TSC1 Tuberous Sclerosis 1

Where is TSC1?

TSC1 Pathway Is a tumor suppressor Part of complex that promotes -growth & proliferation -differentiation -migration Molecular links between PTEN and TSC1/TSC2 are not fully understood. Some studies show that PKB degrades TSC2 but others show that TSC works parallel and independently of insulin receptor pathway. PDK1 activates BOTH S6K and PKB by threonine phosphorylation. CGM http://www.cgm.cnrsgif.fr/montagne/index_en.html

What’s TSC1/Hamartin? Localizes TSC2/tuberin In the cytoplasm- peripheral membrane protein Phosphorylated by… 1. CDK1 2. GSK3β 1. CDK1- 3 residues (site of phosphorylation and level of it may be determining factor in whether Hamartin is stabilized and allowed to work or is inhibited. 2.The hemartin/tubulin complex – are subject to ubiquitination-mediated proteosomal degradation when phosphorylated. Oncogene 2005 Nature Publishing Group

What’s TSC2/Tuberin? Regulated through phosphorylation Forms a heterodimer with TSC1/hemartin Also located in the Golgi apparatus & nucleus RSK1 is ribosomal S6 kinase 1 TOR is a serine-threonine kinase Oncogene 2005 Nature Publishing Group

TSC1/TSC2 complex CGM http://www.cgm.cnrsgif.fr/montagne/index_en.html Oncogene 2005 Nature Publishing Group

What’s Rheb? Ras homolog enriched in brain Heterotrimeric G-protein Activates mTOR by GTP 1.mTOR activation and B-Raf inhibition counterbalance each other limiting Rheb’s ability to induce tumor growth. 2.mTOR induces growth and transcription. 3.B-Raf induces differentiation. http://www.nature.com/nrd/journal/v4/n12/images/nrd1902-f3.jpg

CGM http://www.cgm.cnrsgif.fr/montagne/index_en.html Rheb in the Pathway Oncogene 2005 Nature Publishing Group

Background on Tuberous Sclerosis the Disease Germline mutation Inherited in an autosomal dominant fashion Highly penetrant Manifested in early age Autism or Retardation Tumors are mostly benign Manifestations that occur in certain groups of people. Heart rhabdomyomas are observed in infancy but regress Pulmonary lymphangiomyomatosis (LAM) is exclusively in female patients.

1. Angiomyolipoma (AML) 3.Brain and skin lesions Characteristics 2.Facial angiofibromas 3.Brain and skin lesions

TSC1 gone wrong Nonsense or frameshift mutations LOH No response to amino acid starvation Targets are more phosphorylated Increased expression of 1. Cyclin D1 2. β-catenin TSC1 mutations are less severe than TSC2 Mutations cause premature protein truncation. - mutations cause abnormalities in cell SIZE, #, MORPHOLOGY, and LOCATION 4. Cell migration occurs because tuberin/hamartin activate members of the Rho GTPase superfamily which regulate actin cytoskeleton, morphology, and migration and induce focal adhesions Aberrant migration

How did we know this? TSC1 mouse knockout Mutagenesis screens in Drosophila Biochemical data In vivo studies with Rapamycin 1. TSC1 homozygous mutant died around 10-11 days b/c neural tube didn’t close. 2. TSC1 knockout resembled TSC2 knockout which showed their similarity and pathway, BUT tumor development in mutant TSC1 was slower than in TSC2 mutants. 3. Mutant fly is larger. http://www.bio.davidson.edu/Courses/genomics/method/homoimplant.jpg CGM http://www.cgm.cnrsgif.fr/montagne/index_en.html

Some possible treatments Rapamycin Binding of cellular receptor forms an active toxin of rmTOR through high affinity binding to the FRB domain of mTOR (mammalian target of rapamycin) which is adjacent to its kinase domain Studies showed that p70S6K and S6 were dephosphorylated and the tumor size decreased Cyclin D1 expression and p27 undetection suggests rapamyacin acts independently of cell cycle regulators Observations based on histology and TUNEL staining- increase in caspase 3 activation HOWEVER… because of the feedback inhibition of AKT, treatment with rapamycin raises the concern that malignant tumors may arise again 6.-specific inhibitor -binds the cellular receptor FKBP12 -results in dephosphorylated mTOR targets -cell death observed after 3 daily treatments -cyclin D1 remained highly expressed -reduces proliferation -diminishes tumor size -p27 was undetected CGM http://www.cgm.cnrsgif.fr/montagne/index_en.html http://www.merckbiosciences.co.uk/sharedimages/calbiochem/spotlights/mTOR_fig2.gif

Unanswered Questions? How dRHeb regulates Tor-dependent nutrient sensing. Whether Rheb inhibits other members of the Raf family. Whether hamartin is important for the function of tuberin as a GAP protein. Whether differences in the subcellular localization of hamartin/tuberin reflect 1.cell type 2.cell-specific differential localization 3.antibody nonspecificity