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• W3003641280 abstract "•PTPRA inhibits ligand (GDNF-GFRα1)-mediated RET activity on Ras-MAPK signaling axis•PTPRA dephosphorylate RET on key functional phosphotyrosine sites•PTPRA catalytic (PTPase) domain 1 regulates RET-driven signaling•PTPRA suppresses RET oncogenic mutant MEN2A in both Ras-MAPK and cell invasion models The RET proto-oncogene encodes receptor tyrosine kinase, expressed primarily in tissues of neural crest origin. De-regulation of RET signaling is implicated in several human cancers. Recent phosphatome interactome analysis identified PTPRA interacting with the neurotrophic factor (GDNF)-dependent RET-Ras-MAPK signaling-axis. Here, by identifying comprehensive interactomes of PTPRA and RET, we reveal their close physical and functional association. The PTPRA directly interacts with RET, and using the phosphoproteomic approach, we identify RET as a direct dephosphorylation substrate of PTPRA both in vivo and in vitro. The protein phosphatase domain-1 is indispensable for the PTPRA inhibitory role on RET activity and downstream Ras-MAPK signaling, whereas domain-2 has only minor effect. Furthermore, PTPRA also regulates the RET oncogenic mutant variant MEN2A activity and invasion capacity, whereas the MEN2B is insensitive to PTPRA. In sum, we discern PTPRA as a novel regulator of RET signaling in both health and cancer. The RET proto-oncogene encodes receptor tyrosine kinase, expressed primarily in tissues of neural crest origin. De-regulation of RET signaling is implicated in several human cancers. Recent phosphatome interactome analysis identified PTPRA interacting with the neurotrophic factor (GDNF)-dependent RET-Ras-MAPK signaling-axis. Here, by identifying comprehensive interactomes of PTPRA and RET, we reveal their close physical and functional association. The PTPRA directly interacts with RET, and using the phosphoproteomic approach, we identify RET as a direct dephosphorylation substrate of PTPRA both in vivo and in vitro. The protein phosphatase domain-1 is indispensable for the PTPRA inhibitory role on RET activity and downstream Ras-MAPK signaling, whereas domain-2 has only minor effect. Furthermore, PTPRA also regulates the RET oncogenic mutant variant MEN2A activity and invasion capacity, whereas the MEN2B is insensitive to PTPRA. In sum, we discern PTPRA as a novel regulator of RET signaling in both health and cancer. Protein tyrosine phosphorylation is a prime eukaryotic regulatory step for intracellular signal transduction and is maintained by opposing activities of protein tyrosine kinases (PTKs) and phosphatases (PTPs). Strikingly, the number of PTPs (107) encoded by the human genome roughly matches that of PTKs (90), indicating that PTPs might also have equivalent functional complexity and specificity as their kinase counterparts. However, unlike PTKs, biological circuitry and activity control mechanisms of many PTPs are still undefined. Recently, through global systematic interactome analysis of human protein phosphatases, we have demonstrated that GDNF (glial cell line-derived neurotrophic factor) and GRB2 (growth factor receptor-bound protein 2) form a complex with the protein tyrosine phosphatase receptor-type A (PTPRA) (Yadav et al., 2017Yadav L. Tamene F. Goos H. Van Drogen A. Katainen R. Aebersold R. Gstaiger M. Varjosalo M. Systematic analysis of human protein phosphatase interactions and dynamics.Cell Syst. 2017; 4: 430-444.e5Abstract Full Text Full Text PDF PubMed Scopus (37) Google Scholar). To our interest, GDNF acts as a key homodimeric neurotrophic factor family ligand, which in conjunction with GDNF α-receptors (GFRα1-4; glycosylphosphatidylinositol-anchored proteins) incite RET (REarranged during Transfection) receptor tyrosine kinase via dimerization to prompt Ras-MAPK (mitogen-activated protein kinase) cascade and other signaling pathways (Airaksinen and Saarma, 2002Airaksinen M.S. Saarma M. The GDNF family: signalling, biological functions and therapeutic value.Nat. Rev. Neurosci. 2002; 3: 383-394Crossref PubMed Scopus (1387) Google Scholar, Arighi et al., 2005Arighi E. Borrello M.G. Sariola H. RET tyrosine kinase signaling in development and cancer.Cytokine Growth Factor Rev. 2005; 16: 441-467Crossref PubMed Scopus (331) Google Scholar). Notably, only a few protein phosphatases such as PTPRF (Leukocyte common antigen-related; LAR), PTN6 (Src homology region 2 domain-containing phosphatase-1; SHP1), and PTN11 (Src homology region 2 domain-containing phosphatase-2; SHP2) have been suggested to balance the phosphorylation and oncogenic activity of RET (Hennige et al., 2001Hennige A.M. Lammers R. Hoppner W. Arlt D. Strack V. Teichmann R. Machicao F. Ullrich A. Haring H.U. Kellerer M. Inhibition of Ret oncogene activity by the protein tyrosine phosphatase SHP1.Endocrinology. 2001; 142: 4441-4447Crossref PubMed Scopus (31) Google Scholar, Perrinjaquet et al., 2010Perrinjaquet M. Vilar M. Ibanez C.F. Protein-tyrosine phosphatase SHP2 contributes to GDNF neurotrophic activity through direct binding to phospho-Tyr687 in the RET receptor tyrosine kinase.J. Biol. Chem. 2010; 285: 31867-31875Crossref PubMed Scopus (29) Google Scholar, Qiao et al., 2001Qiao S. Iwashita T. Furukawa T. Yamamoto M. Sobue G. Takahashi M. Differential effects of leukocyte common antigen-related protein on biochemical and biological activities of RET-MEN2A and RET-MEN2B mutant proteins.J. Biol. Chem. 2001; 276: 9460-9467Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar). Similar to PTPRF, PTPRA is a membrane-bound “receptor-type” protein tyrosine phosphatase, with a highly glycosylated ectodomain, a single membrane-spanning region, and two intracellular catalytic phosphatase domains (PTPase; membrane-proximal D1 and -distal D2) (Daum et al., 1994Daum G. Regenass S. Sap J. Schlessinger J. Fischer E.H. Multiple forms of the human tyrosine phosphatase RPTP alpha. Isozymes and differences in glycosylation.J. Biol. Chem. 1994; 269: 10524-10528Abstract Full Text PDF PubMed Google Scholar, Wang and Pallen, 1991Wang Y. Pallen C.J. The receptor-like protein tyrosine phosphatase HPTP alpha has two active catalytic domains with distinct substrate specificities.EMBO J. 1991; 10: 3231-3237Crossref PubMed Scopus (102) Google Scholar, Wu et al., 1997Wu L. Buist A. Den Hertog J. Zhang Z.Y. Comparative kinetic analysis and substrate specificity of the tandem catalytic domains of the receptor-like protein-tyrosine phosphatase alpha.J. Biol. Chem. 1997; 272: 6994-7002Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar). Although ubiquitously expressed, it is particularly abundant in the brain (neurons and glial cells) and insulin target tissues, where it induces cell differentiation, migration, activation of voltage-gated potassium channels, and insulin secretion (Chen et al., 2009Chen S.C. Khanna R.S. Bessette D.C. Samayawardhena L.A. Pallen C.J. Protein tyrosine phosphatase-alpha complexes with the IGF-I receptor and undergoes IGF-I-stimulated tyrosine phosphorylation that mediates cell migration.Am. J. Physiol. Cell Physiol. 2009; 297: C133-C139Crossref PubMed Scopus (10) Google Scholar, Imbrici et al., 2000Imbrici P. Tucker S.J. D'adamo M.C. Pessia M. Role of receptor protein tyrosine phosphatase alpha (RPTPalpha) and tyrosine phosphorylation in the serotonergic inhibition of voltage-dependent potassium channels.Pflugers Arch. 2000; 441: 257-262Crossref PubMed Scopus (26) Google Scholar, Kaplan et al., 1990Kaplan R. Morse B. Huebner K. Croce C. Howk R. Ravera M. Ricca G. Jaye M. Schlessinger J. Cloning of three human tyrosine phosphatases reveals a multigene family of receptor-linked protein-tyrosine-phosphatases expressed in brain.Proc. Natl. Acad. Sci. U S A. 1990; 87: 7000-7004Crossref PubMed Scopus (153) Google Scholar, Kapp et al., 2003Kapp K. Metzinger E. Kellerer M. Haring H.U. Lammers R. The protein tyrosine phosphatase alpha modifies insulin secretion in INS-1E cells.Biochem. Biophys. Res. Commun. 2003; 311: 361-364Crossref PubMed Scopus (10) Google Scholar, Norris et al., 1997Norris K. Norris F. Kono D.H. Vestergaard H. Pedersen O. Theofilopoulos A.N. Moller N.P. Expression of protein-tyrosine phosphatases in the major insulin target tissues.FEBS Lett. 1997; 415: 243-248Crossref PubMed Scopus (51) Google Scholar, Petrone et al., 2003Petrone A. Battaglia F. Wang C. Dusa A. Su J. Zagzag D. Bianchi R. Casaccia-Bonnefil P. Arancio O. Sap J. Receptor protein tyrosine phosphatase alpha is essential for hippocampal neuronal migration and long-term potentiation.EMBO J. 2003; 22: 4121-4131Crossref PubMed Scopus (69) Google Scholar). Nevertheless, several previous studies have disclosed its critical role as a main positive regulator of Src family kinases (Fyn, FAK, and Src) in cell growth and oncogenic transformation (Huang et al., 2011Huang J. Yao L. Xu R. Wu H. Wang M. White B.S. Shalloway D. Zheng X. Activation of Src and transformation by an RPTPalpha splice mutant found in human tumours.EMBO J. 2011; 30: 3200-3211Crossref PubMed Scopus (23) Google Scholar, Tremper-Wells et al., 2010Tremper-Wells B. Resnick R.J. Zheng X. Holsinger L.J. Shalloway D. Extracellular domain dependence of PTPalpha transforming activity.Genes Cells. 2010; 15: 711-724Crossref PubMed Scopus (12) Google Scholar, Zheng et al., 2002Zheng X.M. Resnick R.J. Shalloway D. Mitotic activation of protein-tyrosine phosphatase alpha and regulation of its Src-mediated transforming activity by its sites of protein kinase C phosphorylation.J. Biol. Chem. 2002; 277: 21922-21929Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar). Conforming with this, PTPRA has been suggested to play a dual role in regulating EGFR kinase signaling via Src dephosphorylation and activation (Yao et al., 2017Yao Z. Darowski K. St-Denis N. Wong V. Offensperger F. Villedieu A. Amin S. Malty R. Aoki H. Guo H. et al.A global analysis of the receptor tyrosine kinase-protein phosphatase interactome.Mol. Cell. 2017; 65: 347-360Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar). Despite these findings, relatively few signaling pathways (cell adhesion- and integrin-mediated processes) and cellular targets (or substrates) have been suggested that could unravel the PTPRA-mediated regulation of cellular signaling (Bodrikov et al., 2005Bodrikov V. Leshchyns'ka I. Sytnyk V. Overvoorde J. Den Hertog J. Schachner M. RPTPalpha is essential for NCAM-mediated p59fyn activation and neurite elongation.J. Cell Biol. 2005; 168: 127-139Crossref PubMed Scopus (109) Google Scholar, Truffi et al., 2014Truffi M. Dubreuil V. Liang X. Vacaresse N. Nigon F. Han S.P. Yap A.S. Gomez G.A. Sap J. RPTPalpha controls epithelial adherens junctions, linking E-cadherin engagement to c-Src-mediated phosphorylation of cortactin.J. Cell Sci. 2014; 127: 2420-2432Crossref PubMed Scopus (23) Google Scholar, Yao et al., 2017Yao Z. Darowski K. St-Denis N. Wong V. Offensperger F. Villedieu A. Amin S. Malty R. Aoki H. Guo H. et al.A global analysis of the receptor tyrosine kinase-protein phosphatase interactome.Mol. Cell. 2017; 65: 347-360Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar). In addition to the matching cellular localization, the RET and PTPRA tissue expression also coincide. RET is distinctly expressed in neural tissues (brain and enteric nervous system) and in the developing kidney, where it instigates axonal guidance, neuronal survival, and ureteric bud morphogenesis (Pachnis et al., 1993Pachnis V. Mankoo B. Costantini F. Expression of the c-ret proto-oncogene during mouse embryogenesis.Development. 1993; 119: 1005-1017Crossref PubMed Google Scholar). Its malfunctioning is described in neuroendocrine tumors and diseases (neurocristopathies) such as renal cell carcinoma, multiple endocrine neoplasia type 2 (MEN2), neuroblastoma, and Parkinson's disease (Drinkut et al., 2016Drinkut A. Tillack K. Meka D.P. Schulz J.B. Kugler S. Kramer E.R. Ret is essential to mediate GDNF's neuroprotective and neuroregenerative effect in a Parkinson disease mouse model.Cell Death Dis. 2016; 7: e2359Crossref PubMed Scopus (41) Google Scholar, Mulligan, 2014Mulligan L.M. RET revisited: expanding the oncogenic portfolio.Nat. Rev. Cancer. 2014; 14: 173-186Crossref PubMed Scopus (306) Google Scholar, Schedl, 2007Schedl A. Renal abnormalities and their developmental origin.Nat. Rev. Genet. 2007; 8: 791-802Crossref PubMed Scopus (274) Google Scholar). For example, in MEN2 syndrome, RET carries numerous gain-of-function mutations in extracellular (C634W; MEN2A) and catalytic (M918T; MEN2B) domains, which leads to aberrant kinase activation and, eventually, to pheochromocytomas and medullary thyroid carcinomas (Eng and Mulligan, 1997Eng C. Mulligan L.M. Mutations of the RET proto-oncogene in the multiple endocrine neoplasia type 2 syndromes, related sporadic tumours, and Hirschsprung disease.Hum. Mutat. 1997; 9: 97-109Crossref PubMed Scopus (205) Google Scholar, Santoro et al., 2004Santoro M. Melillo R.M. Carlomagno F. Vecchio G. Fusco A. Minireview: ret: normal and abnormal functions.Endocrinology. 2004; 145: 5448-5451Crossref PubMed Scopus (141) Google Scholar). Importantly, GDNF-GFRα1-activated RET is autophosphorylated at discrete intracellular tyrosine-sites, Y981, Y1015, Y1062, and Y1096 (Y1096 found only in RET51 isoform), which provide docking sites for downstream adaptors or effectors (Src, SHC, GRB2, Enigma, and DOK proteins) and coordinate four key signaling routes: Ras-MAPK, PI3K-AKT, Src, and PLC-γ pathways (Amoresano et al., 2005Amoresano A. Incoronato M. Monti G. Pucci P. De Franciscis V. Cerchia L. Direct interactions among Ret, GDNF and GFRalpha1 molecules reveal new insights into the assembly of a functional three-protein complex.Cell Signal. 2005; 17: 717-727Crossref PubMed Scopus (34) Google Scholar, Besset et al., 2000Besset V. Scott R.P. Ibanez C.F. Signaling complexes and protein-protein interactions involved in the activation of the Ras and phosphatidylinositol 3-kinase pathways by the c-Ret receptor tyrosine kinase.J. Biol. Chem. 2000; 275: 39159-39166Abstract Full Text Full Text PDF PubMed Scopus (197) Google Scholar, Coulpier et al., 2002Coulpier M. Anders J. Ibanez C.F. Coordinated activation of autophosphorylation sites in the RET receptor tyrosine kinase: importance of tyrosine 1062 for GDNF mediated neuronal differentiation and survival.J. Biol. Chem. 2002; 277: 1991-1999Abstract Full Text Full Text PDF PubMed Scopus (90) Google Scholar, Goodman et al., 2014Goodman K.M. Kjaer S. Beuron F. Knowles P.P. Nawrotek A. Burns E.M. Purkiss A.G. George R. Santoro M. Morris E.P. et al.RET recognition of GDNF-GFRalpha1 ligand by a composite binding site promotes membrane-proximal self-association.Cell Rep. 2014; 8: 1894-1904Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar, Melillo et al., 2001Melillo R.M. Santoro M. Ong S.H. Billaud M. Fusco A. Hadari Y.R. Schlessinger J. Lax I. Docking protein FRS2 links the protein tyrosine kinase RET and its oncogenic forms with the mitogen-activated protein kinase signaling cascade.Mol. Cell Biol. 2001; 21: 4177-4187Crossref PubMed Scopus (108) Google Scholar). Among these sites, autophosphorylation of Y1062 is critical for the initiation of Ras-MAPK (GRB2-SOS complex) and PI3K-AKT (GRB2-GAB1 complex) relays in response to GDNF-GFRα1 co-complex during neuronal survival and proliferation (Besset et al., 2000Besset V. Scott R.P. Ibanez C.F. Signaling complexes and protein-protein interactions involved in the activation of the Ras and phosphatidylinositol 3-kinase pathways by the c-Ret receptor tyrosine kinase.J. Biol. Chem. 2000; 275: 39159-39166Abstract Full Text Full Text PDF PubMed Scopus (197) Google Scholar, Coulpier et al., 2002Coulpier M. Anders J. Ibanez C.F. Coordinated activation of autophosphorylation sites in the RET receptor tyrosine kinase: importance of tyrosine 1062 for GDNF mediated neuronal differentiation and survival.J. Biol. Chem. 2002; 277: 1991-1999Abstract Full Text Full Text PDF PubMed Scopus (90) Google Scholar, Kawamoto et al., 2004Kawamoto Y. Takeda K. Okuno Y. Yamakawa Y. Ito Y. Taguchi R. Kato M. Suzuki H. Takahashi M. Nakashima I. Identification of RET autophosphorylation sites by mass spectrometry.J. Biol. Chem. 2004; 279: 14213-14224Crossref PubMed Scopus (64) Google Scholar). To decipher the GDNF-GFRα1-mediated RET-Ras-MAPK signaling-axis regulation by PTPRA, we mapped in-depth molecular interactions and mechanisms involved. Additionally, we focused on the plausible anti-cancer function of PTPRA and, especially, on its role in the regulation of the two oncogenic RET mutants MEN2A (C634W) and MEN2B (M918T) in cancer cessation. The possibility to modulate the oncogenic effects of MEN2A or MEN2B via regulating PTPRA activity would be intriguing and could likely offer novel therapeutic avenues for treating MEN2-type tumors. To expand the analysis of the functional cross talk between PTPRA and the GDNF-induced RET signaling, we applied both affinity purification-mass spectrometry (AP-MS) and proximity-dependent biotin identification (BioID) interaction proteomic approaches, now, using GDNF, RET, and GRB2 as bait proteins (Liu et al., 2018Liu X. Salokas K. Tamene F. Jiu Y. Weldatsadik R.G. Ohman T. Varjosalo M. An AP-MS- and BioID-compatible MAC-tag enables comprehensive mapping of protein interactions and subcellular localizations.Nat. Commun. 2018; 9: 1188Crossref PubMed Scopus (76) Google Scholar, Yadav et al., 2017Yadav L. Tamene F. Goos H. Van Drogen A. Katainen R. Aebersold R. Gstaiger M. Varjosalo M. Systematic analysis of human protein phosphatase interactions and dynamics.Cell Syst. 2017; 4: 430-444.e5Abstract Full Text Full Text PDF PubMed Scopus (37) Google Scholar). The AP-MS allows purification of the intact protein complexes and estimation of their stoichiometry, whereas the BioID enables capturing of extremely transient and close-by interactions (Figure 1A). Hence, for analysis, GDNF, RET, and GRB2 were subcloned into StrepIII-HA and BioID vectors, their corresponding stable, transient, and close-proximity interactors were purified using Strep-Tactin resin, and the interactors were identified with liquid chromatography-mass spectrometry (LC-MS) (Figure 1A) (Liu et al., 2018Liu X. Salokas K. Tamene F. Jiu Y. Weldatsadik R.G. Ohman T. Varjosalo M. An AP-MS- and BioID-compatible MAC-tag enables comprehensive mapping of protein interactions and subcellular localizations.Nat. Commun. 2018; 9: 1188Crossref PubMed Scopus (76) Google Scholar, Yadav et al., 2017Yadav L. Tamene F. Goos H. Van Drogen A. Katainen R. Aebersold R. Gstaiger M. Varjosalo M. Systematic analysis of human protein phosphatase interactions and dynamics.Cell Syst. 2017; 4: 430-444.e5Abstract Full Text Full Text PDF PubMed Scopus (37) Google Scholar). Interestingly, the PTPRA and RET interactomes shared many core proteins that operate in various cell growth events (Figure 1B and Table S1). For example, Ras-MAPK pathway commencing cell-surface receptors (IGF1R, TGFR1, EGFR, and ERBB2), intracellular bona fide docking proteins (SOS1, SOS2, Src, SHB, SHC1, GAB1, GRB2, and FRS2), and other regulators (MARK3, CRK, and MERL) were identified not only in complex with RET but also with PTPRA (Figure 1B). More so, through extended AP-MS analysis of GRB2, as well as of GDNF, we confirmed GDNF-RET, GRB2-PTPRA, and GRB2-SHC1-SOS1-SOS2 associations (Table S1). Consistent with these results, previous studies have described that GDNF-activated RET promotes Ras-MAPK activation, which is essential for development of nervous system (enteric and brain), spermatogenesis, and kidney during embryogenesis (Costantini and Shakya, 2006Costantini F. Shakya R. GDNF/Ret signaling and the development of the kidney.Bioessays. 2006; 28: 117-127Crossref PubMed Scopus (229) Google Scholar, Li et al., 2006Li L. Su Y. Zhao C. Zhao H. Liu G. Wang J. Xu Q. The role of Ret receptor tyrosine kinase in dopaminergic neuron development.Neuroscience. 2006; 142: 391-400Crossref PubMed Scopus (19) Google Scholar, Soba et al., 2015Soba P. Han C. Zheng Y. Perea D. Miguel-Aliaga I. Jan L.Y. Jan Y.N. The Ret receptor regulates sensory neuron dendrite growth and integrin mediated adhesion.Elife. 2015; 4https://doi.org/10.7554/eLife.05491Crossref PubMed Scopus (31) Google Scholar, Xiao et al., 2015Xiao Q. Rongfei W. Lingqiang Z. Fuchu H. The roles of signaling pathways in regulating kidney development.Yi Chuan. 2015; 37: 1-7PubMed Google Scholar). Noticeably, we have also retrieved PTPRA interaction with other receptor tyrosine kinases (RTKs) EGFR and ERBB2 (Figure 1B and Table S1), where PTPRA was earlier shown to dephosphorylate EGFR and subject a positive effect on downstream Ras-MAPK signaling through Src activation (Yao et al., 2017Yao Z. Darowski K. St-Denis N. Wong V. Offensperger F. Villedieu A. Amin S. Malty R. Aoki H. Guo H. et al.A global analysis of the receptor tyrosine kinase-protein phosphatase interactome.Mol. Cell. 2017; 65: 347-360Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar). Therefore, we sought to check the specificity of the derived PTPRA and RET interactomes by comparing them with that of EGFR and ERBB2 along with IGF1R kinase, related to RET in neural oncogenesis (Denardo et al., 2013Denardo B.D. Holloway M.P. Ji Q. Nguyen K.T. Cheng Y. Valentine M.B. Salomon A. Altura R.A. Quantitative phosphoproteomic analysis identifies activation of the RET and IGF-1R/IR signaling pathways in neuroblastoma.PLoS One. 2013; 8: e82513Crossref PubMed Scopus (21) Google Scholar). For this purpose, both AP-MS and BioID approaches were applied to draw the proteomes of these RTKs: EGFR (219 interactions), ERBB2 (111 interactions), and IGF1R (209 interactions) (Salokas et al., Unpublished Data). Upon comparison, PTPRA exhibited <15% common interactions with these RTKs, whereas with RET it shared nearly 49% interactions (Figure S1A and Table S1). Nevertheless, within the Ras-MAPK module, ∼7.3 (average) interactions reoccurred in EGFR, ERBB2, and IGF1R proteomes (Figure S1B). Intriguingly, RET interactome also differed substantially from these RTKs with a similarity of about 11% with EGFR, 5% with ERBB2, and 10% with IGF1R, suggesting their characteristic signaling complexes (Figure S1C). Hence, this comparative analysis data not only verified the distinctiveness of our PTPRA and RET interactomes but also indicated cross talk of PTPRA in regulating RET-mediated Ras-MAPK signaling, by virtue of their converging interaction frameworks (PTPRA-EGFR-FRS2-GRB2-GAB1 and RET-FRS2-GRB2-GAB1-SHC1) (Figure 1B and Table S1). Additionally, overlap of the PTPRA and RET interactomes was also detected with a large cohort of proteins such as UCN5(B-C), NRP1, BASP1, CERS2, VANG2, MARK2, and TULP3, linked to varied aspects of neuronal development, axon guidance, pathfinding, neuronal polarization, and pattern (axis) formation (Figure 1B and Table S1) (Chen et al., 2006Chen Y.M. Wang Q.J. Hu H.S. Yu P.C. Zhu J. Drewes G. Piwnica-Worms H. Luo Z.G. Microtubule affinity-regulating kinase 2 functions downstream of the PAR-3/PAR-6/atypical PKC complex in regulating hippocampal neuronal polarity.Proc. Natl. Acad. Sci. 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Independent mutations in mouse Vangl2 that cause neural tube defects in looptail mice impair interaction with members of the Dishevelled family.J. Biol. Chem. 2004; 279: 52703-52713Crossref PubMed Scopus (129) Google Scholar). Although many of these biological functions are linked to RET, much less is known about PTPRA in regulating these processes. Moreover, the gene expression pattern of PTPRA shows its high levels in the neuroendocrine tissues of the nervous system, kidney, thyroid, and pituitary gland, along with the tumors derived from these tissues (Figure S2). These include astrocytoma, glioblastoma (GBM), oligodendroglioma, mixed glioma, nephroblastoma, and thyroid carcinoma, further implicating the importance of PTPRA in neural development (Figure S2). Therefore, collectively, as our proteomic results point toward physical and functional interaction between PTPRA and RET, we set out to study if RET activity would be PTPRA regulated and if RET would be a direct substrate of PTPRA. The activation of RET by GDNF-GFRα1 complex is the first event in the activation of the Ras-MAPK signaling and acts as a catalyst for the downstream relay. Therefore, to determine the effect of PTPRA on this pathway, we developed a RET-Ras-MAPK activation detection system in HEK293 cells, which included StrepIII-HA-tagged RET (RET9), Elk1-Gal4-binding domain (GBD) effector (pGBD-Elk1), and Gal4-activation domain (GAD) containing Firefly luciferase (pGAD-FR-Luc) reporter along with Renilla luciferase (phR-Luc) control reporter (Figure 2A). Upon activation by GDNF-GFRα1, the reporter system shows >5-fold pathway activity induction. Since HEK293 cells exhibit undetectable levels of endogenously expressed PTPRA (https://amp.pharm.mssm.edu › Harmonizome and Geiger et al., 2012Geiger T. Wehner A. Schaab C. Cox J. Mann M. Comparative proteomic analysis of eleven common cell lines reveals ubiquitous but varying expression of most proteins.Mol. Cell Proteomics. 2012; 11 (M111 014050)Crossref Scopus (543) Google Scholar), this system was then used for assessing the outcomes of transfecting increasing amounts of PTPRA (0, 10, 25, and 50 ng), alongside GFP control, on the RET-Ras-MAPK pathway activity in the presence or absence of soluble GDNF-GFRα1 (100–500 ng/mL; 24 h) ligand complex (Figure 2B). The maximal inhibition on the ligand-activated pathway (orange bar) was achieved with transfection of 50 ng PTPRA (∼2-fold, p = 0.0001) (Figure 2B). Notably, the basal pathway activity (blue bar) was also restricted to a similar extent (∼1.5-fold) (Figure 2B). The detected inhibitory role of PTPRA in RET-Ras-MAPK cascade was further verified using MG87RET reporter fibroblast cells stably expressing RET (Eketjall et al., 1999Eketjall S. Fainzilber M. Murray-Rust J. Ibanez C.F. Distinct structural elements in GDNF mediate binding to GFRalpha1 and activation of the GFRalpha1-c-Ret receptor complex.EMBO J. 1999; 18: 5901-5910Crossref PubMed Scopus (105) Google Scholar). Even under steady RET levels, the PTPRA expression moderated (1.9- to 2.4-fold) the MAPK activation to nearly comparable extents (Figure 2C). This not only validated the attained HEK293 luciferase-reporter assay results but also affirmed the reliability and usability of the RET-Ras-MAPK activation detection system in transiently transfected HEK293 cells. We then followed up the ramification of PTPRA expression on subsequent signaling target MAPKs (ERK1 and ERK2). To do so, HEK293-MSR cell lysates, containing RET (StrepIII-HA-tagged) and increasing amounts of wild-type PTPRA (V5-tagged), were prepared in the absence and presence (15 min stimulation) of GDNF-GFRα1 ligands. The immunoblotting with site-specific p44/42 MAPK (T202/Y204) antibody shows that the phosphorylation of endogenous ERKs (1 and 2) was readily induced by the ligand-activated RET and expression of PTPRA potentiated their phosphorylation, which concluded the PTPRA-facilitated RET-Ras-MAPK inhibition (Figure 3A). Furthermore, from PTPRA and RET complex analysis, we have observed their strong interaction with GRB2, an adaptor signaling protein (Table S1). Previously, GRB2, in complex with other docking (adaptor) proteins including Src kinase, has been reported to bind RET and harmonize the Ras-MAPK cascade (Alberti et al., 1998Alberti L. Borrello M.G. Ghizzoni S. Torriti F. Rizzetti M.G. Pierotti M.A. Grb2 binding to the different isoforms of Ret tyrosine kinase.Oncogene. 1998; 17: 1079-1087Crossref PubMed Scopus (76) Google Scholar, Ohiwa et al., 1997Ohiwa M. Murakami H. Iwashita T. Asai N. Iwata Y. Imai T. Funahashi H. Takagi H. Takahashi M. Characterization of Ret-Shc-Grb2 complex induced by GDNF, MEN 2A, and MEN 2B mutations.Biochem. Biophys. Res. Commun. 1997; 237: 747-751Crossref PubMed Scopus (53) Google Scholar), whereas PTPRA could sequester the SH2 domain of not only GRB2 but also of Src through its" @default.
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• W3003641280 title "PTPRA Phosphatase Regulates GDNF-Dependent RET Signaling and Inhibits the RET Mutant MEN2A Oncogenic Potential" @default.
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