University of Toronto Biochemistry

	Daniela Rotin Professor Ph.D., Toronto, 1988
	Hospital for Sick Children, TMDT-MaRS, Room 11-305 416-813-5098 drotin@sickkids.on.ca

Biochemistry and Function of the Nedd4 Family of Ubiquitin Ligases: The Role of LAR Family Tyrosine Phosphatases in Development.

Research Synopsis

Biochemistry and Function of the Nedd4 Family of Ubiquitin Ligases: My laboratory has been studying the ubiquitin system, particularly the Nedd4 family of E3 ubiquitin ligases, comprised of a C2-WW(n)-HECT domain architecture. We are studying the biochemistry, structure and function of these E3 ligases, as well as develop methodologies to globally identify their substrates. We also study in more detail how they regulate several of these substrates (eg. ENaC, LAPTM5, CNrasGEF). More recently, we have been using mouse knockout models of Nedd4 family members to decipher their biological function in vivo .

LAR family protein tyrosine phosphatases (PTPs):We have been characterizing the LAR family protein PTPsigma both biochemically and developmentally. We knocked out the PTPsigma gene and showed that the mice develop neuroendocrine and neuronal defects. They also exhibit intestinal abnormalities. We recently identified N and E cadherin as in vivo substrates for PTPsigma in the brain and intestine, respectively. We then showed that polymorphism in the PTPsigma gene is associated with ulcerative colitis, and polymorphism in its substrate E-cadherin is associated with Crohn's disease, two IBD illnesses. We are now pursuing studies of function of PTPsigma and its substrates in the intestine and nervous system.

Selected Publications

ROTIN D. and Kumar S. Physiological functions of the HECT family of ubiquitin ligases. Nature Rev. Mol. Cell. Biol. 10:398-409, 2009.

Fouladkou F., Lu C., Jiang C., She YM., Walls, J. Kawabe H., Brose N., Henkelman M., Bruneau B. and ROTIN D. The ubiquitin ligase Nedd4-1 is required for heart development and is a suppressor of Thrombospondin-1. J. Biol. Chem , In press, 2009.

Persaud A., Amsen E., Xiong X, Wasmuth J, Saadon Z, Fladd C, Parkinson J. and ROTIN D. Comparison of substrate specificity between the ubiquitin ligases Nedd4 and Nedd4-2 using proteome arrays. Mol Syst Biol . 5:333, 2009.

Muise AM, Walters TD, Glowacka WK, Griffith AM, Ngan BY, Lan H., Xu W, Silverberg MS and ROTIN D. Polymorphisms in E-cadherin (CDH1) result in a mis-localized cytoplasmic protein that is associated with Crohn's Disease. Gut , 58:1121-1127, 2009.

Trzcinska-Daneluti A., Ly, D., Huynh L., Jiang C., Fladd C and ROTIN D. High content functional screen to identify proteins that correct F508del-CFTR function. Mol.Cell.Proteom .8:780-790, 2009.

Fouladkou F., Landry T., Kawabe H., Lu, C., Brose N., Stambolic V and ROTIN D. The Ubiquitin ligase Nedd4-1 is dispensable for the regulation of PTEN stability, localization or activity. Proc. Natl. Acad. Sci, USA , 105:8585-8590, 2008.

Wiesner S., Ogunjimi A., Wang HR., ROTIN D., Sicheri F., Wrana J., Forman-Kay J. Auto-inhibition of the Hect-type ubiquitin ligase Smurf2 through its C2 domain. Cell , 130:651-62, 2007.

Muise A., Walters T., Wine E., Griffiths A., Duerr R., Turner D., Regueiro M., Ngan BY., Sherman P., Siverberg M. and ROTIN D. Protein Tyrosine Phosphatase sigma is associated with Ulcerative Colitis. Curr. Biol . 17:1212-1218, 2007, (accompanied by a Press Release and media coverage)

Lu C./ Pribanic S., Gaulis A. Jiang C. and ROTIN D. The PY motif of ENaC, mutated in Liddle syndrome, regulate channel internalization, sorting and mobilization from a sub-apical pool. Traffic , 8:1246-1264, 2007.

Gupta R., Kus B., Fladd C., Wasmuth J., Tonikian R., Krogan N., Sidhu S., Parkinson J. and ROTIN D. High throughput ubiquitination screen using protein microarrays for the comprehensive identification of Rsp5 substrates in yeast. Mol Syst Biol . 3:(116) 1-12, 2007.

Siu, R., Fladd C. and ROTIN D. N-cadherin is an in vivo substrate for PTPsigma and participates in PTPsigma-mediated inhibition of axon growth. Mol. Cell Biol . 27:208-219, 2007.

Ing B., Kotler A., Castelli R., Henry P., Pak Y., Stewart B., Boulianne G. and ROTIN D. Ubiquitination of Commissureless by the Ubiquitin Ligase DNedd4 is Required for Neuromuscular Synaptogenesis in Drosophila . Mol. Cell. Biol . 21:481-496,2007.

Staub O. and ROTIN D, The role of ubiquitylation in cellular membrane transport. Physiol. Rev . 86:669-707, 2006.

Pak Y., Glowacka W., Bruce C., Pham N. and ROTIN D. Transport of LAPTM5 to lysosomes requires association with the ubiquitin ligase Nedd4 but not LAPTM5 ubiquitination. J. Cell Biol . 175:61-645, 2006.

Kirkham DL., Axford M, Pacey L., Siu R., ROTIN D, and Doering L. Neural Stem Cells Generate an Altered Neuronal Phenotype in Protein Tyrosine Phosphatase sigma Knockout Mice. BMC Neurosci . 7:50 (1471-2202), 2006.

Kanelis V., Bruce (O'Brien) C., Skrynnikov N. and ROTIN D*/Forman-Kay J*. Structure determinants for high affinity binding in a Nedd4 WW3* domain Comm PY motif complex. Structure , 14:543-553, 2006 (* corresponding authors).

Kus B, Gajadhar A, Stanger K, Cho R, Sun W, Rouleau N, Lee T, Chan D, Wolting C, Edwards A. Bosse R. and ROTIN D. A High Throughput Screen to Identify Substrates for the Ubiquitin Ligase Rsp5. J. Biol. Chem , 280:29470, 2005.

Henry P., Kanelis V., Kim B., O'Brien, Forman-Kay J, C., Schild L. and ROTIN D. Affinity and specificity of interactions between Nedd4 and ENaC. J. Biol. Chem . 278:20019-28, 2003.

Vecchione A, Marchese A, Henry P., ROTIN D. and Morrione A. Grb10/Nedd4 complex regulates ligand-induced ubiquitination and stability of the Insulin-like Growth Factor 1 Receptor. Mol. Cell Biol . 23:3363-3372, 2003.

Pak Y., Pham N. and ROTIN D. Direct binding of the ?1 adrenergic receptor to the cAMP dependent guanine nucleotide exchange factor CNrasGEF leads to Ras activation. Mol. Cell Biol . 22:7942-7952, 2002.

Myat A* / Henry P*., McCabe V., Flintoft L., ROTIN D. and Tear G. Nedd4, a ubiquitin ligase, is recruited by commissureless to control cell surface levels of the roundabout receptor. Neuron 35:447-459, 2002.

Meathrel K, Adamek T., Batt J., ROTIN D. and Doering L. PTP? deficient mice show spinal cord abnormalities and aberrant cytoarchitecture in the cortex, hippocampus and corpus callosum. J. Neurosci. Res . 70:24-35, 2002.

McLean J., Batt J., Doering L., ROTIN D. and Bain JR. Enhanced rate of regeneration and directional errors following sciatic nerve injury in receptor protein tyrosine phosphatase sigma knockout mice. J. Neurosci . 22:5481-5491, 2002.

Batt J., Asa S., Fladd C.and ROTIN D. Pituitary, pancreatic and gut neuroendocrine defects in PTPsigma deficient mice. Mol. Endocrinol ., 16:155-169, 2002.

Kanelis V., ROTIN D. and Forman-Kay, J. Solution structure of a Nedd4 WW domain -bENaC peptide complex. Nature Struct. Biol . 8:407-412, 2001.

Plant P., Lafont F., Lecat S., Verkade P., Simons K. and ROTIN D. Apical membrane localization of Nedd4 is mediated by an association of its C2 domain with Annexin XIIIb . J. Cell Biol. 149:1473-1483, 2000.

Pham N., Cheglakov I., Koch A., de-Hoog C., Moran MF and ROTIN D. The guanine nucleotide exchange factor CNrasGEF activates ras in response to cAMP and cGMP . Curr. Biol .10:555-558, 2000.

Wallace MJ., Batt J., Fladd C., Henderson JT., Skarnes W. and ROTIN D. Neuronal defects and posterior pituitary hypoplasia in mice lacking the receptor tyrosine phosphatase PTPsigma. Nature Genet .,21:334-338, 1999.

Abriel H., Loffing J., Rebhun JF Pratt H, Schild L., Horisberger J-D, ROTIN D. and Staub O. Defective regulation of the epithelial Na+ channel (ENaC) by Nedd4 in Liddle's Syndrome. J. Clin. Invest ., 103:667-673, 1999.

Wallace M, Fladd C. Batt J., and ROTIN D. The second catalytic domain of the tyrosine phosphatase PTP? binds to and inhibits the first catalytic domain of PTPsigma. Mol.Cell. Biol .,18:2608-2616, 1998.

Staub O, Gautschi I., Ishikawa T., Breitschopf K., Ciechanover A., Schild L and ROTIN D. Regulation of stability and function of the epithelial Na channel (ENaC) by ubiquitination. EMBO J . 16:6325-6336,1997.

Staub O, Dho S, Henry P, Correa J.,Ishikawa T., McGlade J, and ROTIN D. WW domains of Nedd4 bind to the proline rich PY motifs in the epithelial Na channel deleted in Liddle's syndrome. EMBO J, 15:2371-2380, 1996.

Department of Biochemistry
University of Toronto
Toronto, Canada

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