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A Shared Molecular Mechanism Underlies the Human Rasopathies Legius Syndrome and Neurofibromatosis-1

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A Shared Molecular Mechanism Underlies the Human Rasopathies Legius Syndrome and Neurofibromatosis-1 Downloaded from genesdev.cshlp.org on September 27, 2021 - Published by Cold Spring Harbor Laboratory Press RESEARCH COMMUNICATION it has been reported to function both upstream of and A shared molecular mechanism downstream from Ras (Wakioka et al. 2001; King et al. underlies the human 2005). Overexpression of Spred1 can increase Raf’s re- cruitment to the plasma membrane, where it associates rasopathies Legius syndrome with Ras without stimulating Raf activation (Wakioka and Neurofibromatosis-1 et al. 2001). Yet, this mechanism has never been fully refined and fails to explain how Spred1 prevents Raf ac- Irma B. Stowe,1 Ellen L. Mercado,1 tivation. A separate report suggests that Spred1 may pre- Timothy R. Stowe,2 Erika L. Bell,1 vent Ras activation, as evidenced by decreased Ras-GTP 3 3 levels when Spred1 is overexpressed (King et al. 2005). Juan A. Oses-Prieto, Hilda Herna´ndez, Heterozygous germline loss-of-function mutations have 3 1,4 Alma L. Burlingame, and Frank McCormick been identified in SPRED1 in Legius syndrome, a develop- 1 mental disorder that shares a number of phenotypes with Helen Diller Family Comprehensive Cancer Center, University Neurofibromatosis-1 (NF1) (Brems et al. 2007). Both syn- of California at San Francisco, San Francisco, California 94158, 2 dromes are characterized as rasopathies, congenital devel- USA; Department of Neurological Surgery, University of opmental syndromes caused by germline mutations that California at San Francisco, San Francisco, California 94143, affect the Ras/MAPK pathway. Legius syndrome has been USA; 3Department of Pharmaceutical Chemistry, University of characterized as a milder form of NF1, with individuals California at San Francisco, San Francisco, California 94143, USA displaying multiple cafe´-au-lait spots, axillary freckling, and macrocephaly, but lacking other common NF1 man- The Ras/mitogen-activated protein kinase (MAPK) path- ifestations such as Lisch nodules, neurofibromas, osseous way plays a critical role in transducing mitogenic signals lesions, or optic pathway gliomas. Initial experiments re- from receptor tyrosine kinases. Loss-of-function muta- vealed that Spred1 mutations were loss-of-function muta- tions in one feedback regulator of Ras/MAPK signaling, tions, incapable of inhibiting the Ras/MAPK pathway SPRED1 (Sprouty-related protein with an EVH1 domain), (Brems et al. 2007). SPRED1 mutations account for at cause Legius syndrome, an autosomal dominant human least 2% of the pathogenic mutations associated with disorder that resembles Neurofibromatosis-1 (NF1). Spred1 patients clinically diagnosed with NF1 (Brems et al. 2007; functions as a negative regulator of the Ras/MAPK path- Messiaen et al. 2009; Pasmant et al. 2009; Spurlock et al. way; however, the underlying molecular mechanism is 2009; Muram-Zborovski et al. 2010; Denayer et al. 2011; poorly understood. Here we show that neurofibromin, the Laycock-van Spyk et al. 2011; Spencer et al. 2011). NF1 gene product, is a Spred1-interacting protein that is NF1, an autosomal dominant, multisystem disorder that affects approximately one in 3500 individuals, was necessary for Spred1’s inhibitory function. We show that the first disorder found to originate from a component of Spred1 binding induces the plasma membrane localiza- the Ras/MAPK pathway. The NF1 gene product neuro- tion of NF1, which subsequently down-regulates Ras-GTP fibromin negatively regulates Ras signaling by functioning levels. This novel mechanism for the regulation of neuro- as a Ras GTPase-activating protein (RasGAP) to accelerate fibromin provides a molecular bridge for understanding the the hydrolysis of active Ras-GTP to inactive Ras-GDP overlapping pathophysiology of NF1 and Legius syndrome. (Martin et al. 1990; Xu et al. 1990). Loss of neurofibromin leads to hyperactive Ras signaling, as observed by elevated Supplemental material is available for this article. Ras pathway activity in cells (Basu et al. 1992; DeClue et al. Received February 28, 2012; revised version accepted May 1992; Bollag et al. 1996). Despite the identification of 25, 2012. neurofibromin as a RasGAP 20 years ago, the regulation of neurofibromin activity remains poorly understood. Neurofibromin has been reported to be positively and negatively regulated by various phosphorylation events, Spred1 (Sprouty-related protein with an EVH1 domain) but the context of this regulation is largely unknown (Feng functions as a negative regulator of growth factor, cyto- et al. 2004; Mangoura et al. 2006; Leondaritis et al. 2009). In kine, and chemokine-induced ERK activation by specifi- addition, neurofibromin protein levels are negatively regu- cally inhibiting the Ras/Raf/MEK/ERK pathway (Wakioka lated by the ubiquitin proteasome system following growth et al. 2001; Miyoshi et al. 2004; Nonami et al. 2004). Spred1 is comprised of an N-terminal Ena/Vasp homology factor stimulation (Cichowski et al. 2003; McGillicuddy 1 (EVH1) domain, a central c-Kit-binding domain (KBD), et al. 2009). However, the exact regulatory mechanism and a cysteine rich C-terminal Sprouty (SPR) domain. that couples receptor growth factor activation to neuro- Mammals contain three homologs, Spred 1, Spred 2, and fibromin’s suppression of Ras activation remains unclear. Spred 3, each of which can negatively regulate the Ras/ Here we report that Spred1 down-regulates the Ras/ Raf/mitogen-activated protein kinase (MAPK) pathway MAPK pathway through an interaction with the NF1 (Kato et al. 2003; King et al. 2006). However, the precise protein neurofibromin. Importantly, this interaction func- mechanism by which the Spreds act remains unclear, and tions to recruit neurofibromin to the plasma membrane. Furthermore, loss-of-function Spred1 mutants observed in Legius syndrome are either unabletobindneurofibromin [Keywords: Legius syndrome; NF1; signal transduction; Ras/MAPK; or incapable of recruiting it to the membrane. Our data Sprouty] provide evidence for a molecular link between the phe- 4Corresponding author E-mail [email protected] notypically overlapping developmental disorders Legius Article is online at http://www.genesdev.org/cgi/doi/10.1101/gad.190876.112. syndrome and NF1. GENES & DEVELOPMENT 26:1421–1426 Ó 2012 by Cold Spring Harbor Laboratory Press ISSN 0890-9369/12; www.genesdev.org 1421 Downloaded from genesdev.cshlp.org on September 27, 2021 - Published by Cold Spring Harbor Laboratory Press Stowe et al. Results and Discussion S1) might similarly affect Ras activity (Fig. 1C). Interest- ingly, we found that ectopic expression of Spred1-bearing Ectopic expression of Spred1 results in suppression of ERK pathogenic missense mutations in the N-terminal EVH1 activation following acute agonist stimulation by various domain and C-terminal SPR domain are defective in sup- growth factors, including EGF and FGF (Supplemental pressing Ras-GTP levels and inhibiting ERK activation Fig. S1; Wakioka et al. 2001). Spred1 has been described as following growth factor treatment. The truncation mu- functioning both upstream of and downstream from Ras tant M266fsX4, which retains the N-terminal EVH1 domain (Wakioka et al. 2001; King et al. 2005). To address these but lacks the SPR and internal Kit-binding domains, was also discrepancies, we tested the hypothesis that Spred1 acts defective in regulating Ras-GTP and phospho-ERK levels. upstream of Ras by examining whether Spred1 could affect The majority of reported Spred1 mutations produce a cellular Ras-GTP levels following growth factor stimula- premature stop codon, resulting in C-terminally truncated tion (Fig. 1A). Inducible expression of Spred1 in T-REx-293 protein products (Brems et al. 2007; Messiaen et al. 2009; cells significantly reduced Ras-GTP levels following EGF Pasmant et al. 2009; Spurlock et al. 2009; Muram-Zborovski stimulation, suggesting that Spred1 might interfere with et al. 2010; Denayer et al. 2011; Laycock-van Spyk et al. Ras regulation. Furthermore, ectopic expression of Spred1 suppressed downstream MAPK signaling, as evidenced by 2011; Spencer et al. 2011). As the SPR domain has previously decreases in phospho-Raf and phospho-ERK. To further been reported to target Spred1 to the plasma membrane via investigate the hypothesis that Spred1 affects Ras regula- interactions with phospholipids and caveolin-1 (Lim et al. tion, we depleted Spred1 from a cancer cell line with 2002; Nonami et al. 2005), we surmised that the truncated reportedly high Spred1 protein levels (Fig. 1B; Li et al. mutants were unable to function as a result of mislocaliza- 2010). Depletion of Spred1 from the PC3 prostate cell line tion. To test this hypothesis, we artificially localized the resulted in elevated levels of Ras-GTP following EGF Spred1 C-terminal truncation mutant (M266fsX4) to the stimulation. Taken together, these data support the model membrane by fusing it to the membrane targeting CAAX that Spred1 can function upstream of Ras to negatively motif of Kras4B (Fig. 2A). Immunofluorescence microscopy regulate its activation, although we cannot exclude the revealed that the mutant Spred1 protein localized primarily possibility that Spred1 may also independently function to the cytoplasm, whereas the mutant CAAX fusion pro- downstream from Ras activation as well. tein was localized to the plasma membrane (Fig. 2B). The We next asked whether SPRED1 loss-of-function mu- mutant CAAX protein also showed a restored ability to tations found in Legius syndrome (Supplemental Table reduce EGF-induced Ras activation and inhibit ERK activa- tion (Fig.
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