InteractiveFly: GeneBrief

SHC-adaptor protein: Biological Overview | References

Receptor tyrosine kinases (RTKs) transduce signals via cytoplasmic adaptor proteins to downstream signaling components. Loss-of-function mutations in have been identified in the Drosophila homolog of the mammalian adaptor protein SHC. A point mutation in the phosphotyrosine binding (PTB) domain of the Drosophila SHC-adaptor protein (Shc) completely abolishes Shc function and provides in vivo evidence for the function of PTB domains. Unlike other adaptor proteins, Drosophila Shc is involved in signaling by only a subset of RTKs: shc mutants show defects in Torso and DER but not Sevenless signaling, as confirmed by epistasis experiments. By double-mutant analysis, the adaptors DOS, DRK, and Shc act in parallel to transduce the Torso signal. These results suggest that Shc confers specificity to receptor signaling (Luschnig, 2000).

During the development of multicellular organisms, receptor tyrosine kinases (RTKs) play an important role in transducing a variety of extracellular signals that trigger cellular responses, such as proliferation, differentiation, or cell survival. RTKs activate a cytoplasmic signaling cascade, the RAS-MAPK pathway, which has been highly conserved from invertebrates to mammals (Luschnig, 2000).

Three Drosophila RTKs have been studied extensively. Torso (TOR) is required for the specification of terminal cell fate at the embryonic poles. Sevenless (SEV) induces R7 photoreceptor cell fate in the developing eye. In contrast to TOR and SEV, each of which being required only for one specific process, the Drosophila EGF receptor (DER) has multiple functions during development. Most of the known components of the RAS-MAPK pathway (CSW, DOS, DRK, KSR, SOS, GAP1, RAS1, DRAF, DMEK, DMAPK) are required for signaling by all three receptors. It is an intriguing problem how different RTKs, which activate one apparently common cytoplasmic signaling cascade, can trigger specific biological responses (Luschnig, 2000).

Upon ligand binding, RTK monomers phosphorylate each other on specific tyrosine residues, thereby creating docking sites for cytoplasmic adaptor proteins with phosphotyrosine (pY) binding modules, such as SH2 (SRC homology 2) or phospho tyrosine binding (PTB). One such adaptor protein, DRK (GRB2 in mammals, SEM-5 in C. elegans) acts as a link between activated RTKs and the Son of Sevenless (SOS) protein, which in turn activates RAS by catalyzing GDP-GTP exchange. The identification of more factors involved in signaling between RTKs and RAS1 has led to the view of a complex signaling network rather than a simple linear cascade. Such factors include Daughter of Sevenless (DOS), which potentially acts as a docking site for several SH2 and SH3 domain proteins, and Corkscrew (CSW), a protein tyrosine phosphatase containing two SH2 domains (Luschnig, 2000).

The maternally derived TOR pathway in the Drosophila embryo is a particularly useful system to study RTK signaling because it marks the first time point in development when the RAS-MAPK pathway becomes essential. The TOR receptor is activated by a spatially restricted ligand at the embryonic poles and regulates, via the RAS-MAPK pathway, zygotic expression of the terminal gap genes, tailless (tll) and huckebein (hkb). Posterior expression of tll and hkb is exclusively regulated by TOR signaling and can serve as a quantitative measure for the strength of signal, while anterior expression depends on additional input by the morphogen Bicoid (Luschnig, 2000).

The loss-of-function phenotypes of signaling components acting between the TOR receptor and DRAF are weaker than those of mutations in TOR or DRAF themselves, and it was shown that DRAF can be activated in the absence of RAS1. This indicates that downstream of the receptor the signal splits into branches and that a RAS1-independent signal can activate DRAF. An analysis of tyrosine phosphorylation sites in the activated TOR receptor revealed that individual sites have distinct positive or negative regulatory effects on signaling, by acting as targets for specific molecules (Luschnig, 2000).

Another adaptor protein, SHC, was suggested by studies in mammalian cells to act in RTK signaling. SHC contains two pY-binding modules, an N-terminal PTB domain, and a C-terminal SH2 domain, separated by a region with similarity to α-1 collagen (Pelicci, 1992; reviewed in Bonfini, 1996 and Cattaneo, 1998). Upon EGF stimulation, SHC binds to EGFR and becomes phosphorylated on a specific tyrosine, which creates a binding site for the SH2 domain of GRB2. Mammalian SHC can act as an adaptor to link RTKs via GRB2 to RAS activation (Rozakis-Adcock, 1992; Luschnig, 2000 and refences therein).

A Drosophila SHC homolog, SHC, has been isolated and characterized biochemically (Lai, 1995; Li, 1996). SHC associates with and becomes tyrosine phosphorylated by activated DER in vivo, and the PTB domain of SHC binds specifically to a NPXpY motif in DER. While the PTB and SH2 domains of SHC show a high degree of conservation between human and Drosophila, SHC lacks the high-affinity GRB2 site present in mammalian SHC, and no binding of SHC to DRK was observed in coimmunoprecipitation experiments. However, the presence of another conserved motif (YYND/S) in all known SHC proteins suggested that SHC could couple RTKs to other downstream targets besides GRB2 and the RAS pathway. Thus far, no functional evidence for the suggested roles of SHC proteins has been shown, since shc loss-of-function mutations have not been reported in any system (Luschnig, 2000).

This work presents functional evidence that a Drosophila SHC homolog is an essential component of distinct RTK signaling pathways in the fly. Mutations affecting either only the PTB domain or eliminating SHC protein cause the same strength of phenotypes in all assays applied. Based on genetic data, it cannot be distinguish whether both domains, PTB and SH2, bind to pY sites on the same activated RTK or on different proteins. DER contains one bona fide binding site for the SHC PTB domain (consensus NPXpY; NPEY¹³⁵⁷ in DER), while in activated TOR two sites with a mismatch at the +2 position are phosphorylated on Tyr (NKGY⁶⁴⁴; NKEY⁶⁹⁸; Gayko, 1999). No motifs for binding by SH2 domains (consensus YIXI) are found in DER, TOR, or SEV (Luschnig, 2000).

The shc^111-40 allele contains a single amino acid exchange (R152 > Y) in the PTB domain. Structural studies have revealed that the corresponding residue (Arg-175) of the human SHC PTB domain is crucial for binding of the PTB domain to pY-containing phosphopeptides: mutating the corresponding Arg-175 of the human Shc PTB domain (to Gln or Lys) completely abolished binding of SHC to the phosphopeptide, while exchanging amino acids at three other structurally relevant positions had less or no significant effect on binding (Zhou, 1995). This work supports the structural data and provides functional evidence for the role of PTB domains in RTK signaling (Luschnig, 2000).

In the absence of SHC protein, TOR and DER signaling is only partially reduced. It has been shown for the TOR pathway that signaling downstream of the receptor does not occur in a simple linear cascade, based on the finding that components acting between TOR and DRAF show weaker loss-of-function phenotypes than TOR or DRAF (Luschnig, 2000).

Double-mutant analysis shows that the TOR signal splits into at least three parallel branches, represented by DOS, DRK, and SHC. Simultaneous removal of DOS and SHC function from the germline does not completely abolish the TOR signal, although it is strongly reduced. Double mutant phenotypes and epistasis experiments suggest that more TOR signal is transmitted via SHC than via DOS or DRK. However, genetic data do not exclude that direct interactions between any of these proteins may exist; for example, DOS contains putative binding sites for the SH2 domains of DRK and SHC (Luschnig, 2000).

It was also shown that DRK and SHC have parallel activities, in agreement with the finding that the unique DRK/GRB2 binding site (YVNV) present in mammalian SHC proteins is missing in Drosophila SHC, and Drosophila SHC does not bind DRK in vitro. While Drosophila SHC lacks the DRK binding site, a central YYND motif is conserved between Drosophila and mammalian SHC proteins and is likely to serve as a docking site for an as yet unknown protein that triggers a RAS-independent signal (Gotoh, 1997). It has been shown that DRAF can be activated in the absence of RAS1, but the molecular nature of a RAS1-independent pathway had remained elusive. Based on genetic results and binding studies, Drosophila SHC is a good candidate to activate downstream targets via a DRK-/RAS1-independent pathway. The identification of signaling components acting independently of RAS will be of great interest for understanding the specificity of RTK signaling, and SHC should provide a useful entry point to study such a pathway (Luschnig, 2000).

It remains a puzzling question why a complex network of adaptor proteins has evolved, if all of these proteins were involved in a single common pathway activated by all RTKs. Interestingly, this study has found that SHC, unlike most other components, functions only in a subset of RTK pathways. DOS, DRK, KSR, and all components downstream of RAS1 are required for signaling by the RTKs TOR, DER, and SEV. Also, unlike all other signaling components, flies lacking SHC can survive to adults, which show a specific phenotype, suggesting that SHC plays a more specific role than other components (Luschnig, 2000).

In the Drosophila eye, DER can replace the function of SEV in R7 determination. This has led to the conclusion that the cytoplasmic signaling pathways triggered by DER and SEV were identical. However, these data are based on the analysis of constitutively activated forms of RTKs that might not faithfully reflect the situation during normal signaling. This study found that SEV signaling appears to be normal in flies lacking SHC protein, and the defects seen in shc mutant eyes appear to be due to impaired DER signaling. The expression of phenotypes caused by activated DER in the eye is very sensitive for the gene dosage of shc. In contrast, dosage-sensitive interactions of shc with activated forms of SEV were not seen, whereas the adaptors DOS and DRK were identified on the basis of their dosage sensitivity for signal transduction by activated SEV. Also, binding of SHC to SEV was not observed, and SEV does not contain consensus binding sites for PTB or SH2 domains of SHC, whereas SHC specifically binds via its PTB domain to DER. Based on these arguments, it is suggested that SHC is not involved in SEV signaling. Even in the absence of SHC protein, the effect of activated SEV was not abolished; however, this study found that the reduction of DER signaling by shc mutations does partially affect the efficiency of SEV-induced overrecruitment of photoreceptors in the anterior of the eye. Accordingly, removing one copy of the DER itself causes partial suppression of the activated SEV phenotype, indicating that induction of photoreceptor cell fate by the DER is necessary before SEV can trigger R7 development (Luschnig, 2000).

The anteriorly-posteriorly graded phenotype of shc mutant eyes can be explained by a common feature of DER signaling: the morphogenetic furrow moves from posterior to anterior across the eye disc and leaves in its wake cells that express Spitz (Spi), an activating DER ligand, and Argos (Aos), a presumptive inhibitory DER ligand. Both Spi and Aos are themselves target genes of DER signaling and are thought to act as diffusible ligands for DER. Hyperactivation of DER by overexpressing activating Spi, or by mutating inhibitory Aos, leads to photoreceptor overrecruitment and eye roughness, which is more pronounced in the posterior of the eye. This graded phenotype is presumably due to accumulation of diffusible Spi in the posterior of the eye disc. It is likely that the same effect accounts for the anterior to posterior graded eye phenotype of shc mutants: although DER signaling efficiency is reduced, Spi can accumulate to sufficient levels in the posterior to allow normal differentiation of photoreceptors. Toward the anterior margin, the level of Spi becomes limiting, and DER signaling activity is reduced below the threshold for photoreceptor differentiation, resulting in the loss of photoreceptors (Luschnig, 2000).

Interestingly, planar polarity defects were found in shc mutant eyes. Frizzled, Notch, and Jak-Stat signaling have been implicated in the establishment of planar polarity in the eye, but to date no role in this process has been assigned to any member of the RAS-MAPK signaling pathway (Luschnig, 2000).

How can different RTKs trigger specific responses by using an apparently common signaling pathway? Several models have been put forward to explain this paradox. In a 'quantitative' model, the kinetics (exact level and duration) of the MAPK signal is critical for the specificity of the response. A 'qualitative' model proposes the existence of cytoplasmic signaling components dedicated to a specific receptor or subset of receptors. Recently, the protein DOF has been shown to act specifically in Drosophila FGFR signaling pathways. Interestingly, DOF is exclusively expressed in cells where also FGFRs are expressed, namely mesoderm and the tracheal system. Tissue-specific expression of a signal transduction component might be one way to create specific responses (Luschnig, 2000).

In contrast, SHC is widely expressed throughout the embryo (Lai, 1995). Specificity appears to be mediated by selective binding of SHC to specific receptors. The curren data show that SHC is functionally dedicated to a subset of RTKs and suggest that the cytoplasmic events triggered by different RTKs (e.g., DER and SEV) can be different. This is a new aspect that could provide an additional explanation for the specificity of RTK signals. It is suggested that different receptors use specific combinations of cytoplasmic adaptor proteins, which have parallel activities, and potentially activate different downstream targets. This could result in quantitatively and/or qualitatively different signal properties. It will be of great interest for the understanding of RTK signaling to elucidate what downstream events are activated by SHC (Luschnig, 2000).

PVR and EGFR signalling during collective migration of border cells

Although directed migration is a feature of both individual cells and cell groups, guided migration has been studied most extensively for single cells in simple environments. Collective guidance of cell groups remains poorly understood, despite its relevance for development and metastasis. Neural crest cells and neuronal precursors migrate as loosely organized streams of individual cells, whereas cells of the fish lateral line, Drosophila tracheal tubes and border-cell clusters migrate as more coherent groups. This study used Drosophila border cells to examine how collective guidance is performed. It is reported that border cells migrate in two phases using distinct mechanisms. Genetic analysis combined with live imaging shows that polarized cell behaviour is critical for the initial phase of migration, whereas dynamic collective behaviour dominates later. PDGF- and VEGF-related receptor and epidermal growth factor receptor act in both phases, but use different effector pathways in each. The myoblast city (Mbc, also known as DOCK180) and engulfment and cell motility (ELMO, also known as Ced-12) pathway is required for the early phase, in which guidance depends on subcellular localization of signalling within a leading cell. During the later phase, mitogen-activated protein kinase and phospholipase Cγ are used redundantly, and it was found that the cluster makes use of the difference in signal levels between cells to guide migration. Thus, information processing at the multicellular level is used to guide collective behaviour of a cell group (Bianco, 2007).

Border cells perform a well-defined, invasive and directional migration during Drosophila oogenesis. They delaminate from the follicular epithelium at the anterior end of an egg chamber and migrate posteriorly, towards the oocyte, as a compact cluster. They then migrate dorsally towards the oocyte nucleus. The border-cell cluster consists of about six outer migratory border cells and two inner polar cells that induce migratory behaviour in the outer cells but seem to be non-migratory. Two receptor tyrosine kinases (RTKs), PDGF- and VEGF-related receptor (PVR) and epidermal growth factor receptor (EGFR), are guidance receptors for border cells. Both receptors act redundantly during posterior migration towards the oocyte, whereas EGFR and its dorsally localized ligand, Gurken, are essential for dorsal migration. Localized signalling from the RTKs is important and actively maintained, especially early in migration. Rac and the atypical Rac exchange factor Mbc (myoblast city, also known as DOCK180) are important effectors. To determine the contribution of Mbc and related proteins, a loss-of-function allele of their common cofactor ELMO (engulfment and cell motility, also known as Ced-12) was generated by homologous recombination. Clusters of elmo mutant border cells arrested early in migration, a defect that could be rescued by expressing elmo complementary DNA. As for mbc, reduction in elmo function suppressed F-actin accumulation caused by constitutive PVR signalling, placing ELMO downstream of the receptor in this respect (Bianco, 2007).

To determine whether later steps in migration also depend on ELMO, mosaic border-cell clusters consisting of wild-type and mutant cells were investigated. If a mutation does not affect migration, mutant cells should be distributed randomly within the cluster. Mutant cells defective in migration would be in the rear, 'carried along' by normal cells. As expected, Pvr and Egfr double mutant cells were in the rear during posterior migration, as were Egfr mutant cells during dorsal migration, reflecting the requirements at each stage. elmo mutant cells were in the rear during the initial migration, but were equally frequent in the leading position during dorsal migration. This indicates that, although ELMO is essential for the early-phase signalling, the later phase of migration does not require the Mbc-ELMO complex (Bianco, 2007).

To understand late guidance signalling, EGFR signalling, on which dorsal migration depends, was dissected. Uniformly activated EGFR, like PVR, dominantly impairs migration. The carboxy-terminal tail of EGFR was essential for this activity. Systematic mutagenesis of all docking tyrosines to phenylalanine identified Y1357 as being critical, with minor contributions from Y1405 and Y1406. Other tyrosines, including Y1095 in the conserved activation loop (phosphorylated in HER2 (Human EGF Receptor 2), were not required. Twenty Src-homology 2- and phosphotyrosine-binding-containing signalling molecules were tested for binding to active EGFR and tyrosine mutants. Y1357 was necessary and sufficient for binding of the adaptor protein Shc and its phosphotyrosine-binding domain. No other tested interactor behaved in this way. Binding was confirmed by immunoprecipitation. Border cells mutant for Shc showed no dorsal migration and, when PVR signalling was also blocked, these cells showed severely impaired posterior migration. This phenotype is identical to that of Egfr mutant cells, suggesting that Shc is essential immediately downstream of EGFR for guidance signalling (Bianco, 2007).

The Shc adaptor protein links EGFR and other RTKs to mitogen-activated protein kinase (MAPK) kinase signalling as well as to other classical downstream pathways. Raf, phospholipase Cγ (PLC-γ) or phosphatidylinositol-3-OH-kinase are not uniquely required for migration; however, the pathways might act redundantly. Simultaneous perturbation of PLC-γ and Raf impaired migration, with no effect of phosphatidylinositol-3-OH kinase. Double mutant border-cell clusters, cell-autonomously lacking PLC-γ and Raf or lacking PLC-γ and MAPK kinase (MAPKK), initiated migration but were delayed later in posterior migration and showed no dorsal migration. This phenotype is more severe than that of Egfr or Shc alone, suggesting that both RTKs might be affected. Prevention of PVR activity in double mutant cells did not block posterior migration, confirming that the requirement for these pathways was stage-specific and not EGFR-specific. Finally, analysis of mosaic clusters showed that Raf/MAPK and PLC-γ were important in late migration, reciprocal to the requirement for elmo. These results genetically define two migratory phases: an early posterior phase requiring ELMO-Mbc and a later posterior and dorsally directed phase requiring Raf/MAPK or PLC-γ. Both RTKs shift effector-pathway-dependency as migration progresses (Bianco, 2007).

To investigate the different migratory phases, border-cell migration was examined via live imaging. Appropriate conditions were establised for culturing and imaging of egg chambers, considering only active, growing ones. Border cells were selectively labelled with green fluorescent protein (GFP) and all membranes were labelled with the vital dye FM4-64. For all 24 wild-type samples, the identity of the front cell changed during the observation period, confirming the inference from fixed samples that cells change position during migration. This indicates that there is no determined front-cell fate. A clear difference was observed in behaviour of clusters during early (first half) and late phases. Early clusters had one, sometimes two, highly polarized cells clearly leading the migration; once these cells delaminated they moved straight and relatively fast. Weakly stained extensions protrude far from delaminating cells and subsequently shorten during movement, suggesting a 'grapple and pull' mechanism. Midway towards the oocyte, strong polarization was lost and cells rounded and started to 'shuffle' while dynamically probing the environment with short extensions. Occasionally the cluster would rotate or 'tumble' completely. This shuffling behaviour still provided effective movement of the cluster towards the oocyte and dorsally, albeit more slowly. Labelling cells with nuclear GFP allowed visualization of changes in positions within the cluster. The front cell exchanged, on average, every 18 min (Bianco, 2007).

As expected, positions corresponding to the second, slower phase of migration were more represented when cluster position along the migratory path was quantified in fixed samples. Also, border cells expressing dominant negative PVR and EGFR were individually active but provided little net cluster movement, as expected from the lack of guidance information. Finally, uniform overexpression of the attractant PVF1 caused an increased shuffling behaviour in the early phase but allowed slow forward movement, resembling normal late migration. This indicates that migrating clusters can interpret a shallow gradient when using the shuffling mode. It also suggests that the normal change in migratory behaviour midway into posterior migration might be triggered by the higher concentration of ligands closer to the oocyte (Bianco, 2007).

The early phase of migration with a highly polarized front cell corresponds temporally to the genetic requirement for ELMO activity. During the later phase, individual elmo mutant cells can alternate with wild-type cells in the lead position. Genetic analysis showed that Raf and MAPKK and, by inference, MAPK activation was sufficient to convey late guidance information. This was puzzling because MAPK activation appeared uniform in migrating border cells, and localized effects are usually a hallmark of guidance signalling. However, signalling that is not localized within an individual cell could still transmit spatial guidance information to the cell cluster if the cell with higher overall signalling indicates the direction of subsequent migration for the whole cluster, as observed for MAPK signalling in border cells. In this 'collective guidance' scenario, each cell of the cluster can be thought of as being analogous to a sector of an individual guided cell. Different levels of signalling in individual cells of the cluster transform into migration vectors because border cells adhere to each other and these contacts differ from substrate contacts. The occasional tumbling of border-cell clusters emphasizes the ability of these cells to behave as a collective unit. Tumbling may help single guided cells to 'reassess' their environment (Bianco, 2007).

To test this model for guidance, the relative levels of signalling in individual cells of the cluster were manipulated. Dynamic shuffling should allow cells to constantly 'compete' for the front position. None of the manipulations discussed below improved migration if all cells in a cluster were affected. Individual border cells with moderately elevated levels of PVR or EGFR were preferentially in the front relative to wild-type cells. Cells with elevated PVR tended to stay in or near the front position, suggesting that they were not competed away by other cells. This bias was ligand-dependent, because reducing PVF1 levels shifted the bias from PVR to EGFR, as was also shown by analysis of dorsal migration. Thus, increased signalling gives a cell-front bias when measuring an informative ligand. Elevating intracellular signalling levels had similar effects, whether by overexpression of an active form of Raf or by preventing downregulation of signalling as in Hrs mutant cells, in which RTK-mediated MAPK signalling is elevated in enlarged endosomes. The more modest front bias in Hrs mutant cells was reflected in behaviour: they could be displaced from the front. The E3 ubiquitin ligase Cbl negatively regulates RTK signalling and is also required to maintain localized RTK signalling within border cells initiating migration. Cbl mutant cells shifted from being preferentially at the back during early stages to being in the front during later migration. This indicates a transition from a mode requiring Cbl-dependent localization of signalling within the leading cell to a mode based on collective decisions within the cluster, in which Cbl mutant cells have an advantage owing to elevated RTK signalling (Bianco, 2007).

It is suggested that guidance of border-cell migration is achieved by two means: signalling localized within the cell, as used in individual migrating cells, and collective guidance, whereby the cluster uses differences in signalling strength among its constituent cells to determine direction. The two modes use the same guidance cues and receptors, but different downstream effectors. Localized signalling is required for the initial, polarized rapid migration, whereas collective behaviour, though observable throughout, dominates in the later phase. Collective decisions on the basis of differences in RTK signalling strength are important in Caenorhabditis elegans vulval development and in branching of Drosophila tracheal tubes, in which they result in specification of discrete cell fates. This differs from the dynamic situation reported in this study, in which the identity of the leading cell constantly changes. Indeed, the frequent exchange of leading cells suggests that front behaviour is normally temporarily restricted, possibly by induced inactivation of signalling. Such dynamics may allow the cluster to better reassess the environment. For guided migration of cell groups, the analysis indicates that sensing and regulation happens both at the single cell level and at the next level-that of collective cell decisions (Bianco, 2007).

SHC-1/p52Shc targets the insulin/IGF-1 and JNK signaling pathways to modulate life span and stress response in C. elegans

Correlative evidence links stress, accumulation of oxidative cellular damage, and aging in several species. Genetic studies in species ranging from yeast to mammals revealed several pathways regulating stress response and life span, including caloric intake, mitochondrial respiration, insulin/IGF-1 (IIS), and JNK (c-Jun N-terminal kinase) signaling. How IIS and JNK signaling cross-talk to defend against diverse stressors contributing to aging is of critical importance but, so far, only poorly understood. This study demonstrates that the adaptor protein SHC-1, the C. elegans homolog of human p52Shc, coordinates mechanisms of stress response and aging. Using genetic and biochemical approaches, it was discovered that SHC-1 not only opposes IIS but also activates JNK signaling. Loss of shc-1 function results in accelerated aging and enhanced sensitivity to heat, oxidative stress, and heavy metals, whereas expression of human p52Shc rescues the shc-1 mutant phenotype. SHC-1 acts upstream of the insulin/IGF receptor DAF-2 and the PI3 kinase AGE-1 and directly interacts with DAF-2. Moreover, SHC-1 activates JNK signaling by binding to MEK-1 kinase. Both aspects converge on controlling the nuclear translocation and activation of the FOXO transcription factor DAF-16. These findings establish C. elegans SHC-1 as a critical scaffold that directly cross-connects the two parallel JNK and IIS pathways and help to explain how these signaling cascades cooperate to ascertain normal stress response and life span in C. elegans (Neumann-Haefelin, 2008).

Characterization of the Phosphotyrosine-binding Domain of the Drosophila Shc Protein

The phosphotyrosine-binding (PTB) domain of Drosophila Shc (SHC) binds in vitro to phosphopeptides containing the sequence motif NPXpY, and physically associates with the activated Drosophila epidermal growth factor receptor homologue (DER) in vivo. The structural elements, specificity and binding kinetics of the SHC PTB domain have now been characterized. The SHC PTB domain appeared similar to the insulin-like receptor substrate-1 PTB domain in secondary structure as suggested by Fourier transform infrared spectroscopy. Surface plasmon resonance measurements indicated that the SHC PTB domain bound with high affinity to phosphopeptides (Der) derived from the Tyr1228 site of the DER receptor. The kinetics of the SHC PTB domain-Der phosphopeptide interaction differed from those of a typical SH2 domain-ligand interaction, in that the PTB domain displayed slower on/off rates. Competition binding assays using truncated versions of the Der peptides revealed that high affinity binding to the SHC PTB domain requires, in addition to the NPXpY motif, the presence of hydrophobic residues at both positions -5 and -7 relative to phosphotyrosine. The SHC PTB domain showed a similar binding specificity to the human Shc (hShc) PTB domain, but subtle differences were noted; such that the hShc PTB domain bound preferentially to a phosphopeptide from the mammalian nerve growth factor receptor, whereas the SHC PTB domain bound preferentially to phosphopeptides from the Drosophila DER receptor. The invertebrate SHC PTB domain therefore possesses a binding specificity for tyrosine-phosphorylated peptides that is optimally suited for recognition of the activated DER receptor (Li, 1996).

A conserved amino-terminal Shc domain binds to phosphotyrosine motifs in activated receptors and phosphopeptides.

Signal transduction by growth factor receptor protein-tyrosine kinases is generally initiated by autophosphorylation on tyrosine residues following ligand binding. Phosphotyrosines within activated receptors form binding sites for the Src homology 2 (SH2) domains of cytoplasmic signalling proteins. One such protein, Shc, is tyrosine phosphorylated in response to a large number of growth factors and cytokines. Phosphorylation of Shc on tyrosine residue Y317 allows binding to the SH2 domain of Grb2, and hence stimulation of the Ras pathway. Shc is therefore implicated as an adaptor protein able to couple normal and oncogenic protein-tyrosine kinases to Ras activation. Shc itself contains an SH2 domain at its carboxyl terminus, but the function of the amino-terminal half of the protein is unknown. This study has found that the Shc amino-terminal region binds to a number of tyrosine-phosphorylated proteins in v-src-transformed cells. This domain also bound directly to the activated epidermal growth factor (EGF) receptor. A phosphotyrosine (pY)-containing peptide modeled after the Shc-binding site in polyoma middle T antigen (LLSNPTpYSVMRSK) was able to compete efficiently with the activated EGF receptor for binding to the Shc amino terminus. This competition was dependent on phosphorylation of the tyrosine residue within the peptide, and was abrogated by deletion of the leucine residue at position -5. The Shc amino-terminal domain also bound to the autophosphorylated nerve growth factor receptor (Trk), but bound significantly less well to a mutant receptor in which tyrosine Y490 in the receptor's Shc-binding site had been substituted by phenylalanine. These data implicate the amino-terminal region of Shc in binding to activated receptors and other tyrosine-phosphorylated proteins. Binding appears to be specific for phosphorylated tyrosine residues within the sequence NPXpY, which is conserved in many Shc-binding sites. The Shc amino-terminal region bears only very limited sequence identify to known SH2 domains, suggesting that it represents a new class of phosphotyrosine-binding modules. Consistent with this view, the amino-terminal Shc domain is highly conserved in a Drosophila Shc homologue. Binding of Shc to activated receptors through its amino terminus could leave the carboxy-terminal SH2 domain free for other interactions. In this way, Shc may function as an adaptor protein to bring two tyrosine-phosphorylated proteins together (van der Geer, 1995).

A Drosophila shc gene product is implicated in signaling by the DER receptor tyrosine kinase

The mammalian EGF receptor can serve as a model for Drosophila signaling. SHC is the Drosophila homolog of a mammalian proto-oncogene that serves as a docking molecule, binding to activated receptors as a prelude to assembling other molecules at the site of the activated receptor. SHCp52 polypeptide has an amino terminal phosphotyrosine binding domain that binds an intracellular phosphorylated residue of activated EGF receptor. SHC is then phosphorylated by the EGF receptor and serves to regulate the RAS pathway through its ability to interact with SOS guanine nucleotide exchange factor (Lai, 1995).

Antibodies to the human SHC adaptor protein were used to isolate a cDNA encoding a Drosophila SHC protein (SHC) by screening an expression library. In flies, the SHC protein physically associates with activated Egfr and is inducibly phosphorylated on tyrosine by Egfr. SHC contains an N-terminal phosphotyrosine-binding domain, which associates in vitro with the autophosphorylated Egfr receptor tyrosine kinase. A potential binding site for the SHC phosphotyrosine-binding domain is located at Tyr-1228 of Egfr (Lai, 1995).

Search PubMed for articles about Drosophila SHC

Bianco, A., et al. (2007). Two distinct modes of guidance signalling during collective migration of border cells. Nature 448: 362-365. PubMed ID: 17637670

Bonfini, L., et al. (1996). Not all Shc's roads lead to Ras. Trends Biochem. Sci. 21: 257-261. PubMed ID: 8755247

Cattaneo, E. and Pelicci, P.G. (1998). Emerging roles for SH2/PTB-containing Shc adaptor proteins in the developing mammalian brain. Trends Neurosci. 21: 476-481. PubMed ID: 9829689

Gayko, U., et al. (1999), Synergistic activities of multiple phosphotyrosine residues mediate full signaling from the Drosophila Torso receptor tyrosine kinase. Proc. Natl. Acad. Sci. 96: 523-528. PubMed ID: 9892666

Gotoh, N., Toyoda, M. and Shibuya, M. (1997). Tyrosine phosphorylation sites at amino acids 239 and 240 of Shc are involved in epidermal growth factor-induced mitogenic signaling that is distinct from Ras/mitogen-activated protein kinase activation. Mol. Cell. Biol. 17: 1824-1831. PubMed ID: 9121430

Lai, K. M.., et al. (1995). A Drosophila shc gene product is implicated in signaling by the DER receptor tyrosine kinase. Mol. Cell. Biol. 15: 4810-4818. PubMed ID: 7651398

Li, S. C., et al. (1996). Characterization of the phosphotyrosine-binding domain of the Drosophila Shc protein. J. Biol. Chem. 271(50): 31855-62. PubMed ID: 8943228

Luschnig, S., Krauss, J., Bohmann, K., Desjeux, I. and Nüsslein-Volhard, C. (2000). The Drosophila SHC adaptor protein is required for signaling by a subset of receptor tyrosine kinases. Mol. Cell 5(2): 231-41. PubMed ID: 10882065

Neumann-Haefelin, E., Qi, W., Finkbeiner, E., Walz, G., Baumeister, R. and Hertweck, M. (2008). SHC-1/p52Shc targets the insulin/IGF-1 and JNK signaling pathways to modulate life span and stress response in C. elegans. Genes Dev. 22(19): 2721-35. PubMed ID: 18832074

Pelicci, G., et al. (1992). A novel transforming protein (SHC) with an SH2 domain is implicated in mitogenic signal transduction. Cell 70: 93-104. PubMed ID: 1623525

Rozakis-Adcock, M., et al. (1992). Association of the Shc and Grb2/Sem5 SH2-containing proteins is implicated in activation of the Ras pathway by tyrosine kinases. Nature 360: 689-692. PubMed ID: 1465135

van der Geer, P., et al. (1995). A conserved amino-terminal Shc domain binds to phosphotyrosine motifs in activated receptors and phosphopeptides. Curr. Biol. 5: 404-412. PubMed ID: 7542991

Zhou, M. M. et al. (1995). Structure and ligand recognition of the phosphotyrosine binding domain of Shc. Nature 378; 584-592. PubMed ID: 8524391

date revised: 1 March 2009

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