L3, Akt; Glucose Storage Flashcards
Detail the steps that leads to Akt activation upon Insulin binding
- Insulin binds to its receptor, which auto-phosph.
- IRS docks to insulin receptor, is phosph.
- PI3K docks to IRS and is activated by phosphorylation
- PI3K converts PIP2 to PIP3
- PIP3 activates PDK1 which phosph.s AKT/PDK
- AKT/PDK fully activated by phosph. by mTORC2`
Give the 3 key downstream effects of Akt activation:
- Gene transcription
- Glycogen Synthesis
- Glucose Transport
How is Glucose stored in cells? How is glycogen produced vs degraded
- Glycogen: Dendromeric structure with protein core
- Branches of alpha-1,4 linked chains and alpha-1,6 linked branches (Key: Stereochemistry)
- Synthesis regulated by Glycogen Synthase (GS)
- Glucose is released by Glycogen Phosphorylase (PP)
How does PP1 operate?
Releases Glc-1-p which intraconverts to Glc-6-P:
- In muscle Glc-6-P enters glycolysis
- In liver, Glc-6-P converted to Glc and exported
- Influenced by phosph. and allosteric regulation
How does GSK3beta influence glycogen synthesis?
- GS is phosph. and inactivated by active GSK3-beta
- PP1 opposes this by dephosphorylating to activate
- GSK3-beta itself is inactivated when phosph. by Akt
- See FC
GSK3: Structure and function
A serine/threonine kinase important in many pathways..
- Two isoforms, alpha and beta which differ by N-terminal domain
- Autophosph. of activation loop pY activates GSK3
- Y279 in alpha, Y216 in beta
- Requires priming phosph. site in recognition motif
- Inhibitory serine phosph. site in N-terminal domain (Ser9 targeted by Akt)
GLUT4 Glucose Transporter: Key Family Members
- Family of facilitative Glucose Transporters, each with 12 transmembrane regions
- GLUT4: Fat and muscle -> insulin response
- Stored in GLUT4 Storage Vesicles (GSVs)
How does insulin affect GLUT4 translocation? (Mechanisms with and without Akt)
- Absence of insulin results in endocytosis of GLUT4 into GSVs
- Insulin action (via Akt phosph of AS160) translocates GSVs to PM, allowing them to import Glc
- Evidence of an additional APS-TC10 mechanism, independent of PI3k-Akt
Describe in detail how Akt activates GLUT4 translocation
- Akt/PKB phosph. and inactivates AS160, a Rab GTPase activating protein (GAP)
- Rab remains active, in GTP-bound form
- Rab translocates GLUT4 to the cell surface
Rab protein recap
- Membrane associated small GTP-binding proteins involved in membrane transpport
- Activated by GEFs, inactivated by GAPs
- Muscle and adipocytes express several Rab isoforms
- Rabs 4, 5, 8, 10, 11 and 31 have been implicated in GLUT4 trafficking
Evidence for the role of AS160
- Insulin treatment induces Akt and AS160 phosph.
- Five AS160 phosph. sites are phosph. by Akt in response to insulin
Describe the 3 steps of GSV exocytosis including a key effector for each:
- Translocation from perinuclear region towards PM (AS160)
- Targeting of GSV to PM (APS-TC10)
- Fusion of GSV with PM (SNARE proteins)
Describe the functions of APS as an adaptor protein:
Binds phosph. Insulin receptor…
- Recruits complex including CAP, Cbl and Crk-C3G
- C3G is a ‘GEF’
- C3G activates TC10 (a Rho GTPase) by exchanging GDP for GTP (bound to PM -> acting as tether for latter steps)
- TC10-GTP recruits the exocyst complex (binds Exo70 subunit) -> RALA on GSV binds Sec5 subunit of Exocsyt complex -> targets GSV to PM via microtubules
How do GSVs fuse with the PM?
- Rab GTP binds Rab effector on PM
- SNARE proteins regulate where fusion occurs, with a coiled coil formation which drives the membrane fusion (requires R-SNARE and two Q-SNARES)
- SNARE complex dissociation driven by ATP
- e.g. R-SNARE = VAMP2, Q-SNARE = Syntaxin-4, SNAP 23
Munc18 involvement in GSV membrane fusion
- Regulates SNARE complex formation
- Binds syntaxin4 (Q-SNARE), preventing both VAMP2 and SNAP23 from binding
- Insulin releases Munc18 from Syntaxin4, permitting membrane fusion
