A Sonic hedgehog coreceptor, BOC regulates neuronal differentiation and neurite outgrowth via interaction with ABL and JNK activation Tuan Anh Vuong a , Young-Eun Leem a , Bok-Geon Kim a , Hana Cho b , Sang-Jin Lee c , Gyu-Un Bae c , Jong-Sun Kang a, ⁎ a Department of Molecular Cell Biology, Sungkyunkwan University School of Medicine, Samsung Biomedical Research Institute, Suwon 16419, Republic of Korea b Department of Physiology, Sungkyunkwan University School of Medicine, Samsung Biomedical Research Institute, Suwon 16419, Republic of Korea c Research Center for Cell Fate Control, College of Pharmacy, Sookmyung Women's University, Seoul 04310, Republic of Korea abstract article info Article history: Received 14 November 2016 Accepted 17 November 2016 Available online 18 November 2016 Neurite outgrowth is a critical step for neurogenesis and remodeling synaptic circuitry during neuronal develop- ment and regeneration. An immunoglobulin superfamily member, BOC functions as Sonic hedgehog (Shh) coreceptor in canonical and noncanonical Shh signaling in neuronal development and axon outgrowth/guidance. However signaling mechanisms responsible for BOC action during these processes remain unknown. In our pre- vious studies, a multiprotein complex containing BOC and a closely related protein CDO promotes myogenic dif- ferentiation through activation of multiple signaling pathways, including non-receptor tyrosine kinase ABL. Given that ABL and Jun. N-terminal kinase (JNK) are implicated in actin cytoskeletal dynamics required for neurogenesis, we investigated the relationship between BOC, ABL and JNK during neuronal differentiation. Here, we demonstrate that BOC and ABL are induced in P19 embryonal carcinoma (EC) cells and cortical neural progenitor cells (NPCs) during neuronal differentiation. BOC-depleted EC cells or Boc -/- NPCs exhibit impaired neuronal differentiation with shorter neurite formation. BOC interacts with ABL through its putative SH2 binding domain and seems to be phosphorylated in an ABL activity-dependent manner. Unlike wildtype BOC, ABL- binding defective BOC mutants exhibit impaired JNK activation and neuronal differentiation. Finally, Shh treat- ment enhances JNK activation which is diminished by BOC depletion. These data suggest that BOC interacts with ABL and activates JNK thereby promoting neuronal differentiation and neurite outgrowth. © 2016 Published by Elsevier Inc. Keywords: BOC Shh coreceptor ABL JNK Neuronal differentiation Neurite outgrowth 1. Introduction Neurite outgrowth is a critical step for neurogenesis and neuronal circuitry formation during neuronal development and regeneration [1]. Axon guidance molecules, such as netrin, slit or Sonic hedgehog (Shh) play important roles in axonal growth and navigation of growth cones to innervate targets which involve remodeling and reorganization of the cytoskeleton [2]. The guidance receptors such as the netrin recep- tor Frazzled/DCC, the slit receptor ROBO or the Shh receptor BOC mediate signals to modulate cytoskeletal remodeling [3–5]. Non-recep- tor tyrosine kinase ABL plays a critical role in nervous system develop- ment [6] and regulates the cytoskeletal rearrangement involved in the Netrin/Frazzled/DCC and Slit/ROBO-mediated axon guidance [7–9]. ABL interacts with these receptors and also phosphorylates ROBO to modulate its function in axon guidance [10–12]. Multiple downstream pathways, such as the Rho family small GTPases and c-Jun. NH2- terminal kinases (JNKs) have been implicated in ABL-mediated axon guidance [7,13,14]. ABL is shown to interact with and phosphorylate JNK-interacting protein 1 (JIP1), which in turn might activate JNK to promote axon outgrowth [15]. JNKs are also involved in diverse process- es of neuronal development, including neuronal differentiation, axon formation/outgrowth, and injury-mediated neuronal degradation [16]. JNK is shown to regulate neuronal differentiation and neurite out- growth induced by nerve growth factor [17–19] and the inhibition of JNK blocks neuronal differentiation of P19 embryonal carcinoma (EC) cells [19,20]. Furthermore, JNK is also implicated in axon guidance me- diated by a netrin receptor DCC/DSCAM complex in the developing ner- vous system [21]. BOC belongs to an immunoglobulin/fibronectin type III (Ig/FNIII) subfamily of cell surface proteins and together with a closely related Cellular Signalling 30 (2017) 30–40 Abbreviations: Shh, Sonic hedgehog; BOC, Brother of CDO; CDO, cell adhesion molecular downregulated by ocogenes; JNK, Jun N-terminal kinase; EC, embryonal carcinoma cell; NPC, neural progenitor cell; DCC, deleted in colorectal cancer; ROBO, roundabout; GLI, glioma-associated oncogene; JLP, JNK-interacting protein 1-like protein; MAPK, mitogen activated protein kinase; SH2, Src homology domain 2; SH3, Src homology domain 3; DCX, doublecortin; MAP2, microtubule-associating protein2; RA, retinoic acid; ITS, insulin/transferrin/selenite; Ngn1, Neurogenin 1; bFGF, basic fibroblast growth factor; EGF, epidermal growth factor; CM, conditioned medium; KD, kinase dead. ⁎ Corresponding author at: Department of Molecular Cell Biology, Sungkyunkwan University School of Medicine, 2066, Seobu-Ro, Jangan-gu, Suwon, Gyunggi-do 16419, Republic of Korea. E-mail address: kangj01@skku.edu (J.-S. Kang). http://dx.doi.org/10.1016/j.cellsig.2016.11.013 0898-6568/© 2016 Published by Elsevier Inc. Contents lists available at ScienceDirect Cellular Signalling journal homepage: www.elsevier.com/locate/cellsig