BIOLOGICAL OVERVIEW

Activation of the Sevenless (Sev) receptor tyrosine kinase (RTK) in the developing Drosophila eye is required for the specification of the R7 photoreceptor cell fate. Daughter of Sevenless (Dos), a putative multi-site adaptor protein, is a substrate of the Sev kinase and is known to associate with the tyrosine phosphatase Corkscrew (Csw). Binding of Csw to Dos depends on the Csw Src homology 2 (SH2) domains and is an essential step for signaling by the Sev RTK. Dos, however, lacks a recognizable phosphotyrosine interaction domain and it was previously unclear how it is recruited to the Sev receptor. The SH2/SH3 domain adaptor protein Drk has been shown to provide this link. Drk binds with its SH2 domain to the autophosphorylated Sev receptor while the C-terminal SH3 domain is able to associate with Dos. The Drk SH3 domain binding motifs on Dos have been mapped to two sites that do not conform to the known Drk SH3 domain binding motif (PxxPxR) but instead have the consensus PxxxRxxKP. Mutational analysis in vitro and in vivo has provided evidence that both Drk binding sites fulfil an important function in the context of Sev and Drosophila Epidermal growth factor receptor mediated signaling processes (Feller, 2002).

A major route by which Sev, as well as RTKs in general, transduce signals from the plasma membrane to the nucleus involves the small GTPase Ras and the MAPK signaling cassette which is comprised of the kinases Raf, Dsor1/MEK and Rolled/MAPK. A direct link between Sev and the Ras/MAPK pathway is provided by Drk, the Drosophila homolog of the SH2/SH3 domain adaptor protein Grb2. The Drk SH2 domain interacts with a specific phosphotyrosine residue (Y2546) in the intracellular domain of the activated Sev RTK while both Drk SH3 domains bind to proline-rich sequences of the guanine-nucleotide exchange factor Sos, which activates Ras by promoting the exchange of GDP for GTP (Feller, 2002 and references therein).

This simplistic view of activation of the Ras/MAPK pathway was questioned by the observation that a Sev protein without a functional Drk binding site can still specify R7 photoreceptor cells in a Drk- and Ras/MAPK-dependent manner, arguing for the existence of alternative ways to activate the Ras/MAPK signaling pathway. Dos, which becomes tyrosine phosphorylated upon Sev activation, could fulfil this function. Similar to the vertebrate Gab and IRS adaptor protein families, Dos contains an N-terminal pleckstrin homology (PH) domain and several tyrosine residues within consensus sites predicted to bind to a variety of SH2 domain containing proteins, including Drk, D-Shc, Phospolipase C (PLC), the tyrosine phosphatase Corkscrew (Csw), and the regulatory subunit of Phosphoinositide 3-kinase (PI3K). However, genetic analyses in flies indicate that with the exception of two phosphotyrosine residues that interact with the Csw SH2 domains, all other predicted SH2 domain binding sites on Dos appear to be dispensable for Dos function or might fulfil redundant functions during signaling. In a similar manner, association of Gab1 (a mammalian protein with structural similarities to Dos) with the vertebrate Csw homolog Shp2, but not with PI3K, Shc or CRKL, has been shown to be essential for c-Met-induced branching morphogenesis of MDCK kidney epithelial cells (Feller, 2002 and references therein).

The requirement of tyrosine phosphorylation of Dos for its in vivo function also raises the question how Dos becomes recruited to the activated RTK. Dos neither has a PTB domain like the IRS proteins nor does it contain sequences homologous to the Met-binding domain (MBD) of Gab1 which mediates interaction with the c-Met RTK. More recently, an indirect, Grb2-dependent mechanism for Gab1 recruitment to the c-Met and EGF RTKs has been described. Grb2 interacts with Gab1 primarily via its C-terminal SH3 domain, leaving the Grb2 SH2 domain free to bind to the autophosphorylated RTK (Fixman, 1997; Nguyen, 1997). Grb2 binding to Gab1 was mapped to two sites: a proline-rich sequence containing the consensus PxxP motif typical for binding SH3 domains and a novel motif (PxxxRxxKP). The PxxxRxxKP sequence motif is also found in Gab2, c-Cbl, SLP-76 and AMSH, which are all adaptor proteins involved in intracellular signal transmission regulation but also in the deubiquinating enzyme UBPY. Several other cases of SH3 domains binding to motifs lacking the canonical PxxP core have also recently been described. Sequence comparison has revealed that Dos contains two PxxxRxxKP motifs and several PxxP sites, possibly indicating an interaction of Dos with SH3 domain proteins (Feller, 2002 and references therein).

Evidence is provided that the two PxxxRxxKP motifs in Dos are functional binding sites for the C-terminal SH3 domain of Drk. Substitution of the conserved arginine residues by lysines interferes with binding of Dos to Drk. To demonstrate a requirement for these binding sites in Sev and Egfr mediated signaling processes, mutant Dos proteins were tested for their ability to substitute the loss of endogenous Dos function in flies. Dos proteins lacking either SH3 domain binding site are impaired in their function in the Sev and Egfr pathway, whereas a Dos protein with both binding sites mutated is largely non-functional. These results support a model in which an important step for signaling by the Sev and Egfr RTKs is to recruit Dos through interaction with Drk (Feller, 2002).

By combining biochemical and genetic approaches, two Drk SH3 domain binding sites have been identified in Dos that play a crucial role in signaling from the Sev and Egfr RTKs (Feller, 2002).

Drk and the vertebrate homolog Grb2 have been shown to bind through their SH3 domains to proline-rich peptide sequences in the C-terminal half of the guanine nucleotide exchange factor Sos; these proline-rich peptide sequences conform to the consensus sequences PxxPxR and adopt a left handed polyproline type II helix conformation. In vitro binding studies indicated that this interaction is mediated primarily via the N-terminal SH3 domain. The results obtained in vitro correlated with in vivo functional studies of the Drk and Grb2 proteins. The ability of Grb2 to support endoderm differentiation is abrogated by mutation of the N-terminal SH3 domain, whereas inactivation of the C-terminal SH3 domain only impairs Grb2 function in this process. Similarly, mutation of the Drk N-terminal SH3 domain completely disrupts the function of Drk in the Sev pathway, whereas mutation of the C-terminal SH3 domain has only a weak effect on Sev signaling. Nonetheless, rescue of the lethality caused by mutation of the endogenous drk gene is dependent on the presence of both SH3 domains in the corresponding drk-transgene, suggesting that the C-terminal SH3 domain of Drk also has important functions in developmental processes controlled by RTKs (Raabe, 1995). Despite these findings, the functions and binding specificities of the C-terminal SH3 domains of Drk and Grb2 remained poorly defined. More recently it became apparent that the C-terminal SH3 domain of Grb2 mediates the interaction with the adaptor protein Gab1. The association between Grb2 and Gab1 maps to two sites, one of which corresponds to the classical SH3 domain binding motif (PxxPxK), the second site defined a novel SH3 domain interaction motif (PxxxRxxKP). Dos is similar to Gab1 in its ability to bind the Drk SH3(C) domain but not the Drk SH3(N) domain. Unlike Gab1, Dos binding to Drk depends only on the two PxxxRxxKP sites (DBS1 and DBS2). Deletion of other putative SH3 binding sites (PxxP) neither affects Drk binding in vitro nor is Dos function impaired in vivo (Herbst, 1999). In contrast, mutation of DBS1 and DBS2 completely abolish Drk binding in coimmunoprecipitation experiments and disrupt Dos function in vivo. Mutation of either binding site alone is sufficient to impair signaling from the Egfr and Sev RTKs (Feller, 2002).

Although the in vitro binding studies indicated that the Dos-Drk interaction is mediated through binding of DBS1 and DBS2 to the C-terminal Drk SH3 domain, genetic analysis indicates qualitative differences between DBS1 and DBS2. Mutation of the DBS2 site induces stronger defects than mutation of DBS1, indicating that the association of Dos with Drk is primarily mediated via the DBS2 site. Alternatively, DBS2 could act in addition also as a docking site for other SH3 domain proteins and therefore deletion of this site would cause more severe defects. In a first attempt to address this question, one putative candidate, PLCgamma was tested. The recruitment of additional R7 photoreceptor cells in PLCgamma-deficient flies is suppressed by mutations in the dos gene (Riesgo-Escovar and T. Raabe, unpublished observation reported in Feller, 2002). However, no interaction of the PLCgamma SH3 domain and Dos has been observed. Dos also contains two tyrosine residues within a consensus sequence predicted to bind to the PLCgamma SH2 domains, but mutation of these tyrosine residues has no effect on Dos function in the Sev or Egfr signaling pathway (Bausenwein, 2000; Herbst, 1999). At present, whether PLCgamma can directly bind to Dos remains unclear (Feller, 2002).

The Drk-Dos interaction provides a new link between Sev and the Ras/MAPK pathway. Dos has been identified as a downstream component of various RTKs in Drosophila, including Sev, Egfr and Torso. In order to fulfil its adaptor protein function for SH2 domain proteins, Dos has to be recruited to the activated RTK to become phosphorylated on specific tyrosine residues. Drk SH3 domain-mediated binding to Dos (Feller, 2002) and association of the Drk SH2 domain with phosphotyrosine residue 2546 on Sev (Raabe, 1995) could provide this link. An important question that arises from these findings is whether this link is sufficient to mediate all biological responses from the activated Sev RTK. In the case of Gab1, there appear to be important differences in the ways it is recruited to different RTKs. Gab1 recruitment to the EGF RTK depends on the association of Gab1 with Grb2 SH3 domains. In contrast, a mutant Gab1 protein lacking both Grb2 SH3 binding sites can still directly associate via its MBD domain with a specific phosphotyrosine residue on the c-Met RTK (Lock, 2000; Schaeper, 2000). Although DBS1 and DBS2 are important for Dos function in the Sev and Egfr signaling pathways, it is likely that other protein-protein interaction sites are also involved in Dos recruitment to Sev. This conclusion can be drawn from genetic analysis using a mutated Sev receptor (Raabe, 1996). Removal of tyrosine 2546 completely abolishes SH2-mediated binding of Drk to Sev and therefore should also block Dos recruitment to Sev. However, specification of the R7 photoreceptor cell fate is only partially impaired and still requires Dos and Drk function. Taking into account these findings it is surprising that a Dos protein lacking both Drk SH3 domain binding sites is non-functional in the Sev pathway. The tyrosine phosphatase Csw might provide an additional way to recruit Dos to Sev. Csw binds to tyrosine phosphorylated Dos by its SH2 domains whereas the interaction between Csw and Sev is independent of both Csw SH2 domains and Sev autophosphorylation. Csw contains a YxN motif indicative of Drk SH2 domain binding, yet a mutant Csw protein lacking this site is not impaired in its function in the Sev signaling pathway (Feller, 2002).

To reconcile these data, the following model is proposed. In the developing R7 cell, the Drk mediated recruitment of Dos to Sev plays the major role while in the absence of the tyrosine 2546 on Sev, Csw binding to Sev and Drk would still allow recruitment and subsequent phosphorylation of Dos by Sev. Only removal of DBSI and DBS2 would completely block the Drk and Csw-Drk mediated recruitment of Dos to Sev and therefore causes a much severe phenotype than blocking either link alone (Feller, 2002).

In conclusion, these data provide evidence that an essential function of Drk is to recruit Dos to the activated RTK. Following tyrosine phosphorylation of Dos, SH2 domain containing proteins can be assembled in the signaling complex. The functional significance of many of these binding sites in the context of signaling by a specific RTK remains to be established (Feller, 2002).

GENE STRUCTURE

cDNA clone length - 3184

Bases in 5' UTR - 45

Exons - 6

Bases in 3' UTR - 502

PROTEIN STRUCTURE

Amino Acids - 878

Structural Domains

Dos contains a N-terminal pleckstrin homology (PH) domain and several tyrosine residues within consensus sites predicted to bind to a variety of SH2 domain containing proteins (Herbst, 1996 and Raabe, 1996)

date revised: 2 January 2023

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