2 Signal transduction 2

• One of the most important genes in development -> mutants produce dramatic phenotypes -> cancer

• Ligands: Delta, Jagged -> large transmembrane proteins

• Notch (=receptor): large extrazellulär domain + linker domain (often mutations in cancer patients)

• PEST domain bound to linker

• Short half life because it’s important

• Ligand binds

• Notch’s extracellular domain is proteolytically cut off by an ADAM Metalloprotease

• Cleaved notch is cubby a secretase-> notch active truncated cytoplasmatic domain (Nicd)

• Nicd migrates into nucleus -> Nicd= co-activator of transcription factor CSL

Tumor growth factor

• ligand TGF -ß-like/ activin binds

• Dimerisation of receptor type I+ type II

• Dimerisation activates type 2: Ser/Thr kinase

• Phosphorylation of receptors

• Receptor phosphorylates SMAD

• SMAD dimerizes and migrates into nucleus where it functions as a TF and regulates the gene expression

• dependent on ligand (TGF, Activin, BMP) different receptor dimers are bound

• Phosphorylation of different SMAD which activate different target gene expressions

• A concentration gradient of a morphogen thus provides position information to each cell along the gradient.

• Exact determination requires a counter gradient

• Different fate for each cell

• necessary for growthof wing disc

• Induces concentration-dependent at least 2 target genes

• Opposite direction : expression of inhibitor: brinker (brk)

Similar to dpp

• Many cytokine receptors (include tyrosines) directly bind to Janus tyrosine Kinase (Jak)

• Citrine binding Jak phosphorylate each other on a tyrosine

• The tyrosine phosphorylated cytokine receptor functions as docking site for

  • STAT protein (via SH2)

• STAT is phosphorylated by JAK (on the tyrosine)

• Phosphorylation causes release and dimer formation of 2 STATs

• STAT dimer migrates into meuchelst

• STAT dimer binds specifically DNA => TF that activates gene expression (in a complex with others)

• TNFa signal molecule binds to receptor

• Indirect activation of Ikk complex

• Active Ikk complex phosphorylates IkB (which is bound to NFkB)

• Phosphorylated IkB releases NFkB and is degraded

• Released NFkB translocates into the nucleus and activates gene expression Of NFkB target genes

• in the target cells without hedgehog- binding: the signal transduction protein smoothened is inhibited by patched protein

• Ci (cubitus interruptus) is bound to microtubules of the cytoskeleton via Fused and Cos2

• Cos2 forms complex of Ci with 4 kinases

• Ci is phosphorylated and processed by slimb

• 1/2 of Ci migrates into the nucleus and acts as a Repressor of the transcription of the target genes

Activation

• Hedgehog binds Patched receptor

• Smoothened not repressed anymore, can inhibit PKA and Slimb

• Ci is not proteolytically processed

• Cos2 phosphorylated releasing the uncut Ci

• During Hedgehog stimulation complete Ci/Gli migrates into the nucleus and activates the expression of target genes with CBP as a co-factor

patched mutation=> gain of function mutation

• Many types of colorectal cancer and tumors of the nervous system are associated with mutations of the patched gene

• Resulting in an activation of the Hedgehog signal transduction pathway leading to uncontrolled cell growth

Without Wg/ Wnt binding

• Cytoplasmic β-Catenin is bound to Axin/APC/GSK3/CK1 (ß-catenin is usually bound to cadherin at the apical surface)

• β-Catenin is phosphorylated at multiple sites marking it for ubiquitination resulting in its degradation

• The downstream transcription factor LEF1/TCF acts as a repressor together with Groucho (because no β-Catenin)

Activation

• Wnt signal binds to frizzled receptor

• Co-receptor LRP and dishevelled is bound to Frizzled

• Dishevelled recruits CK1 and GSK3 kinase

• CK1 and GSK3 phosphorylates LRP Co-receptor

• Axin is recruited to the complex in competition to the degradation complex with APC

• β-Catenin Can’t be degrated because complex is disrupted (auxin)

• Stable b-Catenin translocates into the nucleus

• b-Catenin binding to LEF1/TCF releases the co-

  repressor Groucho

• b-Catenin/LEF1/TCF functions as an activator of expression

• Mutation of APC=> gain of function

• even without Wnt signaling β-Catenin is not bound to the degradation complex, not proteolytically degraded and can translocate into the nucleus

• Resulting in gene activation

  – APC mutant behaves like an

  uncontrolled Wnt gain of function

• One of the target genes of Wnt signaling is Myc regulating cell proliferation

  WSo in APC mutants MYC is ectopically induced causing cancer cell proliferation

• hair growth at a certain position

• Planar polarity is based on the polar localization of two protein groups on the apical side of the cells

• The polar localization of the two protein groups is controlled by Wnt binding to Frizzled

• Nuclear receptors are ZnFinger transcription factors binding DNA as hetero- or homodimers

• Without ligand binding Nuclear receptors are inactive via binding to an inhibitory protein

• Some are retained in the cytoplasm or inactive although bound to DNA

activation

• ligand binds -> inhibitory protein dissociates

• Nuclear Receptors are active and function as transcriptional regulators (activator or repressor) by binding to co-activator proteins or co-repressors

• Broad range of ligands, like steroid hormones, thyroid hormones or metabolites

1. Proteolytic processing, removing parts of the protein by a sequence specific endo-protease

2. Gylcosylation, the addition of short or long sugar side chains

3. Addition of membrane linkers, like fatty acids (palmitoyl-anchor)

4. Ubiquitination or sumilation regulating protein degradation or protein activity

Two kinds of side chains

• N-linked glycosylation via the amino acid asparagine represents the majority

• O-linked via threonine and rarely serine

Localised in the ER

• The asparagine-linked (N-linked) precursor oligosaccharide is added in the rough ER membrane already during synthesis

• Gylcosylation has a major role in the folding of modified proteins in the ER

N-linked oligosaccharides are branched and belong into two classes

Two main classes of asparagine-linked (N-linked) oligosaccharides found in mature glycoproteins

• Complex oligosaccharides with negatively charged sugar moieties

• High mannose oligosaccharides

If protein is not folded properly the sugars won’t stick

Oligosaccharides may promote folding and stability of glycoproteins

• Oligosaccharides also regulate specific transport of proteins towards certain organelles

• Mannose 6-phosphate residues target proteins to lysosomes

• Many organisms form a shield of of glycosylated proteins

• In higher organism mostly proteins of the extracellular matrix

• In some proteins these sugar moieties double the molecular weight of the protein (Bind much water)

Proteoglycans have very long unbranched sugar side chain with a stereotyped organization coupled to the protein at a serine amino acid

• Notch is intensively modified in the Golgi apparatus after its synthesis in the ER By glycolysation

• These modifications mainly involve glycosylations for which two factors are necessary: Fringe and O-Fucosyl- transferase

• These glycosylations affect the binding specificty and stability of Notch

Furin cleave proteins with a specific protein sequence/recognition site in the final step of secretion between the golgi apparatus and the plasma membrane

Example: proinsulin is processed to Insulin

Proteins are attached to a membrane by fatty acid or a prenyl group

The covalent attachment of the membrane anchor can be processed by

• A fatty acid added to the N-terminal glycine (myritosyl anchor)

• A fatty acid can be added to a cysteine (thioester group)

• A prenyl group can be added to a cysteine (thioether group)

In the ER the C-terminus of some proteins is proteolytically cleaved off and the resulting C-terminus is covalently coupled to a glycosylphophstidylinositol (GPI) anchor in the membrane

Hedgehog is activated by processing

• Hedgehog is expressed as a precursor protein

• The carboxyterminal area is proteolytically cleaved off

• the amino-terminal area is modified with a cholesterol residue

• processed Hedgehog is further modified with a fatty acid (palmitic acid)

• Through palmitin modification Hedgehog is associated with membranes (regulation)

• Modified hedgehog is highly hydrophobic and cannot be transported by diffusion in an aqueous environment

• The modified Hedgehog is therefore transported by lipid-protein particles (Vesicle)

Wingless/Wnts is activated by processing

• Wingless/Wnts is expressed as a precursor protein

• Wingless/Wnts is glycosylated in the ER

• Wingless/Wnts is further modified with a fatty

  acid (palmitic acid)

• Modified Wingless/Wnts is transported through Golgi by Wntless/Evi

• Modified Wingless/Wnts is highly hydrophobic and cannot be transported by diffusion in an aqueous environment

• Ubiquitin is a peptide of 76 amino acids

• A single ubiuitin at certein protein position can result in a change of biological

  activity

• A polyubiquitination is normally the signal for protein degradation in the proteasome

• SUMO is a similar peptide linked to lysine with regulatory function

• Notch ligands have to be activated via ubiquitination

• Ubiquitination also serves as a recognition signal for Epsin, which internalizes and degrades the ligands

• Notch ligands have only a short activity time