Lecture 23 Signal Transduction 2

Major Concepts Receptor tyrosine kinases control cell metabolism and proliferation Growth factor signaling through Ras Mutated cell signaling genes in cancer cells are called oncogenes Insulin signaling through PI-3 kinase TNF receptors activate protein complexes that control cell death and survival

Chronic myelogenous leukemia (CML) cells Chromosomal rearrangement leads to expression of a unique signaling kinase (Bcr-Abl) required for the leukemia cells to survive Gleevec inhibits Bcr-Abl kinase, cells die through apoptosis

Receptor Tyrosine Kinases Control Cell Proliferation and Metabolism

Receptor Protein Tyrosine Kinases (EGF, Epidermal growth factor) Courtesy: Roger Miesfeld

Receptor Protein Tyrosine Kinases 1) Receptor tyrosine kinases transmit extracellular signals by ligand-activation of an intrinsic tyrosine kinase function encoded in the cytoplasmic tail of the receptor. 2) Activation of the intrinsic tyrosine kinase activity requires receptor dimerization, which is often stimulated, or at least stabilized, by ligand binding. 3) Autophosphorylation of tyrosine residues within the receptor creates phosphotyrosine docking sites for signaling proteins that establish a relay signal between the receptor and a downstream phosphorylation cascade.

Drosophila eyes and cancer are connected? Activated EGF receptor with intrinsic tyrosine kinase Adaptor protein Grb2 binds EGF receptor dimers Recruitment of SOS Activation of Ras (inactivation by RasGAP) Ras-Raf complex formation Src phosphorylates Raf Raf phosphorylates MEK MEK phosphorylates ERK ERK dimerizes and then phosphorylates ELK SRF binds phosphorylated ELK Initiation of transcription leads to cell proliferation

In “Sevenless” mutant, the R7 photoreceptor does not differentiate properly, leading to no R7 cell, hence the name. SOS (Son of sevenless) is downstream in pathway, interacts with Sevenless BOSS (self explanatory) is ligand from neighboring cell that binds to and turns on Sevenless in developing eye You can read more at: http://www.sdbonline.org/fly/aimorph/eye.htm

Back to Cancer: Receptor Protein Tyrosine Kinases (EGF, Epidermal growth factor) Courtesy: Roger Miesfeld

Grb2 binds to phosphotyrosines on activated EGF receptor (Sevenless) Src homology domain, or SH2 domain phosphotyrosine binding pocket and a separate specificity pocket EGF Receptor (Sevenless class) Courtesy: Roger Miesfeld

SOS (Son of Sevenless) connects Grb2 to Ras, activates Ras SH3 domain Courtesy: Roger Miesfeld SOS is a guanine nucleotide exchange factor (GEF)

Ras/Src activate phosphorylation cascade MAP kinase family: Raf MEK ERK

GTPase activating proteins (GAP) such as RasGAP bind to Ras and stimulate GTP hydrolysis Courtesy: Roger Miesfeld Turn off that signal!!

Oncogenes just turn me on Many “oncogenes” (oncology is the study of cancer), interfere with feedback inhibition of growth factor signaling pathways. Gain of function mutations Ras (rat sarcoma virus oncogene) may be involved in 30% of human cancers Src (Roux sarcoma virus oncogene)

most common Ras activating mutation glycine to valine mutation at codon 12 (G12V) disrupts the intrinsic GTPase activity Courtesy: Roger Miesfeld

Insulin signaling is similar to EGF signaling IRS: insulin receptor substrate proteins Courtesy: Roger Miesfeld

TNF Receptors Activate Proteins That Control Cell Death and Survival

What in the world is TNF? Tumor Necrosis Factor an inflammatory cytokine a signaling molecule that induces apoptosis Apoptosis is cell death through “falling apart” Apoptotic bodies Engulfed by surrounding cells to clean up debris TNF Binds to a trimeric membrane receptor Initiates 2 or more pathways, depending on the cell conditions

To be or not to be… Courtesy: Roger Miesfeld

Signal transduction through TNF receptor family Adaptor complex formation TNF R1 receptor is activated by TNF-alpha 80 amino acid structural motif in the cytoplasmic tail of receptor called a Death Domain (DD) interacts with DDs on other proteins The fate of the cell rests in the relative abundance (and activities) of proteins in two separate, but inter-related, signaling pathways.

Cell survival and cell death are opposing pathways FADD binding to procaspase 8 stimulates an autocleavage reaction leading to cell death TRAF2 binding to TRADD recruits the NFkB-inducing kinase (NIK) leading to cell survival Ratio of FADD/Caspase 8 to TRAF2, RIP and NFkB determines cell fate Courtesy: Roger Miesfeld