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Cooperative Regulation of Ras/MAPK Pathway Signaling by Spredl and Neurofibromin: Insights Into the Moiecuiar Basis of Legius Syndrome

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Cooperative Regulation of Ras/MAPK Pathway Signaling by Spredl and Neurofibromin: Insights Into the Moiecuiar Basis of Legius Syndrome Cooperative regulation of Ras/MAPK pathway signaling by Spredl and neurofibromin: Insights into the moiecuiar basis of Legius syndrome by Irma B Range! Alareon DISSERTATION Submitted in partial satisfaction of the requirements for the degree of DOCTOR OF PHILOSOPHY in Biochemistry and Molecular Biology in the GRADUATE DIVISION of the UNIVERSITY OF CALIFORNIA, SAN FRANCISCO ii For the Sprouty family, without whom this work would not have been possible. iii Acknowledgements I would like to thank my advisor, Frank McCormick, for providing an incredible work environment that is both scientifically stimulating and challenging. Despite my having dropped multiple mass spec gels in the sink, and double-blinding myself by mixing up samples, Frank has a unique skill for keeping his students focused on the positive. Thank you to Amy Young, Cynthia Mysinger, Vernon Phan, Jamie Smyth, Jesse Lyons, Mike Nehil, and Ellen Mercado for incredibly useful scientific discussions and support throughout the years. A special thanks to Amy Young, Cynthia Mysinger, and Jacqueline Galeas for baymate support over the years. Thank you to my thesis committee, Kevin Shannon and Allan Balmain for valuable help and advice. Thank you to my parents, my sisters, and my little brother who have supported me in everything I have ever done (or wanted to try) and for having been committed to my success and happiness above all else. I couldn’t have come close to being where I am today without them. Most importantly, thank you to my husband and best friend, with whom I look forward to many happy years. I adore his creativity and his unique and inspiring approach to life. I am thankful beyond words for his support, companionship, love, and his confidence in me. I can’t imagine a world without him. iv Declaration This work was done under the guidance of Dr. Frank McCormick, at the University of California, San Francisco. Portions of chapter 1 of this dissertation were written for a chapter in Advances in Cancer Research: Young, A., Lyons, J., Miller, A.L., Phan, V.T., Rangel Alarcon, I, McCormick, F. (2009) Chapter 1 Ras signaling and therapies. Advances in Cancer Research 102: 1-17. Portions of chapters 3 and 4 of this dissertation are based on a manuscript that will be submitted for publication, entitled “Spred1 and neurofibromin interact to negatively regulate Ras/Raf/MEK/ERK Signaling.” The following co-authors contributed to this work: Ellen L. Mercado, Timothy R. Stowe, Erika L. Bell, Juan A. Oses-Prieto, Hilda Hernandez, Alma L. Burlingame, and Frank McCormick (UCSF Helen Diller Family Comprehensive Cancer Center, San Francisco, California). v Abstract Cooperative regulation of Ras/MAPK pathway signaling by Spred1 and neurofibromin: Insights into the molecular basis of Legius syndrome. Irma B Rangel Alarcon The Ras/Mitogen Activated Protein Kinase (MAPK) plays a critical role in cell signaling downstream of receptor tyrosine kinases. The conserved Sprouty family proteins function as feedback regulators of Ras/MAPK signaling. Loss of function mutations in one family member, SPRED1, cause Legius syndrome, an autosomal dominant human disorder that resembles neurofibromatosis type 1 (NF1). Spred1 functions as a negative regulator of the Ras/MAPK pathway, however, the underlying molecular mechanism is poorly understood. We found Spred1 functioned by reducing Ras-GTP levels; SPRED1 loss-of- function mutations identified in Legius syndrome patients were unable to regulate Ras activity levels. The N-terminal EVH1 domain of Spred1 was found to be crucial for its inhibitory function (point mutants in the EVH1 domain failed to function, and the EVH1 domain alone was sufficient to decrease Ras-GTP levels when localized to the plasma membrane). Thus, we utilized tandem affinity purification of wild-type Spred1 and EVH1 domain mutants found in Legius syndrome to identify novel binding partners of the N-terminus. Analysis of protein complexes by mass spectrometry revealed neurofibromin, the NF1 gene product, as a novel Spred1 interacting protein. Here we show that neurofibromin is a Spred1 binding partner that is necessary for Spred1’s inhibitory function. Spred1 requires neurofibromin to down-regulate Ras-GTP levels, and we find that the interaction with Spred1 is required for neurofibromin localization to the vi plasma membrane. This novel mechanism for neurofibromin regulation leads to a better understanding of the pathophysiology of these two overlapping developmental disorders. vii Table of Contents Acknowledgements...........................................................................................................iii Declaration.........................................................................................................................iv Abstract...............................................................................................................................v Table of Contents...............................................................................................................vi List of Figures...................................................................................................................vii List of Tables.....................................................................................................................ix Chapter 1: Introduction.......................................................................................................1 Chapter 2: Materials and Methods....................................................................................30 Chapter 3: Investigating the mechanism for Spred1 moderation of Ras/MAPK signaling ……………………………………………………………………...................................40 Chapter 4: Identification of a novel Spred1 interacting partner ...................................... 66 Chapter 5: Future Directions……………………………….............................................93 viii List of Figures Chapter 1 Figure 1.1 Ras signaling pathways ………………………………………………... 19 Figure 1.2 RASopathies associated with the dysregulation of various components of the Ras/MAPK signaling pathway …………………………………………………. 20 Figure 1.3 Spred1 and related proteins ……………….…………………………… 21 Chapter 2 Figure 2.1 Tandem affinity purification scheme …………………...……………… 38 Chapter3 Figure 3.1 Spred1 domain structure and Legius syndrome mutations ………….…. 53 Figure 3.2 Spred1 preferentially interacts with activated forms of Raf …………… 54 Figure 3.3 Ectopic expression of Spred1 results in inhibition of the Ras/MAPK signaling pathway ………………………………………………………………… 55 Figure 3.4 Spred1 interacts with multiple isoforms of Ras …………….…………. 56 Figure 3.5 Spred1 interacts with Nras in a GTP dependent manner …………….... 57 Figure 3.6 Spred1 differentially binds Nras versus Hras ………………...…..… 58 Figure 3.7 Spred1 interacts directly with Ras …………………………….………. 59 Figure 3.8 Spred1 suppresses Ras activity ………………………………….…….. 60 Figure 3.9 Overexpression of Spred1 in Ras mutant cell lines does not inhibit Ras/MAPK signaling …………………………………………………………….… 61 Figure 3.10 Legius syndrome SPRED1 mutants are unable to suppress Ras activity ………...………………………………………………………………. 62 ix Figure 3.11 Spred siRNA depletion increases Ras-GTP and phospho ERK levels following EGF stimulation …………………………………………….. 63 Chapter 4 Figure 4.1 Plasma membrane localization is necessary for Spred1’s function ……. 78 Figure 4.2 Spred1’s inhibitory activity resides in the EVH1 domain ………...…… 79 Figure 4.3 Tandem affinity purification of Spred1 and Spred1 truncation mutants . 80 Figure 4.4 Neurofibromin is a novel Spred1 binding partner ………………..……. 81 Figure 4.5 Multiple Spred isoforms interact with neurofibromin ……………..…... 82 Figure 4.6 Ectopic and endogenous neurofibromin can co-precipitate Spred1 ….... 83 Figure 4.7 Neurofibromin is necessary for Spred1’s inhibitory function …….…… 84 Figure 4.8 Neurofibromin is necessary for Spred1’s inhibitory function in NF1-/- cells .…………………………………………………………………..…….. 85 Figure 4.9 Spred1 recruits neurofibromin to the plasma membrane ……………… 87 Figure 4.10 EGF stimulation increases Spred1 and neurofibromin localization to the plasma membrane ……..……………………………………….……… 88 x List of Tables Chapter 4 Table 4.1 SPRED1 mutations identified in Legius syndrome used in this study …….. 89 Table 4.2 Mass spectrometry data for Spred1 and EVH1 protein binding complexes .. 90 xi Chapter 1: Introduction 1 Ras Proteins Ras genes were initially discovered as the transformative factors in the Harvey and Kirsten rat sarcoma retroviruses [1, 2]. Molecular techniques in the late 1970s and early 1980s allowed researchers to identify the ras genes as the initiating genes in experimental transformation and human tumor pathogenesis. The three ras genes were found to encode four highly homologous 21 kD proteins: Hras, Nras, and Kras4B and Kras4A (two proteins that result from alternative splicing) and were subsequently observed to be mutated in many human cancer cell lines [3]. It is now estimated that approximately 30% of all human tumors contain an activating mutation in one of the ras genes [4]. Specific ras oncogenes are preferentially mutated in distinct types of tumors: Kras mutant alleles are commonly found in pancreatic, lung and colon carcinomas, Hras in bladder carcinomas, and Nras in lymphoid malignancies, hematopoietic tumors and malignant melanomas [5]. The Ras proteins are critical regulators of cellular biology, relaying receptor activation to downstream effector pathways that control a diverse array of cellular fates, including proliferation, differentiation, migration,
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