Jade Buchanan-Carter Aaron Buechlein Leo Senderowicz School of Informatics

1. PEA-15 and its Association with Diabetes and Cancer Jade Buchanan-CarterAaron BuechleinLeo SenderowiczSchool of Informatics © 2006 Bioinformatics Indiana University April, 24th 2006 1

2. Outline • Introduction • Background and Motivation • Association to Disease • Potential for Drug Therapy • Catpa • Summary © 2006 Bioinformatics Indiana University April, 24th 2006 2

3. Introduction > Introduction > Background & Motivation • Phosphoprotein enriched in astrocytes 15 • Located on Chromosome 1 specifically at 1q21.1 • Originally identified by Chneiweiss and colleagues. • Most highly expressed in the nervous system • Particularly high levels in astrocytes and neurons of the hippocampus > Disease Association > Therapeutic Agent > Pathway > Catpa > Summary © 2006 Bioinformatics Indiana University April, 24th 2006 3

4. > Introduction > Background & Motivation > Disease Association > Therapeutic Agent > Pathway > Catpa > Summary © 2006 Bioinformatics Indiana University April, 24th 2006 4

5. What are Astrocytes? > Introduction > Background • Astrocytes are the most common type of glial cells in the nervous system • They have numerous projections that anchor neurons to their blood supply. • They regulate the external chemical environment of neurons by removing excess ions, notably potassium, and recycling neurotransmitters released during synaptic transmission. • Actually outnumber neurons ten to one. & Motivation > Disease Association > Therapeutic Agent > Catpa > Summary © 2006 Bioinformatics Indiana University April, 24th 2006 5

6. Scop Classification > Introduction > Background & Motivation > Disease Class: All Alpha Fold: DEATH domain Superfamily: DEATH domain Family: DEATH effector domain Association > Therapeutic Agent > Catpa > Summary © 2006 Bioinformatics Indiana University April, 24th 2006 6

7. Death Effector Domain >Introduction >Background & Motivation > Disease • Part of the Death effector Domain (DED) protein family which is generally associated with regulation of apoptosis. • CASP8 • FADD • CFLAR • Caspase 10 • DEDD • DEDD2 Association > Therapeutic Agent > Catpa > Summary © 2006 Bioinformatics Indiana University April, 24th 2006 7

8. Binding > Introduction > Background & Motivation • PEA15 contains two phosphorylation sites. • Ser104 and Ser116 • Ser116 is phosphorylated by calcium-calmodulin kinase II and also by Akt (protein kinase B) • Ser104 is phosphorylated by protein kinase C • PEA15 is reported to bind FADD via its death domain, blocking its ability to recruit caspases thus preventing cell death. • PEA15 is also reported to bind ERK1/2, a MAP kinase, retaining ERK in the cytoplasm. > Disease Association > Therapeutic Agent > Pathway > Catpa > Summary © 2006 Bioinformatics Indiana University April, 24th 2006 8

9. PEA-15 Phosphorylation > Introduction > Background & Motivation > Disease • Unphosphorylated PEA15 is reported to be apoptotic, but the phosphorylated form is believed to be antiapoptotic. • Phosphorylated at both Ser104 & 116, PEA15 blocks its interaction with ERK1/2 • Phosphorylated at Ser104, PEA15 blocks ERK binding • Phosphorylated at Ser116, PEA15 promotes FADD binding. Association > Therapeutic Agent > Pathway > Catpa > Summary © 2006 Bioinformatics Indiana University April, 24th 2006 9

10. DiseaseAssociation > Introduction > Background & Motivation > Disease Association > Therapeutic • Type II Diabetes • Over expression of PEA15 has been found to inhibit • insulin-stimulated glucose transport. • Cancer • Found to be overexpressed in cell lines associated with • Breast Cancer, Glioma, and Squamous Carcinoma. • Hypothesized to prevent cellular apoptosis and/or • promote cell proliferation. Agent > Pathway > Catpa > Summary © 2006 Bioinformatics Indiana University April, 24th 2006 10

11. Clinical Background of Diabetes > Introduction > Background & Motivation > Disease • Previously called non–insulin-dependent diabetes mellitus (NIDDM) or adult-onset diabetes. • Accounts for about 90% to 95% of all diagnosed cases of diabetes. • Associated with older age, obesity, family history of diabetes, history of gestationaldiabetes, impaired glucose metabolism, physical inactivity, and race/ethnicity. • In type 2 diabetes, not enough insulin produced or body ignores insulin action Association > Therapeutic Agent > Pathway > Catpa > Summary © 2006 Bioinformatics Indiana University April, 24th 2006 11

12. Diabetes type II study > Introduction > Background & Motivation > Disease • Overexpression of the ped/pea-15 gene causes diabetes by impairing glucose-stimulated insulin secretion in addition to insulin action. • Overexpression of the ped/pea-15 gene is a common feature of type 2 diabetes. • Stable overexpression of ped/pea-15 in the glucose-responsive MIN6 beta-cell line also caused protein kinase Calpha activation and a marked decline in glucose-stimulated insulin secretion. • Antisense block of endogenous ped/pea-15 increased glucose sensitivity by 2.5-fold in these cells • Cells rescued by the protein kinase C inhibitor bisindolylmaleimide (Ser104). Association > Therapeutic Agent > Pathway > Catpa > Summary © 2006 Bioinformatics Indiana University April, 24th 2006 12

13. PEA15 associated cancers > Introduction > Background & Motivation > Disease • A glioma is a type of primary central nervous system (CNS) tumor that rises from glial cells. • Squamous Cell Carcinoma: affects tissues such as skin, the esophagus, the lungs, and the cervix. - 200,000 people in the United States alone every year. Smoking is a significant risk factor. • Breast Cancer - Most common form of cancer in females (worldwide) - affects one out of eleven women in the western world. - genetic factors (i.e. DNA damage of BRCA1,BRCA2) and environmental factors (alcohol, birth control pills). Association > Therapeutic Agent > Pathway > Catpa > Summary © 2006 Bioinformatics Indiana University April, 24th 2006 13

14. Therapeutic Agent(s) > Introduction > Background • Dominant negative expression of AKT increases apoptosis • in vitro • Thus, promoting apoptosis via inhibition of AKT • phosphorylation can be used as drug therapy • Targeting the PI3K(phosphoinositol-3-kinase) pathway is the • strategy available in literature • LA (leuprolide acetate) inhibits phophorylation of AKT which • leads to inhibition of phosphorylation on Ser116 of PEA15 • which increases apoptosis • Rapamycin also targets PI3K pathway and slows down • tumor growth without significant side effects in animal • models & Motivation > Disease Association > Therapeutic Agent > Pathway > Catpa > Summary Healthy Unhealthy © 2006 Bioinformatics Indiana University April, 24th 2006 14

15. Phosphorylation at Ser-116 by CaKII or Akt Omi/HtrA2 > Introduction Phosphorylation at Ser-104 by PKC Anti-Apoptotic Activity > Background & Motivation Increase in cell surface Glut1 > Disease PEA-15 Association Overexpression > Therapeutic Agent Inhibits glucose transport due to impairment of insulin-dependent Glut4 > Pathway FADD > Catpa ERK > Summary Elk-1 Fas CELL PROLIFERATION Type 2 Diabetes mellitus Caspase 8/10 CREB mediated transcription APOPTOSIS © 2006 Bioinformatics Indiana University April, 24th 2006 15

16. Curation Alignment Tool for Protein Analysis > Introduction > Background & Motivation > Disease CATPA Association > Therapeutic Agent > Pathway CATPA DATA?? > Catpa > Summary © 2006 Bioinformatics Indiana University April, 24th 2006 16

17. Summary > Introduction > Background & Motivation > Disease • Excess of PEA15 in cells reduces apoptosis and promotes cell proliferation via binding to FADD and ERK1/2 respectively • Study of these pathways may lead to discovery of therapeutic agents for treatment of cancer and diabetes Association > Therapeutic Agent > Pathway > Catpa > Summary © 2006 Bioinformatics Indiana University April, 24th 2006 17

18. Citations • Tibbetts, M., Zheng, L., Lenardo, M. The death effector domain protein family: regulators of cellular homeostasis. Nature 4, 404-409 (2003). • Ramos, J.W., PEA-15 Phosphoprotein: A potential cancer drug target. Hawaii Med. J. 64, 77-79 (2005). • Kitsberg, K. et al. Knock-Out of the neural death effector domain protein PEA-15 demonstrates that its expression protects astrocytes from TNFα-induced apoptosis. The J. of Neuroscience 19, 8244-8251 (1999). • Underhill, D.A., Vogan, K.J., Underhill, T.M., Gros, P., Identification of a novel, alternatively spliced isoform and single nucleotide polymorphisms in the murine Pea-15 gene. Mammalian Genome 12, 172-174 (2001). • Renganathan, H., Vaidyanathan, H., Knapinska, A., Ramos, J.W., Phosphorylation of PEA-15 switches its binding specificity from ERK/MAPK to FADD. Biochem. J. 390, 729-735 (2005). • Wolford, J.K., Bogardus, C., Ossowski, V., Prochazka, M., Molecular characterization of the human PEA15 gene on 1q21-q22 and association with type 2 diabetes mellitus in Pima Indians. Gene 241, 143-148 (2000). • Condorelli, G., Vigliotta, G., Iavarone, C., Caruso, M., Tocchetti, C.G., Andreozzi, F., Cafieri, A., Tecce, M.F., Formisano, P., Beguinot, L., Beguinot, F., PED/PEA-15 gene controls glucose transport and is overexpressed in type 2 diabetes mellitus. The EMBO J. 17, 3858-3866 (1998). © 2006 Bioinformatics Indiana University April, 24th 2006 18

19. Questions © 2006 Bioinformatics Indiana University April, 24th 2006 19