Exam Study Notes

Signal Transduction

  • Juxtacrine Signaling: Requires physical contact between cells.
  • Receptor Inhibitors: Compounds preventing ligand binding are antagonists.
  • G-Protein Activation:
    • When a G-protein-coupled receptor (GPCR) activates a G protein, the α subunit exchanges GDP for GTP.
  • Second Messengers:
    • Adenylyl cyclase is NOT a second messenger; it produces cAMP, which is a second messenger.
  • Cholera Toxin:
    • Targets Gs protein and inhibits its GTPase activity, locking it in the GTP-bound form.
  • Protein Kinase A (PKA) Activation:
    • PKA is activated when the regulatory subunits bind to cAMP, releasing the active catalytic subunits.

Adrenaline and Muscle Cells

  • Adrenaline binding activates the Gs-signaling pathway, increasing intracellular glucose levels by breaking down muscle glycogen.
  • Cholera Toxin Effect:
    • Muscle cells treated with cholera toxin (which inhibits Gs GTPase activity) will have higher intracellular glucose levels compared to normal cells when adrenaline is present.
  • cAMP Phosphodiesterase Overexpression:
    • Cells overexpressing cAMP phosphodiesterase (which breaks down cAMP) will have lower intracellular glucose levels compared to normal cells when adrenaline is present because cAMP levels will be reduced.
  • Mutant PKA:
    • If PKA catalytic subunits cannot associate with regulatory subunits, the intracellular glucose levels will be higher because the catalytic subunits will always be active even without cAMP.

GPCR in the Brain

  • Gq Pathway and CaM-Kinase:
    • A GPCR in the brain enhances learning and memory via the Gq pathway, leading to CaM-kinase activation.
    • Mice lacking CaM-kinase expression show learning and memory defects.
  • Other Mutant Mice:
    • Mice lacking the IP3 receptor would likely exhibit similar defects because the Gq pathway activates phospholipase C, which produces IP3, leading to calcium release and CaM-kinase activation. Blocking IP3 production or reception would disrupt this pathway

Thyroid Cells

  • Gq Proteins and Thyroid Hormone:
    • Stimulation of adrenergic receptors activates Gq proteins, resulting in thyroxine secretion and cell proliferation.
  • GTPγS Treatment:
    • Treatment with GTPγS (a non-hydrolyzable GTP analog) increases both cell proliferation and thyroxine secretion.
  • PKC Inhibition:
    • Inhibiting PKC decreases thyroxine secretion without affecting cell proliferation.
  • A23187 Treatment:
    • Increasing intracellular Ca++ increases cell proliferation without affecting thyroxine secretion.
      • Thyroxine secretion is induced by DAG production.
      • Cell proliferation is induced by Calcium.

IP3 Receptors

  • IP3 receptors are associated with ER membranes.

RTK Signaling in Cockroaches

  • RTKX and Ras:
    • Mutant male cockroaches with loss-of-function mutations in RTKX (a receptor tyrosine kinase) or Ras are oblivious to female pheromones.
  • Restoring Function:
    • Additional mutations that increase the function of protein Z restore the ability to respond to females in Ras loss-of-function mutants.
    • Z could be Ras-GAP (GTPase activating protein).

PI3-Kinase Pathway

  • EGF-Induced Cell Survival:
    • The PI3-kinase pathway mediates EGF-induced cell survival and is activated when PI3-kinase binds to phosphorylated tyrosine 134 on the EGF receptor.
  • Blocking PI3-Kinase Recruitment:
    • To block the PI3-kinase pathway, replace tyrosine 134 with alanine. Alanine cannot be phosphorylated, preventing PI3-kinase binding.

Superchick Receptor

  • RTK Mutations and Cell Proliferation:
    • A mutation that causes RTK to spontaneously dimerize leads to uncontrolled cell proliferation, promoting tumor formation, as it activates the receptor without needing the growth factor.

PH Domain

  • PH Domain Containing Protein:
    • PI3-kinase is likely to contain a PH domain, which binds to phosphoinositides.

PDK

  • PDK phosphorylates Akt.

Dominant Negative Effect

  • Mutant Proteins:
    • A mutant PI3-kinase protein that binds to active RTKs but lacks kinase function would have a dominant negative effect, preventing the normal PI3-kinase from being activated.

Phosphoinositides

  • Preferred Substrates:
    • Phosphoinositide B is the preferred substrate for Phospholipase C.
    • Phosphoinositide A is the preferred substrate for PI3-kinase.
    • Phosphoinositide E is the preferred substrate for PTEN.

PTEN

  • PTEN is a phosphatase.

Growth Factor Z

  • Protein X and Protein Y work sequentially in the growth factor Z-mediated signal transduction.
    • Following growth factor Z receptor activation, Protein X activates Protein Y to induce cell proliferation.
    • Protein X is upstream of Protein Y.
      • The fibroblasts will proliferate even when growth factor Z is added
    • The fibroblasts will not proliferate even when growth factor Z is added

Cell Cycle

  • Cyclins:
    • Cyclins modulate cell cycle progression by activating protein kinases (Cdks).
    • Cyclin presence is required for Cdk activation
  • Cdk Activity:
    • Cdk phosphorylation, Cdk dephosphorylation, and cyclin synthesis and degradation is involved in regulating mitotic Cdk activity.
  • p53 and Rb Mutations:
    • Mutations in p53 and Rb are commonly loss-of-function mutations.
  • Retinoblastoma (Rb) Protein:
    • The Retinoblastoma (Rb) protein blocks cells from entering the cell cycle by inhibiting G1/S- and S-cyclin expression.
  • Rb Phosphorylation:
    • Rb gets phosphorylated upon Erk activation.
  • UV Irradiation Response:
    • UV irradiation leads to C (activation of ATM/ATR), D (activation of Chk1/Chk2), B (p53 accumulation), and then A (expression of a Cdk inhibitor).
  • Gain-of-Function Mutations:
    • Gain-of-function mutations in G1-cyclin can increase cell proliferation.
  • Mitotic Cdk: M-CDK is present during M stage.
  • Mitotic cdk-cyclin activity is inhibited by Wee1.
  • Mutations in Mad/Bub result in uneven distribution of chromosomes after cell division because spindle assembly checkpoint is disrupted.
  • DNA contents in terminally differentiated cells in G0 phase are 2n.

Yeast Cell Cycle Mutant

  • G2 to M Transition Failure:
    • If yeast cells fail to transition from G2 to M phase despite normal M cyclin-Cdk complex formation, inactivation of Cdc25 (a phosphatase that activates M-Cdk) is likely responsible.

APC and Degradation

  • APC Targets:
    • APC directly targets securin for degradation.

Nocodazole Treatment

  • Spindle Formation Inhibition:
    • Cells treated with nocodazole (inhibits spindle formation) arrest in metaphase, and APC is not activated.

Yeast Protein Homologs

  • Yeast vs. Mammalian Proteins:
    • Cut1 corresponds to separase.
    • Cut2 corresponds to securin.
    • Cut4 corresponds to APC.

FACS Analysis

  • After treatment with UV, cells will be arrested so that FACS analysis shows a higher peak (higher cell population) at 2n, indicative of G1 arrest.
  • If p53 cannot be phosphorylated (Cell line A), cells will progress into S phase because DNA damage checkpoint control is not working properly to arrest the cells in G1. So, FACS analysis shows no clear separation between 2N and 4N peaks.
  • If cells cannot expression p21 (Cell line B), cells will progress into S phase because DNA damage checkpoint control is not working properly to arrest the cells in G1. So, FACS analysis shows no clear separation between 2N and 4N peaks.

Brainy Receptor

  • If Drug X increases Smarty expression when Brainy is activated, it is likely an activator of PI3-kinase, PDK1, or Akt, any of which would enhance the PI3-kinase pathway.
  • If Drug Y inhibits Smarty expression when Brainy is activated, it's likely an inhibitor of PI3-kinase, PDK1, or Akt, disrupting the pathway.