Lecture 15- Signaling 2 Flashcards
Phosphorylation
- Covalent attachment of a phosphate group.
- Protein kinases add phosphates to proteins.
- Phosphates may be added to serine, threonine, or tyrosine residues
Protein phosphatase
-Removes the phosphate group
G-Protein
- GTP bound state changes conformation so can interact with other proteins (signal-on state)
- Hydrolysis of GTP to GDP returns it to the signal-off state. Some G proteins will remain on without the assistance of a GAP (GTPase activating protein)
- Re-activation of the G-protein requires release of GDP and binding of GTP. Ususally requires a GEF (GTP exchange factor) protein
Two classes of G-Protein
Trimeric and monomeric
Trimeric G-Protein Coupled Receptors
- Three subunits (alpha, beta, gamma). Alpha has guanine nucleotide binding sites.
- Linked to cytosolic face of PM by lipid tails
Trimeric G-Protein Coupled Receptors Steps
1) Ligand binds the GPCR Receptor
2) Trimeric G-Protein binds the intracellular part of that receptor
3) GDP-GTP exchange is stimulated (receptor itself does not bind GTP)
4) G alpha GTP subunit separates from beta and gamma
5) The free alpha GTP and beta/gamma dimer bind to and alter activities of membrane-associated enzymes or ion channels.
6) Bound GTP is slowly hydrolyzed to GDP, GDP dissociates slowly from alpha chain
Gs family of trimeric G-proteins
- Activation of Gs family members stimulates adenylate cyclase, raising cAMP concentrations
- Increased cAMP concentration stimulate protein kinase A, a Ser/Thr kinase
- Binding of alpha s to adenylate cyclase stimulates alpha s GTPase activity, shutting off the signal
PKA
- Has 2 catalytic subunits and 2 regulatory subunits
- When no cAMP present the catalytic subunits are inhibited by the regulatory subunits
- When cAMP binds to the regulatory subunits they dissociate from the catalytic subunits, permitting high levels of activity.
- Destruction of cAMP by phosphodiesterase leads to re-association of regulatory and catalytic subunits inhibiting PKA
Gi family of trimeric G-proteins
- Activation of Gi family members inhibits adenylate cyclase, reversing effects of Gs
- Activation also alters K+ levels in some cells
Gq family of trimeric proteins
- Activation of Gq stimulates phospholipase Cbeta which cleaves PIP2 into IP3 and DAG.
- DAG remains in the membrane and IP3 diffuses into cytoplasm
- IP3 opens gated Ca channels in ER which then stimulates CAM kinases
- DAG activates protein kinase C by increasing its affinity for Ca
Tyrosine kinases basic mechanism of activation
- Many growth factor receptors
- Receptor dimerization causes autophosphorylation of tyrosine residues in the receptor itself and stimulates kinase activity (transphosphorylation)
- SH2 domains on cytoplasmic proteins bind to phosphotyrosine groups on the receptor, this activates a sort of signal relaying
Second messenger pathway of receptor tyrosine kinases–phospholipase Cgamma
Binding of phospholipase Ggamma by its SH2 domain to a phosphorylated receptor stimulates its enzymatic activity and activates signaling though IP3 and DAG
Second messenger pathway of receptor tyrosine kinases–SH2-SH3 adaptor proteins
Binding of SH2-SH3 adaptor proteins indirectly activates monomeric G-proteins of the ras family
- SH2-SH4 recruite GEFs for ras family members
- Exchange of GTP for GDP on ras occurs very slowly in the absense of a GEF
- Ras inactivated by GTP hydrolysis aided by a GAP
Second messenger pathway of receptor tyrosine kinases–MAP kinase cascade
- Activated Ras stimulates a MAP Kinase cascade
- Map-kinase-kinase phosphorylates Map-kinase-kinase which phosphorylates Map-kinase.
- Map kinase important in growth control
Tyrosine kinase-liked receptors
- Receptor lacks kinase activity but ligand binding causes dimerization of receptors and activates a separate protein tyrosine kinase
- Mutated forms associated with tumor formation
