CELLULAR SIGNAL TRANSDUCTION 2) From the membrane to the nucleus TEIL F TRANSCRIPTION FACTORS AND THE REGULATION OF TRANSCRIPTION Rainer deMartin / Erhard Hofer Institute of Vascular Biology and Thrombosis Research Vienna Competence Center Lazarettgasse 19, 1090 Wien

Erhard Hofer activation of transcription factors by surface receptors Summary selected examples CREB, SRF, NFAT, SMAD additional specific example: - Regulation of signaling pathways/transcription factors by proteolytic cleavage: WNT, NOTCH (embryonic development, adult stem cells) Nuclear import, -export Chromatin Transcription initiation complexes Rainer deMartin: Principals of transcriptional regulation Structural features of transcription factors Basic mechanisms of transcriptional regulation

SMAD SMAD/Co-SMAD SMAD STAT A-CyclasePLC Ras STAT cAMP Signaling pathways: Receptor to transcription factors PKA SRF IKKK NFAT / NF  B

CREB, SRF, NFAT, SMAD

Gene regulation by PKA: CREB bound to CRE Is phosphorylated by PKA, activates transcription without effect on DNA binding Example 3

The phosphorylated MAPK ERK is transported into the nucleus and phosphorylates the transcription factor TCF on the promoter ERK: extracellular signal regulated kinase TCF: ternary complex factor SRF: serum response factor SRE: serum response element (DNA binding sequence for TCF and SRF in promoter of various genes) Genes for Cell cycle/ Proliferation early response genes, c-fos or: PLC-  Raf MEK

Ca++ Calmodulin Calcineurin NFAT PIPI Ca ++ Signaling pathway - Gene regulation the Phosphatase Calcineurin dephosphorylates NFAT NFAT translocates Into nucleus NFAT= transcription factor (nuclear factor activated T cell) Kern P

Transport of phosphorylated SMADs into nucleus

Regulation of transcription factors by proteolytic cleavage

WNT signaling pathway Secreted signaling peptide, important in embryonic development Mutatios on proteins of wnt signaling pathway frequent in cancer Example 4a Wnt Wingless (Drosophila) Int-1 (breast cancer oncogen) (detected experimentally by virus Integration) In signalling pathway: APC (adenomoteous polyposis coli) mutated in adenoma of colon and 80 % of colon cancer induces myc gene and proliferation

(LDL rceptor related protein) (Signaling protein) (Corepressor)  -Catenin signaling pathway: w/o signal:  -catenin is continously phosphorylated, ubiquitinylated, degraded in proteasom Wnt-signal: Kinase is inhibited, non-phosphorylated  -Catenin transported into nucleus, aktivates transcription by competing of a corepressor (  -Catenin Coaktivator) Phosphorylation, Ubiquitinylation, degradation in proteasom

components of the WNT signaling pathways e.g. important for maintaining the stem cell population in gastro-intestinal tract, Over-activation by APC mutation - cancer

Notch hedgehog 1- Embryonic development E.g. nerve cells Drosophila, Delta on nerve cells signals to neighbouring cell: Do not differentiate to nerve cell, Peptide translocates into nucleus and converts CSL to become an activator 2- Angiogenesis: Tip versus stalk cell, tip cell signals stalk cell not to become another tip cell example 5b

Nuclear import / -export

Nuclear membrane: Double membrane with nuclear pores

26 nm

Nuclear pore complex innere Seite

The nuclear localization signal is a basic amino acid sequence Model of nuclear import Mediated by the small G-protein Ran

Model of nuclear import and export

CHROMATIN

Summary Interphase Metaphase

Eigenschaften von Insulatorelementen model

Change of chromatin structure by activator protein

Ac HAT HDAC condensed CHROMATIN no TRANScRIPTION Z.B. HETEROCHROMATIN loose CHROMATIN TRANCRIPTION Z.B. EUCHROMATIN HAT = HISTONACETYLTRANSFERASE ACTIVATOR OF TRANSCRIPTION HDAC = HISTONDEACETYLASE REPRESSOR of TRANSCRIPTION DNA NUCLEOSOM CHROMATIN GENEXCS18

A REPRESSION ACTIVIERUNG HDAC X Y HAT Target GENE Z REGULATION OF TRANSCRIPTION BY CHANGE OF CHROMATIN STRUCTUR condensed Chromatin open Chromatin Repressor Aktivator GENEXCS19

Gene regulatory proteins form frequently large complexes

DNA Looping Mediator

Gene expression