Meeting 21, 23, 22

• (1) Jak/STAT signaling pathway
• (2) RTK’s (EGF, FGF): mutations in cause almost all different types of cancer
• (3) TGFβ
• (4) Wnt: involved in B.cancer
• (5) Hedgehog: involved in brain tumors (when hedgehog pathway is activated)
• (6) Notch

• domain found in Src-oncoprotein and in many other intracellular signal-transducing proteins
• -the domain recognizes a phosphorylated tyrosine (Y-PO4) on another protein
• -its presence on a protein helps that protein "find" said other protein
• -ONLY useful if tyrosine on other protein has a phosphate attached

• •SH1 domain: catalytic kinase domain
• •SH2: binds Y-PO₄ peptides with consensus
• •SH3: Interacts with Proline-rich areas (mediate protein-protein interactions)
• •SH4: myristylation (membrane-localization signal)

• HERE'S ANOTHER SEPERATE ONE:
• *PH domains recognize PIP₂ + phosphates

• • Jak (just another kinase) is soluble: not attached to membrane, free to move inside the cytoplast; has an affiinity for α-interferon receptors though
• • ligand (α-interferon) binds to α-interferon receptor, causing them to dimerize
• • overall: leads to cross activation of Tyk2 & Jak1; the two add phosphates to each other and become activated; (slight tyrosine kinase activity causes phosphorylation of Jak, which in turn phosphorylates Tyk)
• • phosphorylation of Tyk turns it into phosphotyrosine, which in addition to phosphorylating Jak, also recruits STAT, a TX factor
• -STAT's SH2 domain is why it has a high binding affinity for phosphotyrosine
• • STAT protein gets phosphorylated
• -2 STAT proteins dimerize if they're e/c attached to a phsophate ---> can now activate Tx
• • interferon system in our body fights viral infections (+ MS); also important for response to cytokines: signals produced by immune system
• -most likely STAT dimer activates genes that tell immunce cells to proliferate

• • receptor tyrosine kinases (*kinases add phosphates to hydroxyl group in serine, threonine or tyrosine)
• -tyrosine acts on top (beginning) of the pathway, serine kinases act more downstream

• a large family of single-pass transmembrane receptors

• often growth factor receptors (ex. EGF, Insulin)

• cytoplasmic domain: contains protein tyrosine kinase; homology among all RTKs b/c they all serve the same purpose, to activate tyrosine kinase

• extracellular domain varies greatly: responsible for recognition of different ligands

• activation involves trans-phosphorylation of specific tyrosine residues for downstream recruitment & stimulation of kinase activity

• •ligand binds to receptor, causes dimerization of thereceptor
• •results in phosphorylation by cross-phosphorylation
• • mutating one Y-kinase domain on one receptor: there’s NO kinase activation ---> shows that the two domains act on each other & phosphorylate cross-activationally (cannot activate themselves)
• - to treat a cancer caused by the activation of these receptors, could add mutant receptors (specifically mutated in their protein Y-kinase catalytic region) to prevent activation/phosphorylation of such receptors

• • EGFs (epidermal growth factors) act as ligands to their RTK receptors
• • GRB2 binds to an activated (phsophorylated) RTK (has a SH2 domain) & brings along w/ it a Sos protein
• -Sos protein has GEF activity; when in proximity to Ras it ACTIVATES it (by swapping its GDP for GTP)

• • activated Ras (GTP bound) goes ON to:
• 
• • relieve inhibition of Raf; leads to Raf kinase activity
• • specifically phosphorylates MAK2K (3K?)
• • MAP2K activates (phosphorylates) MAPK
• • MAPK (MAP kinase) translocates to nucleus where it activates numerous TX factors

• a preformed signaling complex located on a scaffold
• 
• -oftentimes the way all these proteins interact with a receptor in line is b/c activated receptor recruits their SH2 activity

• -stands for Wingless; like a drosophila mutant w/out wings
• -controls development of MANY different systems in different animal development

-ex. in Hydra, it controls tentacle formaiton

• transmembrane proteins that ensure cells w/in tissues are bound together (cell adhesion)
• -depend Ca2+ ions to function
• -name = shortening of "calcium-dependent adhesion"

• (just when you thought all hope was lost)
• • Wnt molecule acts as a ligand
• - there are 2 possible scenarios for the path, depending on whether Wnt is present

• 1) NO Wnt PRESENT
• • without Wnt, β-catenin complexes w/ 3 proteins: Axin, APC, & GSK3
• - Axin: scaffolding protein that mediates the formation of this complex
• - GSK3: phosphorylates β-catenin, tagging it for degradation
• - *also TCF: TX factor in the nucleus represses target genes without modification

• 2) Wnt PRESENT!
• • Fz (Frizzled) = Wnt receptor
• -when bound to Wnt ligand, it triggers phosphorylation of LRP receptor (just another receptor) by GSK3 & another kinase
• • phosphorylated/activated LRP binds Axin
• -this disrupts the formation of that β-catenin + 3 other protein complex, preventing (!) phosphorylation & degredation of β-catenin [by GSK3]
• • β-catenin accumulates in cell
• • β-catenin translocates into nucleus
• • binds to TCF, and allows activation of formerly repressed genes

*non-autonomy: Wnt can act at a distance; diffuses through cells

Canonical: previously described Wnt pathway; prevents inhibition by TCF of necessary genes

• Non-canonical: polarizes the cell
• - doesn't change the cell's fate like canonical does (via β-catenin), just determins its planar cell polarity (PCP)
• - PCP: which direction is R & which is L (opposite of apical/basal!)

explains strabismus, or cross-eyedness

• -perpendicular to apical-basolateral polarity
• -PCP comes about because of non-canonical Wnt pathway

• -just key signaling factors
• • Fz (frizzled): lalalalalala
• • Dgo (Diego): interacts with Dsh
• • Stbm: interacts with Pk
• • Dsh: Interacts with Dgo
• • Pk (Prickle): interacts wwith Stbm

• - appears there are intracellular antagonistic interactions btwn Fz/Dsh & Stbm/Pk complexes
• 
• - MAYBE THAT'S HOW THE CELL IS POLARIZED

• • signaling pathway involved in lateral inhibition
• • both Delta & Notch are embedded in the membrane
• • in the absence of Delta, the extracellular subunit of Notch on a responding cell is associated w/ its transmembrane cytosolic subunit
• • when Notch binds to Delta, it's cleaved
• -its extracellular & cytosolic portions are released
• - cytosolic part travels to nucleus to direct TX factors (in example, prevents expression of genes that might turn a cell 'blue')

*cleavage of cytosolic portion of Notch is accomplished by γ-secretase

• •They're going to ask you a question: if one cell has higher Delta activity, and another has higher Notch, which will remain a ______ cell and which will likely be converted into a ______ (ex. nerve) cell?
• -DELTA CELL will be converted to nerve; occurs by random jostling if both Delta & Notch receptors are expressed in both cells

• (1) if cells only express delta & notch is malfunctioning, then ALL cells will differentiate to become nerve cells, or something
• • nothing to prevent transition

• (2) by cutting Notch from outside to inside (?), can introduce forced processing of Notch receptor
• - makes cells think they’ve received the Notch signal, meaning EVERYONE's inhibited (aka remains an epidermal cell, no differentiation)

• • Notch (N) is a recessive gene; only one copy needed to function
• • pink piece of tissue is heterozygous for Notch (N-/N+); is normal
• • sometimes cells get 2 copies of mutated Notch (-/-), or 2 copies of WT notch (+/+) when mitosis occurs
• • in the absence of Notch (N-/N-) all cells are normal

• • at the border of N+/N+ and N-/N+: neuroblasts form, always on the +/- side because....
• -heterozygotes have fewer notch proteins
• -a cell w/ only one notch copy recieves less signal & is less repressed --> more likely to differentiate into nerve cell

• • notch is short range & stochastic (random)
• • is autocatalytic: the reaction product itself (repression of differentiation) is the catalyst for that reaction

• - ZPA (zone of polarizing activity): located in posterior mesoderm
• - signals produced by organizer change gene expression in receiving cells, leading to differentiation of particular structures
• - signal produced in ZPA is SHh (hedgehog)
• - the same signal exists in the spinal cord
• - Hh mutants are cyclops' & also has implications in limbs (ex. cats have too many digits, polydactyl)

• • in the absence of Hh
• - basically a TX factor, Ci, is 1st phosphorylated then cleaved, resulting in repression of Hh-responsive genes

• • when Hh is present
• - the factors that phosphorylate/cleave Ci are 'distracted', meaning it isn't cleaved and can activate TX (when complexed w/ CREB-binding protein (CRB))

• *if you remove Ci, the cell will NOT have the same phenotype as if there weren't Hh (b/c fragmented Ci acts as repressor)
• -you'd fail to repressed & fail to activate

-might recieve less input from other signaling pathways (unlike others studied), b/c it's more DIRECT

• Hh comes from: floor plate
• BMP comes from: head plate

-both induce formation of MNs (?), motor neurons

probably how Hh diffuses, even though there are still some examples of it diffusing via relay

• • French flag is used to represent the effect a morphogen has on cell differentiation
• • morphogen affects cell states based on concentration, & states are represented by the different colors of the French flag
• -high concentrations activate a "blue" gene
• - middle concentrations activate a "white" gene
• - low concentrations activate a "red" gene

• • can be applied to Hh, the level of which determines type of neuron development
• -High levels (blue): motor neurons form (MNs)
• -Medium levels (white): interneurons form
• -Low levels (red): sensory neurons form

• • posterior compartment of normal wing contains engrailed gene
• (can also visualize it used apterous, marks posterior of wing)

• • where sources of morphogens are located
• - ex. Dpp

• similar to Jak/STAT pathway: both recruit + phsophorylate TX factor, arrive @ target gene & activate transcription

• (1) TGFβ binds to RII (TGFβ) receptor
• - TII = a constantly active kinase
• (2) ligand bound RII recruits/activates RI
• (3) activated RI phosphorylates Smad3, unmasking its NLS (nuclear localization sequence)
• (4) complex is formed of
• - 2 Smad3's, Smad4 & Importin-β (Imp-β)
• (5) complex is translocated to nucleus
• (6) Imp-β dissociates
• (7) remaining Smad3/Smad4 complex associates with TX factor (TFE3), activating TX of target genes