Transcript Enzyme Catalysis

Signal Transduction
Biochemistry – February 23, 2005
Chapter 12 – parts 12.3, 12.4
Signaling Characteristics
Types of Receptors
Receptor tyrosine kinases
(RTKs)
• ligand-binding domain, single transmembrane domain,
conserved intracellular domain
• typical ligands are peptide hormones and growth factors – all
will stimulate cell division
– Insulin - released by pancreas
in the presence of high blood
[glucose]
– Vascular endothelium growth
factor
– Insulin-like growth factor
– Platelet-derived growth factor
– Epidermal growth factor
Receptor tyrosine kinases (RTKs)
• Ligand binding causes dimerization, cross-phosphorylation on tyrosine
• Phosphorylated tyrosines shift position of activation loop, exposing
substrate binding site – making it active.
• Regulatory mechanism is seen in many protein kinases
Insulin Receptor Tyrosine
Kinase activity
• Insulin receptor phosphorylates
Insulin receptor substrate-1 (IRS-1)
on specific tyrosine residues
SH2 domains
• Phosphotyrosine recognized by SH2 (src-homology)
domain found on many signaling proteins
–
–
–
–
–
non-receptor tyrosine kinases (src)
Phospholipase C-  (PLC-),
GTPase activating proteins (GAPs)
Phosphoinositide-3 kinase (PI-3K)
Adapter proteins such as Grb2
Response to Insulin
•
•
•
Phosphorylated IRS-1 bound by Grb2,
which binds Sos (son of sevenless)
Sos is a guanine nucleotide exchange
factor (GEF), activates ras by triggering
release of GDP, binding of GTP by ras
Ras is a member of the small GTPase
family – myristylation associates ras with
membrane
ras signaling
• activated ras binds raf, a S/T kinase,
localizing it to the plasma membrane
• raf phosphorylates MEK (MAP and ERK
kinase) (Y/T kinase),
• MEK phosphorylates ERK & MAP kinase
(S/T kinase)
• ERK & MAP kinase phosphorylate many
different proteins involved in cell
division and response to insulin
– Transcription factors
– Cell cycle regulators such as Cyclin
dependent kinases (cdk’s)
Insulin response
•
•
•
•
Phosphorylated IRS-1 activates PI-3K
PI-3K phosphorylates PIP2 to form PIP3
PIP3 activates PDK1 which activates protein kinase B (PKB)
PKB phosphorylates Glycogen Synthase Kinase 3 (GSK3) to INACTIVATE
it, preventing phosphorylation of Glycogen Synthase (GS)
• Phosphatases can dephosphorylate
GS to activate it
• GS uses available glucose to
produce glycogen, a storage form
of glucose.
• End result – high blood [glucose]
leads to glycogen synthesis
Stop here!
7TM receptors
• Receptors bind extracellular ligand,
such as epinephrine, triggering
conformational change. Change
allows interaction with “downstream
effectors” – often a G-protein.
• Receptors often referred to as
GPCR’s – G-protein
coupled receptors.
G-proteins
• Activated receptor functions as GEF –
guanine nucleotide exchange factor
• GDP released by heterotrimeric Gprotein, GTP bound by a subunit,
conformational change
causes dissociation
from b subunits.
• Activated (GTP bound)
a-subunit interacts with
downstream effectors
b-adrenergic receptor
stimulates Gs
• Activated Gsa activates Adenylate cyclase which produces cAMP
• Adenylate cyclase acts as a GAP – GTPase activating protein
– Hydrolysis of GTP inactivates Gsa subunit
cAMP activates protein kinase A
• cAMP activates protein kinase A by binding to regulatory
subunit, which then releases active catalytic subunit
• PKA is a S/T kinase that phosphorylates many proteins,
triggering a variety of responses
– metabolic changes
– changes in gene expression
– changes in ion transport
Response to
hormones
• Epinephrine (adrenalin) is the
fight or flight hormone - energy
reserves must be mobilized in
preparation for “action”
• Glucagon is produced by
pancreas in response to low
blood [glucose]
• Liver and muscle respond by
making energy reserves
available
Turning off response
• Receptor turned off by phosphorylation on intracellular
domain and binding of b-arrestin
– result is that more hormone binding to other receptors is
required for response.
Phospholipase C (PLC)
• Some 7TM receptors activate specific G-proteins that activate
phospholipase C b(PLCb).
• PLC cleaves PIP2 (phosphatidyl inositol-4,5-bisphosphate) present
in the cytosolic leaflet of the plasma membrane) to IP3 (inositol
trisphosphate) and DAG (diacylglycerol).
Phospholipase C
• PH and C2 domains bind
lipids, associate protein
with membrane
• EF hand domains bind
Calcium
• Catalytic domain
catalyzes reaction
PIP2 IP3 + DAG
• Regulatory domain
interacts with activator
Inositol Lipid Signalling
• IP3 diffuses to ER membrane, binds ion channel
receptor, causing release of calcium
– calcium triggers exocytosis, smooth muscle
contraction
– calcium is bound by calmodulin, which activates
cAMP phosphodiesterase (turning off cAMP
pathway) and specific protein kinases
Protein Kinase C
• calcium binds C2 domain of protein kinase C
- causes association with membrane
• DAG binds C1 domains of protein kinase C,
removing pseudosubstrate from active site
• PKC phosphorylates specific proteins to
cause a cellular response
• DAG is also a precursor of
arachidonic acid and
prostaglandins
Calcium signaling
• Can alter and monitor levels
– A23187 - Ca ionophore
– EGTA - Ca chelator
– fura-2, fluo-3, aequorin - Ca sensitive
fluorescent dyes, proteins
Calcium effectors
• Calmodulin binds 4 Ca ions
• Ca-Calmodulin binds basic amphipathic
helices on target proteins, triggering
conformational changes that activate the
protein
– Ca-ATPase to restore Ca levels
– Calmodulin dependent protein kinase II
(CaM kinase II) - different targets in
different cell types
G-protein families
Growth Factor Signalling
• often SH2 proteins also have SH3 domain
(drk-Grb2 adaptors)- involved in
interactions with other proteins, such as
Guanine nucleotide exchange factors
(e.g. sos)
• GTP binding activates ras (a small
monomeric GTPase) which
activates a protein kinase
cascade.
ras pathway
• ras pathway involved in many growth and development pathways
– Drosophila R7 photoreceptor pathway
• mutants lack R7 cell – sevenless
– sos = son of sevenless
– boss = bride of
sevenless
Adapter Proteins
• SH2 and SH3 domains of
different proteins have different
specificities for target proteins
based on sequence surrounding
phosphotyrosine (SH2) or
conserved prolines (SH3)
ras activation
• GRB2 (adaptor protein)
interacts with RTK and
Sos (son of sevenless)-like
protein
• GEF activity of Sos protein
causes ras to release GDP, bind GTP
and become activated.
• ras involved in cell growth/development
signalling pathways
• constitutive ras mutations found in up
to 50% of human cancers
ras signaling
• 14-3-3 proteins inhibit raf
activity, ras causes the
proteins to dissociate
• Ksr required for proper
interaction between raf,
14-3-3, MEK, MAP
kinase.
MAP kinase pathway
evolution
• single-celled eukaryotes do not
depend on growth signals from other
cells but do use MAP kinases to
respond to major changes in
environment
• Scaffold proteins homologous to ksr
link specific effector kinases
to signal perception
• specificity of response
dependent upon specific
binding of effectors.
Transcriptional activation
• In response to growth factor induction of MAP kinase,
pp90rsk and MAP kinase activate serum response
factors, which induce expression of fos and jun.
• fos/jun complex (AP-1) activates expression of genes
necessary for
progression
through cell
cycle.
Growth Hormone
Receptor
• Hormone binding causes
dimerization
• dimerized receptor phosphorylated
by JAK (Janus Kinase)
• JAK SH2 domains bind
phosphotyrosine on receptor
• JAK phosphorylates target
proteins (e.g STATs)
• STATs activate
transcription
Signal Amplification
• Signal transduction cascades
amplify a signal
• ligand-receptor complex can
activate many Ga
• each Ga can activate adenyl
cyclase such that many cAMP
are produced
• each cAMP activates cAPK
which can phosphorylate
multiple proteins
• each protein can then affect
multiple downstream
effectors