Transcript Signaling by Serine/Threonine Kinase Receptors

Signaling by Serine/Threonine
Kinase Receptors
Major Classes of Protein Ser/Thr Kinases
(not a complete list)
• 2nd-Messenger-dependent protein kinases
cAMP-dependent protein kinase
cGMP-dependent protein kinase
Ca2+/CaM-dependent protein kinases
Protein kinase C
• 2nd-Messenger-independent protein kinases
MAPK kinase cascade and target kinases
Raf, MEK kinases, MEKs, SEKs, ERKs, JNKs, SAPKs, RSKs
Major Classes of Protein Ser/Thr Kinases (cont’d)
• Cyclin dependent kinases (CDKs) and CDK regulating
kinases
Cdc-2, CAK, CAK kinase
• GPCR kinases
GRK2 (βARK1), GRK3 (βARK2), GRK5, GRK6
• P21-activated kinases (PAK)
• Kinases involved in cytoskeletal organization and
development
ROCK/Rho kinase
• Transmembrane receptor protein ser/thr kinases
TGFβ receptor protein kinase
• Casein kinases
CK1, CK2
Receptors with intrinsic serine/threonine kinase activity
Smad 6/7 are inhibitory - they act as decoys and
occupy the sites that would normally be
utilized by other activating Smads.
Other proteins such as noggin and chordin bind
BMPs and inhibit their ability to activate
receptors
Follistatin inhibits signaling via activins
Ser/Thr Protein Kinases
2 Major Types:
1. 2nd-messenger-dependent protein kinases.
2. 2nd-messenger-independent protein kinases.
2nd-Messenger-Dependent Protein Kinases
All kinases in this category share common design:
Several functional domains that can reside on the
same pp chain or on separate ones.
Each kinase has a catalytic domain (intrinsically
active), which is kept inactive by a regulatory
domain.
Regulatory domain have auto-inhibitory regions and
binding sites for 2nd messengers.
Interaction with the 2nd messenger dissociates the
auto-inhibitory site from the cat domain  disinhibiting it.
Additional regions of the kinases may be
responsible for oligomerization or for targeting
the kinases to distinct cellular locations.
PKA
The function of cAMP
Targeting PKA (cyclic-AMP-dependent protein kinase A)
Schematic view of
the different domains
of 2nd-messengerdependent protein
kinases. The different
kinases contain a
regulatory domain
that encodes
specialized functional
domains and a
conserved cat domain
kept inactive by the
auto-inhibitory
region (black).
PKA
• Binding affinities of reg subunits to cAMP:
RIIβ < RIIα < RIα < RIβ
Necessary for decoding the cAMP signals that
differ in intensity and duration:
PKAI activated transiently by weak cAMP signals
PKAII activated by persistent and strong cAMP
levels.
RIα may be protective against proteolytic
degradation (as in, say, when one of the other
subunits are removed (gene targeting)).
Ca2+/CaM kinases (next slide)
(A) Domains and holoenzyme structure of CaMKII.
(B) Mechanism of autophosphorylation.
Thr286.
Displacement of the autoinhibitory domain by
calmodulin exposes T286, which can then be
phosphorylated if its proximate neighbor is
active.
(C) Trapping of calmodulin to the CaMKII subunits
increases the P(autophosphorylation) during
successive Ca2+ spikes at high frequency.
Ca2+/CaM kinases
Autophosphorylation of Thr286/287 has 2 consequences:
1. Calmodulin remains bound to the phosphorylated subunit
for extended periods of time even at low [Ca2+] (trapped
state) because the autophosphorylation greatly decreases
the calmodulin dissociation.
2. Autophosphorylated α and β subunits are rendered
Ca/CaM-independent (autonomous), but still retain
substantial kinase activity.
Both these consequences (calmodulin trapping and
autonomy) allow the phosphorylated kinase subunits to
remain active beyond the limited duration of a Ca2+ spike.
Transgenic mice carrying Thr286A1a point mutation in
αCaMKII do not exhibit LTP.
NMDAR subunits (NR2B) remain active even after dissociation
of Ca2+/CaM.
PKC
Family consists of at least 10 structurally related
phospholipid-dependent protein kinases.
Most are expressed in brain.
All isozymes grouped into 3 subclasses according to
their regulatory properties (conferred by specific
domains of the protein):
“Conventional”: (cPKCs; α, βI, βII, γ) are regulated
by Ca2+, DAG, or phorbol esters and PS.
“Novel”: (nPKCs; δ, ε, θ, η) activated by DAG or
phorbol esters and PS, but are Ca2+-independent.
“Atypical”: (aPKC; μ) unresponsive to DAG, Ca2+, or
phorbol esters.
PKC
Refer to Schematic view of PKC (earlier slide):
Single polypeptide chain = N-term regulatory
domain and C-term cat (kinase) domain.
Regulatory: auto-inhibitory or pseudo-substrate
site and 1-2 membrane targeting motifs (C1
and C2).
C1 binds phorbol esters and DAG.
C2 binds acidic phospholipids and Ca2+ (in the
cPKCs).
PKC
Regulation of PKC isozymes: requires removal of
the auto-inhibitory pseudo-substrate from the
active site (high specific binding of DAG and PS
to the C1 and C2 domains)  conformational
Δ
This allows translocation of PKCs from cytoplasm
to the membrane  enzyme is maximally
activated.
But, both phosphorylation of PKCs and specific
protein-protein interactions are also
important for their activation.
PKC
One critical protein-protein interaction is with PDK1:
PDK-1 phosphorylates PKC  auto-phosphorylation
of 2 additional residues.
Necessary for the newly synthesized PKC isozymes
to achieve catalytically competent conformation.
Inactive conformation: binds AKAPs (anchoring kinase
associated proteins) and 14-3-3.
Active conformation: RACKs (receptors for activated C kinase)
Both kinds of binding proteins aid in shuttling PKC
to specific compartments.
PKG
A dimer of 2 identical subunits.
Compared to the other kinases, very limited
cellular distribution.
The 2nd messenger, cGMP, very limited in its
actions.
2nd-MessengerIndependent
Protein Kinases
MAPK Cascade.
See next slide for
repertoire of
neuronal GEFs
that convey on
Ras and Rap the
extranuclear
signals leading to
MAPK activation
and gene
expresion.
Neuronal Substrates of Kinases
Neurotransmitter Release
E.g., PKA and CaMKII phosphorylate synapsin I in
the N-terminus.
CaMKII phosphorylates synapsin I in the Cterminus.
ERKs phosphorylate synapsin I in both N- and
C-terminus.
Neuronal Substrates of Kinases
Ligand-gated ion channels and K+ channels
E.g., CaMKII phosphorylates AMPARs (LTP).
E.g., PKA phosphorylates GluR1 subunits . incr
P(opening) of AMPARs.
E.g. PKC phosphorylates GluR2 subunits 
differentially regulates its interaction with PDZ
domain proteins, GRIP1 and PICK1.
E.g., PKC, PKA, CaMKII, and ERK all phosphorylate
Kv4.2 (VG K+ channel).
E.g., PKC and PKA phosphorylate the Kir1 channel.
Neuronal Substrates of Kinases
Transcription Factors
Many Different Kinases Phosphorylate CREB
CREB – end-point of several signaling pathways
Role of the Kinases in Synaptic
Transmission and Cross-Talk
Between the Different kinase
Pathways
Role of Kinases in Synaptic Plasticity
•
•
•
•
PKA
CaMKII
PKC
MAPKs
Post-synaptic Signaling by Glutamate Release in an Excitatory
Synapse
Serine Threonine Phosphatases
• Critical for signaling and regulating complex
neuronal functions (e.g., synaptic plasticity)
activated by phosphorylation.
• Arise from different genes with no homology.
• Impossible to i.d. consensus sequences for their
specificity of substrate recognition.
• The same phosphatases can dephos substrates
that have been phos by different kinases.
• A limited number of multifunctional
phosphatases (PP1 and PP2B) account for most
phosphatase activity.
Serine Threonine Phosphatases
Protein Phosphatase 2B (PP2B, Calcineurin)
• Ca/CaM-dependent phosphatase.
• Highly enriched in brain.
• Heterodimer [A (60 kDal) and B (19 kDal )
subunits].
• A subunit: N-term cat domain and C-term reg
domain.
• B subunit: CaCaM binding domain.
• PP2B activated by low [Ca2+].
Serine Threonine Phosphatases
Protein Phosphatase 2B (PP2B, Calcineurin)
• At least 3 PP2B binding proteins have been
identified as inhibitors of the enzyme: CAIN,
AKAP 79, and FKBP12.
• E.g., FKBP12 promotes the assoc of PP2B to
ryanodine and IP3 receptors allowing PP2B to
regulate [Ca2+]free cyto.
• E.g., AKAP 79 binds both PP2B and PKA,
bringing them close together for the
regulation of receptors and ion channels.
Serine Threonine Phosphatases
Protein Phosphatase 2B (PP2B, Calcineurin)
• PP2B expressed highly in hipp: in dendritic
spines, but is virtually absent in glia and
interneurons.
• PP2B is specific for CREB’s ser133.
• In mice over-expressing the auto-inhibitory
domain of PP2B, LTP, at subsaturating
tetanization, but not saturating conditions,
was enhanced 
• Inhibition of PP2B facilitates LTP formation.
Serine Threonine Phosphatases
Protein Phosphatase 1 (PP1)
• Several subunit catalytic isoforms, α, β, γ1,
and γ2, interacts with other proteins in
subcellular targeting.
• E.g., PP1 binds Yotiao, which, in turn, also
binds NR1 receptors and the RII reg subunit of
PKA so that PP1 and PKA activity are target to
the NMDARs.
• PP1 activity can be regulated in various ways,
including direct inhibition:
Serine Threonine Phosphatases
Protein Phosphatase 1 (PP1)
• There are at least 4 known PP1 inhibitory
proteins: Inhibitor-1 (I1), inhibitor-2 (I2),
dopamine, and cAMP-regulated phosphoprotein
(DARP-32), and nuc inhibitor (NIPP1).
• PP1 activity is differentially regulated by the
phosphorylation of proteins:
Phos of I1 and DARP-32 inhibits PP1 activity;
Phos of I2 and NIPP1 activate PP1.
Gating Mechanism for PP1 Regulation
PKA
PP2B
ATP
ADP
ADP
ATP
I1 (T35) and
DARP-32 (T34)
cAMP pathway
activation would
phosphorylate and
activate DARP32
Ca2+ pathways activation
would dephosphorylate
and inactivate DARP-32
Signals