Isolation of Proteins that Interact with the Signal Transduction Molecule Dof and Identification of a Functional Domain Conserved between Dof and Vertebrate BCAP

doi:10.1016/S0022-2836(03)00489-3

Journal of Molecular Biology

Volume 329, Issue 3, 6 June 2003, Pages 479-493

https://doi.org/10.1016/S0022-2836(03)00489-3 Get rights and content

Abstract

Dof is a large molecule essential for signal transduction by the two FGF receptors in Drosophila. It contains two ankyrin repeats and a coiled-coil region, but has no other recognisable structural motif. Dof shares these features with its closest vertebrate relatives, the B-cell signalling molecules BCAP and BANK. In addition, this family of proteins shares a region of homology upstream of the ankyrin repeats, which we call the Dof/BCAP/BANK (DBB) motif. We have identified 44 proteins that interact with Dof in a yeast two-hybrid screen. These include the Drosophila FGF-receptor Heartless and Dof itself. We show that the integrity of the DBB motif is required both for Dof and for BCAP to form dimers. Analysis of the interactions between a set of deletion constructs of Dof and the panel of interactors suggests that Dof may adopt different conformations, with a folded conformation stabilized by interactions between the DBB motif and the C-terminal part of the protein.

Introduction

FGF-receptors (FGFRs) are members of the large family of receptor tyrosine kinases (RTKs) that control morphogenetic and differentiation processes by activating a variety of downstream signalling modules. Grb2/Drk is one of the main adaptor molecules linking the MAPK cascade to activated RTKs, but in the case of the FGFR it is not able to bind directly to phosphotyrosine residues in the cytoplasmic domain of the receptor. Instead, an additional link is provided in vertebrates by the membrane-anchored adapter protein FRS2/SNT-1.1., 2., 3. FRS2 binds to the human FGFR-1 via its PTB domain, which recognizes 12 residues in the juxtamembrane portion of the receptor, independent of the phosphorylation state of the receptor.4., 5., 6., 7.

Drosophila has two FGF-receptor homologs and one FGF homolog, which are required for the differentiation and morphogenesis of a variety of cell types. The two FGFRs, Heartless and Breathless, share 32% and 27% identity with human FGFR-1. However, the intracellular domains are highly divergent from FGFR-1 at the juxtamembrane region, the region used by FGFR-1 to recruit FRS2. A distant relative of FRS2 exists in Drosophila, but is not required for the transduction of the FGF signal (A. Michelson, personal communication). By contrast, the Drosophila cytoplasmic molecule Downstream of FGFR (Dof, also known as Heartbroken or Stumps)8., 9., 10. is a potential adapter for the FGFR, since it is essential for signal transmission by FGFRs and acts upstream of Ras. The most closely related vertebrate protein identified so far is the B lymphocyte signal transduction molecule BCAP,¹¹ which has been found in chicken, human and mouse, and acts as an adapter in B-cell receptor signal transmission. It becomes phosphorylated by Syk and Btk in response to B-cell receptor activation and, in its phosphorylated form, recruits PI3-kinase. BCAP, as well as another signalling molecule in B cells, BANK,¹² share the overall structure of Dof. Like Dof, both have ankyrin repeats consisting of only two repeats and a region predicted to form a coiled-coil structure approximately 330 amino acid residues downstream of the ankyrin repeats. However, the similarity between Dof, BCAP and BANK extends beyond the ankyrin repeats. A stretch of approximately 140 amino acid residues upstream of the ankyrin repeats shows 27 (19%) identities and 21 conservative substitutions (15%) between Dof and BCAP (Figure 1).

In the Drosophila FGF pathway, a constitutively active Ras molecule can compensate, at least partly, for the absence of Dof, whereas a constitutively active FGFR requires Dof for signal transmission.8., 9., 10. Although the epistatic relationships would be consistent with a function for Dof as a classical adaptor protein for FGFR signaling to the MAPK pathway, other functions, for example in facilitating receptor dimerization or receptor trafficking to the membrane, cannot be excluded. To study the role of Dof in FGFR signal transduction, we have identified a set of interaction partners of Dof using the yeast two-hybrid system. Here, we use them to probe structural and functional properties of Dof.

Section snippets

Results

A yeast two-hybrid screen based on the Gal4 system and using a triple selection method in the yeast strain PJ69-4a was carried out to identify interaction partners of Dof.¹³ As the bait, we used a C-terminally truncated form of the Dof protein (Dof1-802, Figure 1), which was found to be fully functional in an in vivo assay (R.W., unpublished results). We constructed the yeast strain AR2.9, which contains the construct for the Gal4 fusion protein DB-Dof1-802, as a chromosomally integrated

Discussion

We have identified cDNA fragments of 44 genes whose products interact in the yeast-two-hybrid assay with Dof. They may, on the one hand, contain physiological interaction partners that will eventually allow us to determine the precise role of Dof in the FGF signal transduction pathway. This will require much additional detailed analysis of the individual genes. On the other hand, irrespective of their physiological functions, and irrespective of the question of whether the clones express

Expression vectors

A NotI site was introduced into the unique BglII site in the expression vectors pGAD-c(x), pGBD-c(x) and pGBDU-c(x) (gifts from Phillip James) to generate pGAD-c(x) NotI, pGBD-c(x) NotI and pGBDU-c(x) NotI. The NotI site was introduced by filling in the BglII site and adding a NotI linker (NEB1126). The expression vector pODB-8 was a gift from Marc Crouzet. A231 was made from pGBD-C3 NotI by ligating an AvrII linker (NEB 1123) to a filled-in NotI site and an NcoI linker (NEB 1151) to a

Supplementary Files

Acknowledgements

We thank Steve Elledge for the Drosophila 0–18hours cDNA library in pACT, Mark Crouzet for the pODB8 vector, Phillip James for the PJ69-4a strain and the vectors pGBD-c(x), pGAD-c(x), and pGBDU-c(x), Denise Montell for the lambda-breathless construct, Christa Kalmbach-Zürn and Michael Reth for reagents and help with the Schneider S2 transfection system and Frank Sprenger, Greg Beitel and Jürgen Dohmen for comments on the manuscript. We are grateful to Norbert Lehming for advice, and to Heidi

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As signaling molecules, in addition to effector enzymes, adaptor proteins, which lack intrinsic enzymatic activity, have emerged to regulate various signaling events and their biological consequences by recruiting effector enzymes for integrating or diversifying multiple pathways (Bishop, 2004; Kurosaki, 2002b; Qian et al., 2004). BANK, a recently described adaptor molecule, shares overall structural features with its relatives BCAP and Dof; they consist of an ankyrin-repeat-like region and a coiled-coil domain (Battersby et al., 2003; Yokoyama et al., 2002). Functionally, BANK has been suggested to participate in BCR-mediated calcium homeostasis through acting on a calcium release channel, inositol 1,4,5-trisphosphate receptor, but its physiological role has not been directly approached (Yokoyama et al., 2002).
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^†: Present address: A. Battersby, School of Biological Sciences, University of Wales-Bangor, Deiniol Road, Bangor LL57 2UW, UK.

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Journal of Molecular Biology

Isolation of Proteins that Interact with the Signal Transduction Molecule Dof and Identification of a Functional Domain Conserved between Dof and Vertebrate BCAP

Abstract

Introduction

Section snippets

Results

Discussion

Expression vectors

Supplementary Files

Acknowledgements

Cell

J. Biol. Chem.

J. Biol. Chem.

J. Biol. Chem.

Mol. Cell

Immunity

Biochim. Biophys. Acta

Mol. Cell

Cell

Mol. Cell

Broadly expressed SNT-like proteins link FGF receptor stimulation to activators of Ras

Oncogene

Binding of Shp2 tyrosine phosphatase to FRS2 is essential for fibroblast growth factor-induced PC12 cell differentiation

Mol. Cell. Biol.

FRS2 proteins recruit intracellular signaling pathways by binding to diverse targets on fibroblast growth factor and nerve growth factor receptors

Mol. Cell. Biol.