The Gtpase-Activating Protein Rap1gap: a New Player to Modulate Ret Signaling

The GTPase-activating protein Rap1GAP: A new player to modulate Ret signaling

Gustavo Paratcha1, 2, Fernanda Ledda1, 2

1Division of Molecular and Cellular Neuroscience, Institute of Cellular Biology and Neuroscience Prof. Dr. E. De Robertis (IBCN)- CONICET, School of Medicine, University of Buenos Aires, Buenos Aires, Argentina; 2Laboratory of Molecular and Cellular Neu- roscience, Department of Neuroscience, Karolinska Institute, Stockholm, Sweden Cell Research (2011) 21: 217-219. doi:10.1038/cr.2010.143; published online 19 October 2010

Glial cell line-derived neurotrophic ent signaling pathways, including the active GTP-bound state. While guanine factor (GDNF) plays a critical role Ras-Raf-MAPK (ERK1/2) cascade, the nucleotide exchange factors (GEFs) in orchestrating the development and phosphatidylinositol-3-kinase (PI3K)- stimulate the exchange of GDP to GTP maintenance of different populations of Akt, the PLC-γ and the Src signaling to generate the activated form of Rap1, central and peripheral neurons. GDNF pathways [3]. GTPases activating proteins, like Rap- was initially discovered by its abil- To gain a better understanding of 1GAP, increase the intrinsic rate of GTP ity to promote the survival of ventral Ret signaling, Jiao et al. [4] performed hydrolysis inactivating Rap1. Thus the midbrain dopaminergic neurons [1]. In a yeast two-hybrid assay to screen for activation state of Rap1 depends on addition, GDNF promotes the survival novel Ret interactors using the intracel- the balance between Rap1GEFs and and control the differentiation of motor lular domain of Ret as bait. In this paper, Rap1GAPs. neurons and many peripheral neurons, the authors describe the identification Despite considerable advances in including sensory and sympathetic neu- of Rap1GAP, a GTPase-activating understanding the signaling pathways rons. Moreover, it is required to induce protein (GAP) for Rap1, as a novel through which Ret controls neuronal proliferation, migration and survival of Ret-binding protein. Interestingly, this survival and proliferation, the molecular enteric neurons. GDNF promotes these result provides the first evidence for a determinants underlying Ret-mediated trophic effects through the activation direct interaction between Rap1GAP neuronal differentiation are still much of the receptor tyrosine kinase (RTK) with a RTK. In this elegant study, the less known. It has been established that Ret. A distinctive feature of the receptor authors also demonstrate that endog- ERK1/2 activation by growth factors complex for GDNF is the requirement enous Rap1GAP specifically associate can induce cell growth and/or differen- of two types of receptors, one special- with Ret in midbrain and spinal cord in tiation. Although transient activation of ized in GDNF binding, represented vivo. By systematically comparing five ERK1/2 is thought to promote prolifera- by the glycosyl-phosphatidyl inositol different tyrosine phosphorylation sites tion, its sustained activation appears to (GPI)-linked co-receptor GFRα1, and (Tyr905, Tyr981, Tyr1015, Tyr1062 and induce morphological differentiation. another involved in transmembrane Tyr1096) within the intracellular do- In the PC12 cell line, the neurotrophin signaling, represented by the RTK Ret main of Ret (RetICD), Jiao et al. identify nerve growth factor (NGF) promotes or the neural cell adhesion molecule the Tyr981 as the crucial docking site a robust neurite outgrowth through a NCAM [1, 2]. Following homodi- for Ret-Rap1GAP association. sustained ERK1/2 activation mediated meric GDNF binding to GFRα1, Ret Rap1 is a Ras-like small GTPase, by the activation of Rap1 [6]. Taking becomes dimerized and tyrosine phos- which is activated by different extra- advantages of this cellular model, Jiao phorylated, and triggers many differ- cellular factors and has been involved et al. show that Rap1GAP inhibits in diverse processes, such as cell adhe- GDNF/Ret-induced neurite outgrowth sion, cell-cell junction formation and by restricting both the activation of neuronal differentiation [5]. Rap1, as Rap1 (Rap1-GTP) and the sustained Correspondence: Fernanda Ledda E-mails: [email protected]; mledda@fmed. others small GTPases cycles between activation of ERK1/2 [4]. These find- uba.ar an inactive GDP-bound state and an ings are in agreement with previous data npg 218

Figure 1 Model describing the proposed role of Rap1GAP in the control of GDNF-induced Ret signaling. After activation of Ret by the heterocomplex GDNF/GFRα1, Ret becomes phosphorylated at different tyrosine sites. In particular, the Tyr1062 has been described to mediate the activation of Dok-4, which then triggers a sustained activation of Rap1-ERK1/2 pathway and neurite outgrowth. At the same time, Ret activation results in phosphorylation of the Tyr981 (Y981), which is the docking site involved in the recruitment of the negative signaling regulator Rap1GAP. Thus, Rap1GAP counterbalances the Dok-4/Rap1/ ERK1/2 pathway and controls neurite outgrowth. The figure also shows a possible role of PKA in the inhibition of Rap1GAP activity (dashed line), and indicates the potential involvement of Rap1 in the modulation of neurite outgrowth through the con- comitant activation of Vav2 and/or Tiam1, two GEFs for Rac1 and Cdc-42 (dashed arrow).

showing that GDNF-promoted neurite mediates the recruitment of Rap1GAP, phosphorylation (Figure 1). outgrowth during neuronal develop- a negative regulator of Rap1 signaling While the study by Jiao et al. pro- ment involves prolonged activation of (see Figure 1). vides new insights into the molecular Rap1-ERK1/2 pathway via the adaptor A recent study identified Rap1GAP mechanisms through which Rap1GAP protein, Dok-4, which is phosphorylated as a prominent PKA substrate. In limits Ret-induced neurite outgrowth, after Ret activation [7]. Interestingly, this study, the authors show that the some questions still remain unanswered. Dok-4 was identified as a direct interac- phosphorylation of Rap1GAP by PKA Thus, how the Rap1GAP activity is tor partner of Ret by yeast two-hybrid is associated with the inhibition of modulated upon binding to Ret and screen [8]. In this work, the authors Rap1GAP activity [9]. As it is known, how Rap1GAP competes with other show that Dok-4 can directly associate activation of Ret by GDNF elevates adaptor proteins, like Src, which has with Tyr1062. Together, these results cytoplasmic levels of cAMP and acti- been described to bind Tyr981 [11], are indicate that after ligand binding, Ret vates PKA in neuronal cells [10]. These important questions that require addi- becomes phosphorylated in different observations suggest that Ret induces a tional analysis. Interestingly, Rap1 plays sites including Tyr1062 and Tyr981. sustained Rap1-ERK1/2 pathway prob- an important role in the regulation of While phosphorylation of Tyr1062 ably by a dual mechanism that involves the cytoskeleton by activating the Rho mediates the activation of Dok-4, which (i) Dok-4 mediated Rap1GEF activation GTPase family members Rac1/Cdc42 then triggers a sustained activation of and (ii) inhibition of Rap1GAP activity via the binding and recruitment of the Rap1-ERK1/2 pathway and neurite by sequestering and blocking its GAP GEFs Tiam1 and Vav2 [12]. Therefore, outgrowth, phosphorylation of Tyr981 activity through a PKA-dependent another issue that would need to be

Cell Research | Vol 21 No 2 | February 2011 npg 219 addressed is whether Rap1GAP may adds a new physiological mechanism neurite outgrowth through downstream also collaborate in the control of addi- to negatively regulate signaling and activation of Rap1 and mitogen-acti- tional downstream signaling pathways biological responses induced by GDNF vated protein kinase. J Cell Sci 2006; 119:3067-3077. involved in Ret-dependent neurite and Ret in neuronal cells. In particular, 8 Grimm J, Sachs M, Britsch S, et al. outgrowth, such as activation of Rho the fact that Ret is coexpressed with Novel p62 family members, dok4 and GTPases. Rap1GAP in midbrain dopaminergic dok5, are substrates of the c-Ret recep- The pleiotropic roles played by Ret neurons together with the association tor tyrosine kinase and mediate neu- during development and the complex between both molecules in midbrain ronal differentiation. J Cell Biol 2001; intracellular network of signaling tissue argue that this mechanism could 154:345-354 pathways triggered by Ret, suggested have a potential therapeutic value for 9 McAvoy T, Zhou M, Greengard P, that Ret as other RTK, requires the treatment of PD. Nairn AC. Phosphorylation of Rap- 1GAP, a striatally enriched protein, by existence of cellular mechanisms that protein kinase A controls Rap1 activity control its activity, avoiding thereby References and dendritic spine morphology. Proc abnormal cellular behavior and disease. Natl Acad Sci USA 2009; 106:3531- Indeed, the picture that arise from the 1 Lin LF, Doherty DH, Lile JD, Bektesh 3536. last years indicates that this receptor S, Collins F. GDNF: a glial cell line- 10 Cai D, Shen Y, De Bellard M, Tang S, is tightly regulated in a cell-specific derived neurtrophic factor for midbrain Filbin MT. Prior exposure to neurotro- dopaminergic neurons. Science 1993; manner through the coordinated action phins blocks inhibition of axonal re- 260:1130-1132. generation by MAG and myeling via a of different protein inhibitors that func- 2 Paratcha G, Ledda F. GDNF and cAMP-dependent mechanism. Neuron tion at multiple levels of the signaling GFRalpha: a versatile molecular com- 1999; 2:89-101. cascade and at different time points plex for developing neurons. Trends 11 Encinas M, Ceowder RJ, Milbrandt J, after receptor engagement. Examples Neurosci 2008; 8:384-391. Johnson EM Jr. Tyrosine 981, a novel of Ret negative modulators include the 3 Airaksinen MS, Saarma M. The GDNF ret autophosphorylation site, binds c- ERK1/2 antagonist, sprouty [13] and the family: signaling, biological functions Src to mediate neuronal survival. J Biol and therapeutic value. Nat Rev Neuro- leucine-rich repeat and Ig-like protein, Chem 2004; 279:18262-18269. sci 2002; 5:383-394. 12 Arthur WT, Kuilliam LA, Kooper JA. Lrig1 [14]. 4 Jiao L, Zhang Y, Hu C, Wang YG, Huang Rap1 promotes cell spreading by local- The physiological requirement of Ret A, He C. Rap1GAP interacts with Ret izing Rac1 nucleotide exchange factor. signaling for the survival, maintenance and suppresses GDNF-induced neurite J Cell Biol 2004; 167:111-122. and regeneration of the dopaminergic outgrowth. Cell Res 2011; 21:327-337. 13 Ishida M, Ichihara M, Mii S, et al. system supports additional investigation 5 Kooistra M, Dubé N, Bos JL. Rap1: a Sprouty2 regulates growth and differ- toward optimizing the ongoing GDNF key regulator in cell-cell junction for- entiation of human neuroblastome cells clinical trials for Parkinson disease mation. J Cell Sci 2007; 120(Pt 1):17- through Ret tyrosine kinase. Cancer 22. (PD) using activators or reducing the Sci 2007; 98:815-821. 6 York RD, Yao H, Dillon T, et al. Rap1 14 Ledda F, Bieraugel O, Fard SS, Vilar effects of endogenous antagonists of mediates sustained MAPK activation M, Paratcha G. Lrig1 is an endogenous Ret signaling. Unlike Ret positive sig- induced by nerve growth factor. Nature inhibitor of Ret receptor tyrosine ki- naling effectors, which are relatively 1998; 392:622-626. nase activation, downstream signaling, well understood, signaling attenuation 7 Uchida M, Enomoto A, Fukuda T, et and biological responses to GDNF. J is under intensive study. In this regard, al. Dok-4 regulates GDNF-dependent Neurosci 2008; 28:39-49. the study performed by Jiao et al.