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IGP Signaling Course 2002

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1) T. Pawson and J. Scott (1997) Signaling through scaffold, anchoring, and ... Light induction of rhodopsin activates a G-protein (Gaq), which activates a PLC. ... – PowerPoint PPT presentation

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Title: IGP Signaling Course 2002


1
IGP Signaling Course - 2002 Wednesday, January
30 900-1000 am Scaffold and Anchoring
Proteins Brian Wadzinski Department of
Pharmacology Office 424 RRB Email
brian.wadzinski_at_vanderbilt.edu
Readings 1) T. Pawson and J. Scott (1997)
Signaling through scaffold, anchoring, and
adaptor proteins. Science 2782075-2078. 2) M.
Colledge and J. Scott (1999) AKAPs From
structure to function. Trends in Cell Biology
9216-221.
2
Signaling through scaffold, anchoring, and
adaptor proteins
Mechanisms for recruiting/localizing signal
transduction complexes. (A) Assembly of modular
signaling mole-cules on an activated receptor
tyrosine kinase. (B) A localized signaling
complex of three anchored signaling enzymes.
Bound enzyme is inactive in response to the
appropriate signal, the enzyme is released and
activated.
Protein modules for the assembly of signaling
complexes. Modular domains recognizing specific
sequences on their target acceptor protein are
depicted.
3
Kinase anchoring and scaffold proteins (
molecular glue)
4
Identification of A-kinase anchoring proteins
  • Two hormones that produce a similar elevation in
    cAMPi do not always produce the same
    physiological response.
  • Experiment Incubate isolated rabbit
    cardiomyocytes with isoproterenol (ISO) or
    prostaglandin E1 (PGE1) (both ligands activate
    Gs) prepare extracts assay cAMP levels, PKA
    activity, and glycogen phosphorylase activity.
  • cAMP PKA activity Phosphorylase activity
  • Control 4.0 0.15
    0.10
  • PGE1 9.0 0.42
    0.10
  • ISO 8.0 0.45 0.30
  • Possible interpretation
  • PGE1 and ISO (via their receptors and Gs) induce
    similar increases in cAMP levels, but in
    different parts of the cardiomyocyte. Each pool
    of cAMP activates a portion of cellular PKA (to a
    similar extent), but only one of these pools of
    PKA is functionally coupled to glycogen
    phosphorylase.
  • How can cellular PKA be divided into distinct
    pools coupled to different physiological
    responses?

Different
Similar
Similar
5
R subunits of PKA copurify with other proteins on
cAMP-agarose
Identification of R subunit interacting proteins
(gel overlay)
Tissue extracts (50 mg) were fractionated by
SDS-PAGE. After electrotransfer, RII-binding
proteins were visualized by gel gel overlay
(using radiolabeled RII) and autoradio-graphy.
Two identical blots were incubated with either
32P-RII (A) or 32P-RII in the presence of 0.4 mM
Ht31 peptide (B).
Incubate brain extracts with cAMP agarose, wash
resin with 2 M NaCl, elute proteins from resin
with cAMP, and analyze eluted proteins by
SDS-PAGE and Coomassie Blue staining.
cAMP, 0 hr
cAMP, 2 hr
cAMP, 3 hr
6
Common features of A-kinase anchoring proteins
  • Many AKAPs identified by copurification with PKA
    RII subunits, or by screening cDNA expression
    libraries for RII-binding proteins.
  • Characterization of the RII binding domains from
    these proteins revealed no sequence homology, but
    did reveal a conserved secondary structure an
    amphipathic a-helix.
  • Ht31 peptide inhibits AKAP-RII interaction (used
    in the analysis of AKAP-mediated functions).

7
Mechanism of RII binding to AKAPs
PKA holoenzyme
8
Schematic representation of a prototypic AKAP
signaling complex
9
AKAPs have diverse subcellular targets
A schematic representation of the subcellular
localization of various AKAPs. A selection of
AKAPs, the signaling molecules that they bind,
and their subcellular location are depicted.

Compartmentalization of AKAPs. Targeting of
various AKAPs to different cellular compartments
is illustrated. In each panel, the location of
the AKAP was visualized by immunofluorescent
labeling using antibodies specific for the
indicated AKAP. In all cases, the green label
indicates the AKAP.
10
Targeting of AKAPs to subcellular structures
  • AKAP79 targeting
  • Positive () targeting interactions between PS
    and PIP2, with PKC and AKAP79 basic regions
    (A,B,C).
  • Possible negative (-) regulation of targeting by
    protein phosphorylation (PKA and PKC) and
    calmodulin (Cam).
  • Other ways AKAPs are targeted
  • Fatty acid modifications (e.g. AKAP15/18 -
    targeted to plasma membrane).
  • Consensus sequences (e.g. D-AKAP1 - targeted to
    outer mitochondrial membrane).
  • Spectrin-like repeats (e.g. mAKAP - targeted to
    perinuclear locations).
  • Interactions with other proteins (e.g. AKAP79 -
    targeted to PSDs via interaction with MAGUK
    proteins).

11
Targeting of PKA to GluR through a MAGUK-AKAP
Complex
  • Compartmentalization of GluRs with signaling
    enzymes that modulate their activity is crucial
    for normal synaptic transmission.
  • Two classes of binding proteins organize these
    complexes the MAGUK proteins that cluster GluRs
    and the AKAPs that anchor kinases and
    phosphatases.
  • MAGUKs (membrane-associated guanylate kinases)
  • Modular proteins composed of three N-terminal
    PDZ domains, followed by a src homology 3 (SH3)
    domain and a guanylate kinase-like (GK) domain.
  • The C-terminal tails of the NMDA receptor and
    the AMPA receptor mediate high affinity binding
    to the PDZ domains PSD95 and SAP97, respectively
    (clustering of GluRs).
  • Colledge, et al. (Neuron 27107, 2000) recently
    reported that GluRs and PKA are recruited into
    macromolecular signaling complexes via a direct
    interaction between the MAGUK protein and AKAP79
    (the SH3 and GK regions of the MAGUKs mediate
    binding to AKAP79).
  • Cell-based studies indicate that phosphorylation
    of AMPA receptors is facilitated by a
    SAP97-AKAP79 complex that directs PKA to the
    GluR.
  • MAGUK-AKAP complex may be centrally involved in
    the control of synaptic activity.

12
The AKAP79 signaling complex
PKA
PP2B
AKAP79
PKC
Postsynaptic density
  • AKAP79 is targeted to the plasma membrane and
    postsynaptic density.
  • Directly binds PKA, PKC, and PP2B (calcineurin)
    enzymes are inhibited when bound.
  • Positions the kinases and phosphatase in close
    proximity to target proteins.
  • Efficient means of controlling phosphorylation
    state of a given protein in response to multiple
    intracellular signals.
  • AKAP79 also interacts with b2-adrenergic
    receptor (unidentified domain).

13
Targeting proteins for serine/threonine kinases
and phosphatases
14
Scaffold proteins for mitogen-activated protein
kinases (MAPKs)
15
Examples of MAPK scaffolds
Figure 2 Protein scaffolds. The scaffolding
protein for each example is shaded. (a) The
yeast signal transduction pathway involved in the
mating response uses Ste5 as a scaffolding
protein to bind the members of the MAPK module,
Ste11 (MKKK), Ste7 (MKK), and Fus3 (MAPK). Ste20
is an MKKKK in this pathway. Activation of Ste11
and Ste20 occurs with pheromone binding to the
seven-transmembrane protein pheromone receptor,
which then leads to dissociation of the Ga
subunit from the Gbg subunit. The Gbg subunit
then activates Ste11 and Ste20 10 11 12
13. (b) The high osmolarity response pathway.
In this pathway, the same MKKK (Ste11) is used.
PBS2 acts as both the MKK and the scaffold
protein. Hog 1 acts as the MAPK 18. (c) MP1
is a recently described scaffolding protein which
binds to MEK1 (an MKK) and ERK1 (a MAPK),
enhancing the efficiency of MAPK cascades
involving these proteins 19. (d) JIP-1 binds
HPK1 (an MKKKK), MLK3 or DLK (MKKKs), MKK7, and
JNK (a MAPK), leading to enhanced JNK activation
22. (e) MEKK1, in addition to acting as a
kinase for MKK4, is able to bind JNK, allowing it
to act as a scaffold to bring together all three
components of this MAPK module 26.
16
b-Arrestin 2 a receptor-regulated MAPK scaffold
for JNK activation
McDonald, et al. (2000) Science 2901574-1577
  • b-arrestins involved in GPCR desensitization
    (bind GRK phosphorylated GPCR, facilitate
    clathrin-mediated GPCR endocytosis).
  • Recently, c-Jun amino-terminal kinase 3 (JNK3)
    identified as a binding partner for b-arrestin.
  • Apoptosis signal-regulating kinase (ASK1) and
    MKK4, also found in complex.
  • GPCR activation triggers colocalization of
    b-arrestin 2 and active JNK3 to intracell.
    vesicles.
  • b-arrestin 2 acts as scaffold, which brings the
    spatial distribution and activity of a MAPK
    module under the control of a GPCR.
  • Docking site in b-arrestin 2 for binding JNK3
    and stimulating the phosphorylation of JNK3 by
    MKK4.

17
Signaling via scaffolded protein kinases is a
question of balance
  • If there is too little scaffold, signaling will
    be low (left).
  • At an intermediate concentration of scaffold
    (stoichiometric with kinase), signaling will be
    high (middle).
  • Once the concentration of scaffold exceeds that
    of the kinase it binds, the signaling begins to
    decrease (right).
  • Adding too much kinase can decrease the output
    of a scaffolded cascade (right), just as having
    too little kinase can (left).

18
INAD is a scaffold for different components of
the Dros. phototransduction pathway
  • Phototransduction in Drosophila
  • G-protein-coupled PLC signaling pathway.
  • Light induction of rhodopsin activates a
    G-protein (Gaq), which activates a PLC.
  • PLC catalyzes the hydrolysis of PIP2 into IP3
    and DAG, leading to the opening and modulation of
    light-activated ion channels (e.g. TRP).
  • Calcium-dependent regulatory processes,
    including activation of an eye-specific PKC and
    CaM, mediate deactivation of the light response.
  • Properties of Dros. phototransduction 1)
    fastest known G-protein signaling cascade 2)
    pathway displays tremendous sensitivity to light
    changes. How is speed and sensitivity achieved?
  • INAD (inactivation-no-afterpotential D)
  • Multivalent PDZ protein (5 PDZ domains).
  • Scaffold for the assembly of the
    phototransduction pathway into a macromolecular
    transduction complex.
  • Assembles PLC, TRP, and an eye-PKC.
  • This macromolecular organization endows
    photoreceptors with many of their signaling
    properties, including high sensitivity, fast
    activation and deactivation kinetics, and
    exquisite regulation by small localized changes
    in Ca2i.
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