Meeting 21, 23, 22

  1. Major pathways that control development (6)
    • (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
  2. SH2 (Src Homology 2) domain
    • 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
  3. Other SH Src-homology regions
    • •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)

    • *PH domains recognize PIP2 + phosphates
  4. The Jak/STAT pathway: type of Cell Surface Receptor pathway
    Image Upload 1
    • • 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
  5. RTKs are...
    • • 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
  6. Image Upload 2
    • •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
  7. The Ras Pathway
    Image Upload 3
    • • 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:
    • Image Upload 4
    • • 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
  8. Signalosome
    • a preformed signaling complex located on a scaffold
    • Image Upload 5
    • -oftentimes the way all these proteins interact with a receptor in line is b/c activated receptor recruits their SH2 activity
  9. overview
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  10. Wnt Signaling
    • -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
  11. Cadherins
    • transmembrane proteins that ensure cells w/in tissues are bound together (cell adhesion)
    • -depend Ca2+ ions to function
    • -name = shortening of "calcium-dependent adhesion"
  12. The Wnt pathway
    Image Upload 7
    • (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
  13. Canonical vs. Non-canonical Wnt pathway
    Image Upload 8
    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!)
  14. planar cell polarity defects in the mouse cochlea
    explains strabismus, or cross-eyedness
  15. Planar cell polarity (PCP): plane of an epithelium
    Image Upload 9
    • -perpendicular to apical-basolateral polarity
    • -PCP comes about because of non-canonical Wnt pathway
  16. Core PCP factors
    Image Upload 10
    • -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
    • Image Upload 11
  17. The Notch/Delta Signaling Pathway
    Image Upload 12
    • • 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
  18. Malfuncting Notch Can Result in (2) Different Mutations
    • (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)
  19. Dosage dependence of Notch and Delta (Blob!)
    Image Upload 13
    • • 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
  20. limb-bud organizer
    • - 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)
  21. Hedgehog (Hh) Signaling Pathway
    • • 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
  22. Hh comes from:
    BMP comes from:
    • Hh comes from: floor plate
    • BMP comes from: head plate

    -both induce formation of MNs (?), motor neurons
  23. relay signalingImage Upload 14
  24. gradient mechanism
    Image Upload 15
    probably how Hh diffuses, even though there are still some examples of it diffusing via relay
  25. the French flag model
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    • • 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
  26. Image Upload 17
    • • posterior compartment of normal wing contains engrailed gene
    • (can also visualize it used apterous, marks posterior of wing)
  27. compartment boundaries
    • • where sources of morphogens are located
    • - ex. Dpp
  28. TGFβ/BMP/Dpp Signaling Pathway
    Image Upload 18
    • 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
Card Set
Meeting 21, 23, 22
Jak/Stat, RTKs, Wnt, HdgHg,