is conserved, integrated and interpreted in
the complex milieu of the cell. The challenge for future Shc
investigations will be completing and compiling the simple
characterizations of these proteins, and applying this knowledge
to explore the dynamic contributions of this adaptor family to cell
homoeostasis and disease.
Shc family adaptor protein signalling
FUNDING
This work was supported by the Natural Sciences and Engineering Research Council of
Canada (NSERC) [grant number RG327372 (to N.J.)]. N.J. is also the recipient of an
NSERC University Faculty Award and a Tier II Canada Research Chair. M.K.B.W. holds
a Canadian Institutes of Health Research Vanier Canada Graduate Scholarship and the
University of Guelph Brock Doctoral Scholarship.
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Received 9 May 2012/5 July 2012; accepted 6 July 2012
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