What is the mechanism of phosphorylation in signaling?
- Phosphorylation = covalent attachment of a phosphate group
- Phosphates may be added to the hydroxyl groups of serine, threonine, or tyrosine residues in proteins
- Phosphorylation can change protein conformation, activity, or binding affinity
- To reverse the regulation: protein phosphatase removes the phosphate group
What is the mechanism of G-protein signaling?
- G-proteins are slow GTPases that work as molecular ON/OFF switches
- The GTP-bound conformation is the 'ON' position; hydrolysis of GTP turns the switch 'OFF'
- Reactivation requires GDP-GTP exchange, which usually requires a GTP exchange factor (GEF)
- 2 classes: trimeric and monomeric
What are the properties of trimeric G-proteins?
- Trimeric G-proteins have α (contains guanine nucleotide binding sites), β, and γ subunits
- α/β/γ-GDP trimer = inactive
- Linked to the cytosolic face of the plasma membrane by lipid tails
- All known trimeric G-proteins are associated with cell surface receptor signaling
- Receptors = 7-pass transmembrane proteins that bind extracellular ligands
What is the basic mechanism of trimer G-protein coupled receptor (GPCR) regulation?
- Ligand binds to GCPR → trimeric G-protein binds to intracellular part of receptor → GTP-GDP exchange on α subunit
- After GTP binding: α/β/γ-GTP → activated α-GTP + activated β/γ complexes
- Both complexes are linked to membrane but can diffuse laterally; they bind to and activate enzymes and channels (below, β/γ activation of amplifying K+ channel)
- GTP → GDP + Pi (very slowly); activating subunits dissociate from target and rebind with the other complex
What is the function of the Gs family of trimeric G-proteins?
Activation of Gs family members stimulates adenylate cyclase → cAMP concentrations rise → stimulation of PKA
Binding of αs to adenylate cyclase also stimulates αs GTPase activity, shutting off the signal
What is protein kinase A (PKA, A-kinase), and how is it activated/inactivated?
A serine/threonine kinase with 2 catalytic subunits and 2 regulatory subunits.
- No cAMP present → reg. subunits inhibit cat. subunits (PKA inhibited)
- cAMP binds to reg. subunits → reg. subunits dissociate → high cat. subunit activity (PKA activated)
- Phosphodiesterase destruction of cAMP → reg. and cat. subunits reassociate (PKA inhibited)
PKA can activate transcription of genes that are regulated by the cAMP response element binding protein (CREB).
What is the function of the Gi family of trimeric G-proteins?
- Activation of Gi family members inhibits adenylate cyclase, reversing effects of Gs
- Also alters K+ channel conductance in some cells
What is the function of the Gq family of trimeric G-proteins?
Activation of Gq stimulates phospholipase C-β, which cleaves phosphoinositol bisphosphate (PIP2) into inositol trisphosphate (IP3) and diacylglycerol (DAG), beginning the process of protein kinase C (PKC, C-kinase) activation.
How is PKC activated?
- Gq activated →
- Phospholipase C-β stimulated →
- PC-β generates IP3 and DAG →
- DAG diffuses along the membrane and activates PKC by increasing its Ca2+ affinity →
- IP3 binds to gated channels in the ER and releases Ca2+ →
- Ca2+ stimulates activated PKC (+CAM kinases)
How is calmodulin involved in the transduction of Ca2+ signals?
Calmodulin is a CAM kinase regulatory subunit that dramatically changes conformation when Ca2+ ions bind to its four calcium binding sites. In the presence of Ca2+, calmodulin activates serine-/threonine-specific CAM kinases such as myosin light chain kinase (MLCK).
What are receptor tyrosine kinases, and how are they activated?
Receptor tyrosine kinases are common receptors for growth factors and often regulate choices between cell division and differentiation. It is activated in the following way:
- Ligand (usu. a GF) binds to an RTK →
- Receptor dimerization →
- Transphosphorylation (each receptor molecule phosphorylates tyrosines on its partner in the dimer)
Phosphotyrosines bind to SH2 domains on cytoplasmic receptor proteins.
What are some of the second messenger pathways of receptor tyrosine kinases?
Signaling via IP3 and DAG: SH2 domain binds phospholipase C-γ to a phosphorylated receptor
- Monomeric ras G-proteins → MAP kinase cascade
How do receptor tyrosine kinases activate monomeric G-proteins?
- SH2 domains of SH2-SH3 adaptor proteins bind and indirectly activate the ras monomeric G protein pathway
ras (→ monomeric G proteins) is activated by GTP Exchange Factors (GEFs) and turned off by GTPase Activating Proteins (GAPs), along with GTP hydrolysis
What is the MAP kinase cascade?
Activated (GTP-bound) ras activates the MAP kinase cascade:
MAP-kinase-kinase-kinase → MAP-kinase-kinase + Pi → MAP-kinase + Pi → changes in protein activity/gene expression
What are tyrosine kinase-linked receptors?
TK-linked receptors have a similar pathway to RTKs, but no kinase activity of their own.
Ligand binding → receptor dimerization → activation of a separate protein tyrosine kinase assoc. with the receptor protein → kinase transphosphorylation and receptor phosphorylation.
Other proteins with SH2 groups bind the phosphotyrosines, just as with receptor tyrosine kinases.
ex) JAK-STAT pathway