1-Docking properties of the phosphatidylinositol 5-phosphatase type II (SHIP2) a negative regulator of the PI3 kinase cascade Phosphoinositides (PI) are crucial for recruitment and regulation of proteins in different cellular membranes and play essential role in cell signaling and membrane dynamics. Among them PI(3,4,5)P3, important for cellular survival, is produced by the PI 3 kinase (PI3K) the most frequently known activated aberrant pathway in human cancers. SHIP2 contributes to the negative regulation of this pathway by converting PI(3,4,5)P3 in PI(3,4)P2, another important PI involved in cell adhesion and migration. The PI(3,4,5)P3 phosphatase activity of SHIP2 is not the only functional element of the protein as it is also involved in a network of protein-protein interactions that could influence its cellular location and indirectly its activity. Using two-hybrid screening, we previously identified potential proteic partners for SHIP2 that suggest a role of the protein in cell physiology. Among the identified partners c-Cbl, Intersectin (ITSN), JIP1 or APS are involved in vesicular traffic mechanisms, adhesion processes or EGF or insulin receptor regulation (Xie et al., 2013). Studying the physiological and cellular roles of SHIP2, particular interest has been attached to a new potential regulation mode of SHIP2 : ubiquitination (De Schutter et al., 2009). SHIP2 is notably the object of phosphorylations on tyrosine, serine and threonine whose impact on its cellular localization has already been well documented. We have shown that SHIP2 can also be the subject of mono-ubiquitination, which is particularly important in trafficking and endocytotic mechanisms. Faced with all these converging results that suggest an implication of SHIP2 in the regulation of receptor endocytosis and cellular adhesion and migration, we continue to attempt to better define the molecular mechanisms controlled by SHIP2 as a scaffold protein and its role in the ubiquitination signaling pathways. RhoGTPases regulate most cellular processes that need cytoskeleton or vesicular trafficking regulation and play specific roles in cancer development through activation of specific effectors. Among these effectors, Rhophilin-1 and Rhophilin-2 (Rhpn1/2) are specific partners of activated Rho proteins and are overexpressed in various cancer. Rhophilin-2 is an effector of RhoB and RhoB KO mice are more susceptible to induced skin cancer. Less is known about Rhophilins biological mechanisms of action or physiological roles. In the present project, we propose to investigate the role of Rhpn1/2 in cellular migration processes of skin cancer cells. Rhpn stable knock-down in cancer cell lines (skin and breast) will allow to study the role of Rhpns in cell migration and invasion. These cells metastatic properties will be assessed through xenografts experiments in zebrafishes. By generation of KI zebrafish models expressing fluorochrome reporters under the control of Rhpn promoters, we will detail tissue and subcellular expression profiles of Rhpn1/2. Inducing melanoma in knock out fishes compared to wt will allow us to evaluate the role of Rhpn in tumor growth and metastasis. This will be completed by biochemical experiments to unravel the proteic partners of RHPN2 to decipher the molecular function of this isoforms. Our previous two-hybrid work also allowed the characterization of potential interactors of RHPN2 but they are not yet characterized biochemically. Nevertheless, all of them are crucial proteins involved in vesicular trafficking, a main physiological event in cancer progression. Interestingly, we showed that SHIP2 and Rhophilin-2 share some binding partners among which the RhoGTPases. Rho proteins are important regulators of the cytoskeleton and are of major importance in membranar trafficking in cancer cells underlining the importance of studying Rhophilin involvement in cancer development. Even if most vertebrate G protein-coupled receptors (GPCR) have been functionally characterized some remained however orphan until now. Among these are a subgroup of three orphan G protein-coupled receptors characterized by an exceptionally high conservation across vertebrate species. These so called SREB receptors are expressed in the central nervous system, but also in specific leukocyte populations different for each subtype. The objective of the present project is to delineate the signaling and pharmacological properties and the role of the three SREB receptors using human, mouse and zebrafish model systems. By expressing the three human and zebrafish receptors transiently or stably in cell lines, we will determine whether they exhibit constitutive activity and through which pathways, determine their subcellular localization, their spontaneous trafficking and whether they are able to hetero-oligomerize with each other. We will search for natural agonists in extracts from mammalian and fish organs, including inflammatory tissues and fluids, lymphoid organs and brain regions. In parallel, transgenic fish lines will be generated for studying more precisely the expression of the three receptors in leukocyte populations and elsewhere, and their regulation along developmental stages and in the adult. Knock in and knock out lines are also being generated, allowing to complement our distribution studies and determine the potential role of the receptors.
Isabelle Pirson, PI Lab Members Jade Rager (Master thesis) Camille Perazzolo (Technician) Alumni Sarah Duerinckx (co-direction)
Publications Duerinckx, S., Jacquemin, V., Drunat S., Vial Y., Passemard S., Perazzolo C., Massart A, Soblet, J., Racapé J., Desmyter L., Badoer C., Papadimitriou S., Le Borgne Y-A., Lefort A., Libert F., De Maertelaer V., Rooman M., Costagliola S., Verloes A., Lenaerts, T., Pirson I. & Abramowicz M. (2019, in printing) Digenic inheritance of human primary microcephaly delineates centrosomal and non centrosomal pathways.  Hum Mutat. 2019 Nov 7. doi: 10.1002/humu.23948. Duerinckx, S., Meuwissen, M. E. C., Perazzolo, C., Desmyter, L., Pirson, I., & Abramowicz, M. (2018, avril). Phenotypes in siblings with homozygous mutations of TRAPPC9 and/or MCPH1 support a bifunctional model of MCPH1. Molecular genetics & genomic medicine. doi:10.1002/mgg3.400 Duerinckx, S., Verhelst, H., Perazzolo, C., David, P., Desmyter, L., Pirson, I., & Abramowicz, M. (2017, mai). Severe congenital microcephaly with AP4M1 mutation, a case report BMC medical genetics, 18(1), 48. doi:10.1186/s12881-017-0412-9 Ansseau, E., Gerbaux, C., Cloet, S., Wauters, A., Zorbo, S., Meyer, P., Pirson, I., Laoudj-Chenivesse, D., Wattiez, R., Harper, S. S., Belayew, A., Eidahl, J. J., Coppee, F., Lancelot, C., Tassin, A., Matteotti, C., Yip, C., Liu, J., Leroy, B., & Hubeau, C. (2016, janvier). Homologous transcription factors DUX4 and DUX4c associate with cytoplasmic proteins during muscle differentiation PloS one, 11(1), e0146893. doi:10.1371/journal.pone.0146893 Xie, J., Erneux, C., & Pirson, I. (2013). How does SHIP1/2 balance PtdIns(3,4)P2 and does it signal independently of its phosphatase activity? BioEssays, 35(8), 733-743. doi:10.1002/bies.201200168 Genin, A., Désir, J., Lambert, N., Biervliet, M., Van Der Aa, N., Pierquin, G., Killian, A., Tosi, M., Urbina, M., Lefort, A., Libert, F., Pirson, I., & Abramowicz, M. (2012). Kinetochore KMN network gene CASC5 mutated in primary microcephaly. Human molecular genetics, 21(24), 5306-5317. doi:10.1093/hmg/dds386 Drielsma, A., Jalas, C., Simonis, N., Désir, J., Simanovsky, N., Pirson, I., Elpeleg, O., Abramowicz, M., & Edvardson, S. (2012). Two novel CCDC88C mutations confirm the role of DAPLE in autosomal recessive congenital hydrocephalus. Journal of medical genetics, 49(11), 708-712. doi:10.1136/jmedgenet-2012-101190 Burniat, A., Pirson, I., Vilain, C., Kulik, W., Afink, G., Moreno Reyes, M. R., Corvilain, B., & Abramowicz, M. (2012). Iodotyrosine deiodinase defect identified via genome-wide approach. The Journal of clinical endocrinology and metabolism, 97(7), E1276-E1283. doi:10.1210/jc.2011-3314 Azizieh, N.-R., Orduz Perez, D., Van Bogaert, P., Bouschet, T., Rodriguez, W., Schiffmann, S. N., Pirson, I., & Abramowicz, M. (2011). Progressive myoclonic epilepsy-associated gene KCTD7 is a regulator of potassium conductance in neurons. Molecular neurobiology, 44(1), 111-121. doi:10.1007/s12035-011-8194-0 Vilain, C., Rens, C., Aeby, A., Balériaux, D., Van Bogaert, P., Remiche, G., Smet, J., Van Coster, R., Abramowicz, M., & Pirson, I. (2011). A novel NDUFV1 gene mutation in complex I deficiency in consanguineous siblings with brainstem lesions and Leigh syndrome. Clinical genetics, 82(3), 264-270. doi:10.1111/j.1399-0004.2011.01743.x De Schutter, J., Guillabert, A., Imbault, V., Degraef, C., Erneux, C., Communi, D., & Pirson, I. (2009). SHIP2 (SH2 domain-containing inositol phosphatase 2) SH2 domain negatively controls SHIP2 monoubiquitination in response to epidermal growth factor. The Journal of biological chemistry, 284(52), 36062-36076. doi:10.1074/jbc.M109.064923 Onnockx, S., Xie, J., Degraef, C., Erneux, C., & Pirson, I. (2009). Insulin increase in MAP kinase phosphorylation is shifted to early time-points by overexpressing APS, while Akt phosphorylation is not influenced. Experimental cell research, 315(15), 2479-2486. doi:10.1016/j.yexcr.2009.06.006 Xie, J., Vandenbroere, I., & Pirson, I. (2008). SHIP2 associates with intersectin and recruits it to the plasma membrane in response to EGF. FEBS letters, 582(20), 3011-3017. doi:10.1016/j.febslet.2008.07.048 Xie, J., Onnockx, S., Vandenbroere, I., Degraef, C., Erneux, C., & Pirson, I. (2008). The docking properties of SHIP2 influence both JIP1 tyrosine phosphorylation and JNK activity. Cellular signalling, 20(8), 1432-1441. doi:10.1016/j.cellsig.2008.03.010 Onnockx, S., De Schutter, J., Blockmans, M., Xie, J., Jacobs, C., Vanderwinden, J.-M., Erneux, C., & Pirson, I. (2008). The association between the SH2-containing inositol polyphosphate 5-Phosphatase 2 (SHIP2) and the adaptor protein APS has an impact on biochemical properties of both partners. Journal of cellular physiology, 214(1), 260-272. doi:10.1002/jcp.21193Our research
In humans, gene mutations of SHIP2 cause opsismodysplasia, a pathology of skeletal bone formation (Below et al., 2013; Huber et al., 2013). In living cells, SHIP2 appears to be involved in developmental processes (proliferation, adhesion and cell migration) and in metabolic functions (insulin signaling and obesity).
2-Rhophilin2 (RHPN2) a RhoB GTPase effector
3- SREBs GPCR characterization
Group members
(ilpirson@ulb.ac.be)
Phone #: +32 (0) 2 555 41 37
Jeanne Pauwels (Master thesis)
Valérie Jacquemin (PhD student) co-direction
Abeer Kaafarani (PhD student) co-direction
Romain Darche (PhD student) co-direction
Mathieu Antoine (PhD student)
Isabelle Vandenbroere
Christine Jacobs
Tatjana Arsenijevic
Séverine Steuve
Sheela Onnockx
Jingwei Xie
Julie DeSchutterPublications
Igoillo Esteve, M., Genin, A., Lambert, N., Désir, J., Pirson, I., Abdulkarim, B., Simonis, N., Drielsma, A., Marselli, L., Marchetti, P., Vanderhaeghen, P., Eizirik, D. L., Wuyts, W., Julier, C., Chakera, A. J., Ellard, S., Hattersley, A. T., Abramowicz, M., & Cnop, M. (2013). tRNA methyltransferase homolog gene TRMT10A mutation in young onset diabetes and primary microcephaly in human. PLoS genetics, 9. doi:10.1371/journal.pgen.1003888
Erneux, C., Elong Edimo, W., Deneubourg, L., & Pirson, I. (2011). SHIP2 multiple functions: a balance between a negative control of PtdIns(3,4,5)P₃ level, a positive control of PtdIns(3,4)P₂ production, and intrinsic docking properties. Journal of cellular biochemistry, 112(9), 2203-2209. doi:10.1002/jcb.23146
Montagut, G., Onnockx, S., Vaqué, M., Bladé, C., Blay, M., Fernández-Larrea, J., Pujadas, G., Salvadó, M. J., Arola, L., Pirson, I., Ardévol, A., & Pinent, M. (2009). Oligomers of grape-seed procyanidin extract activate the insulin receptor and key targets of the insulin signaling pathway differently from insulin. Journal of nutritional biochemistry. doi:10.1016/j.jnutbio.2009.02.003
