The neurofibromin recruitment factor Spred1 binds to the GAP related domain without affecting Ras inactivation Theresia Dunzendorfer-Matta,1, Ellen L. Mercadob,1, Karl Malyc, Frank McCormickb,2, and Klaus Scheffzeka,2 aDivision of Biological Chemistry, Biocenter, Medical University of Innsbruck, 6020 Innsbruck, Austria; bHelen Diller Family Comprehensive Cancer Center, University of California, San Francisco, CA 94158; and cDivision of Medical Biochemistry, Biocenter, Medical University of Innsbruck, 6020 Innsbruck, Austria Contributed by Frank McCormick, May 10, 2016 (sent for review November 3, 2015; reviewed by Jonathan Licht and Nancy Ratner) Neurofibromatosis type 1 (NF1) and Legius syndrome are related glycerophospholipid binding Sec14-like domain associated with a diseases with partially overlapping symptoms caused by alterations previously undetected C-terminal pleckstrin homology (PH)-like of the tumor suppressor genes NF1 (encoding the protein neurofibro- domain (13–15). Although a number of interaction partners of min) and SPRED1 (encoding sprouty-related, EVH1 domain-containing neurofibromin have been reported, the significance of the un- protein 1, Spred1), respectively. Both proteins are negative regulators derlying protein–protein interaction is not clearly established in of Ras/MAPK signaling with neurofibromin functioning as a Ras- many cases, as reviewed in refs. 2 and 16. specific GTPase activating protein (GAP) and Spred1 acting on hitherto The recently described Legius syndrome (LS) shares the undefined components of the pathway. Importantly, neurofibromin milder features with NF1, including café au lait macules, axillary has been identified as a key protein in the development of cancer, as freckling, and sometimes macrocephaly, but does not appear to it is genetically altered in a large number of sporadic human malig- include the more severe clinical manifestations of NF1 such as nancies unrelated to NF1. Spred1 has previously been demonstrated neurofibromas, optic pathway gliomas, or osseous lesions (17). LS is caused by germ-line mutations in the SPRED1 gene to interact with neurofibromin via its N-terminal Ena/VASP Homol- - - ogy 1 (EVH1) domain and to mediate membrane translocation of its (17, 18) encoding the Sprouty related, EVH1 domain containing target dependent on its C-terminal Sprouty domain. However, the protein 1 (Spred1), which has been demonstrated to specifically inhibit the Ras/MAPK pathway in response to growth factor-, region of neurofibromin required for the interaction with Spred1 has – remained unclear. Here we show that the EVH1 domain of Spred1 cytokine-, and chemokine-induced ERK activation (19 22). In pediatric acute myeloblastic leukemia, Spred1 acts as a tumor binds to the noncatalytic (GAPex) portion of the GAP-related domain suppressor (23). The 50-kDa protein comprises an N-terminal (GRD) of neurofibromin. Binding is compatible with simultaneous Ena/VASP Homology 1 (EVH1) domain (24) and a C-terminal binding of Ras and does not interfere with GAP activity. Our study Sprouty-related domain (SPR) separated by a central c-Kit bind- points to a potential targeting function of the GAPex subdomain of ing domain (19). EVH1 domains are PH-like protein–protein neurofibromin that is present in all known canonical RasGAPs. interaction modules (25, 26) that bind to proline rich sequence stretches of the type FPPPP (27, 28). Most of the reported in- – cancer | signal transduction | protein protein interaction | RASopathy | teraction partners of Spred1 involve binding the C-terminal SPR nontruncating mutations domain with unclear function or significance (29). Most of the eurofibromatosis type 1 (NF1) is an autosomal dominant Significance Ngenetic disorder with an incidence of 1 in 3,500 newborns and full penetrance. Patients with NF1 have an increased risk of Neurofibromatosis type 1 (NF1) and Legius syndrome (LS) are developing the disease-typical neurofibromas consisting of benign disorders characterized by deregulation of the Ras/MAPK path- and malignant tumors of the nervous system. Additional symptoms way, which is central to a variety of cellular processes, including include characteristic pigmentation anomalies (café au lait mac- growth and differentiation. Both neurofibromin protein and ules, axillary freckling), melanocytic hamartomas of the iris (Lisch Sprouty-related, EVH1 domain-containing protein 1 (Spred1) in- nodules), bone deformations, and learning disabilities, altogether hibit the Ras/MAPK pathway, and loss-of-function alterations in defining a clinical picture that is far from being well understood their encoding genes cause NF1 or LS, respectively. Such alter- and for which no cure is available (1). The tumor suppressor gene ations are also often found in sporadic tumors unrelated to NF1 or NF1 encodes the giant signal regulatory protein neurofibromin and LS. An earlier report described a cellular complex containing is mutated in NF1 patients. The reported alterations (currently neurofibromin and Spred1; the details of these interactions BIOCHEMISTRY >2,000 mutations are listed in the Human Gene Mutation Data- remained unknown. Here we show that the EVH1 domain of base) include splice variants, nonsense and missense mutations, Spred1 binds to the GTPase activating protein-related domain of insertions and deletions, duplications and rearrangements, as well neurofibromin without interfering with its catalytic activity. We as repeat variations (www.hgmd.cf.ac.uk/ac/index.php). About further describe how a disease associated mutation in neuro- 10% of the mutations comprise nontruncating (in-frame) de- fibromin prevents Spred1 binding. letions or missense mutations that are in principle compatible with the translation of the protein product (2). Author contributions: T.D.-M., E.L.M., F.M., and K.S. designed research; T.D.-M. and E.L.M. Neurofibromin is a 320-kDa Ras-specific GTPase activating performed research; K.M. contributed new reagents/analytic tools; T.D.-M., E.L.M., K.M., protein (RasGAP) that uses the central GAP-related domain and F.M. analyzed data; and T.D.-M., E.L.M., F.M., and K.S. wrote the paper. (GRD) to enhance the GTPase activity of the small guanine nu- Reviewers: J.L., University of Florida; and N.R., Cincinnati Children’s Hospital. cleotide binding protein Ras, thereby down-regulating its biological The authors declare no conflict of interest. activity (3–5). The NF1 gene has gained considerable impact as it is Freely available online through the PNAS open access option. frequently mutated in a variety of very aggressive human malig- 1T.D.-M. and E.L.M. contributed equally to this work. nancies, including glioblastoma, lung adenocarcinoma, acute my- 2To whom correspondence may be addressed. Email: [email protected] or eloid leukemia, and ovarian and breast cancers (6–12). Using [email protected]. structural biology approaches, we previously discovered a bipartite This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. module located C-terminal to the GRD that is composed of a 1073/pnas.1607298113/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1607298113 PNAS | July 5, 2016 | vol. 113 | no. 27 | 7497–7502 Downloaded by guest on October 1, 2021 LS-associated mutations found in the SPRED1 gene result in pre- homogeneity and analyzed their interaction with the EVH1 do- mature stop codons, presumably associated with truncated trans- main of Spred1 using an in vitro affinity purification approach. lation products, and the majority of the identified nontruncating Immobilized recombinant Spred1(EVH1) captured all neuro- missense mutations are located within the EVH1 domain (18). fibromin protein variants containing the GRD including the Using MS combined with a tandem affinity purification (TAP) isolated GRD (Fig. 1B). Two LS patient-derived pathogenic approach, neurofibromin was identified as a Spred1-interacting missense mutations T102→R and W31→C (18) located on the protein (30, 31). Stowe and coworkers performed a detailed anal- surface of the EVH1 domain prevented the interaction with ysis using overexpressed as well as endogenous proteins to suggest GRD-containing fragments (24) (Fig. S1). This result was sup- a model for the role of Spred1 in Ras/MAPK inhibition, in which ported and confirmed by analysis of several Flag-tagged neuro- the EVH1 domain serves as the binding site for neurofibromin; the fibromin constructs (Fig. 1A) for their ability to coimmunopre- complex is then translocated to the plasma membrane via the cipitate with Spred1 from human embryonic kidney cell lysates Sprouty domain of Spred1. However, it was not clear from that overexpressing both proteins (Fig. 1C). The observed failure of study which region of the 2,818 residues comprising neurofibromin the constructs containing the N- and C-terminal portions of is necessary for Spred1 recognition and whether the interaction was neurofibromin to bind but not of those containing the GRD direct or mediated by another cellular factor (31). strongly suggests that no other Spred1 binding site is present on In this study, we tested binding of purified recombinant neurofibromin. Membrane localization, which is a requirement Spred1(EVH1) to different segments of neurofibromin that were to inactivate membrane-anchored Ras, is mediated by Spred1/2 prepared following recombinant expression in Escherichia coli or (30). To determine whether membrane recruitment of neuro- in insect cells. We find that the EVH1 domain binds to the GRD fibromin