IGP Cell Signaling Section 2004

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IGP - Cell Signaling Section 2004 Monday,
February 9 1000-1100 am Lecture 2 Scaffold
and Anchoring Proteins Brian E.
Wadzinski Department of Pharmacology Office 424
RRB Email brian.wadzinski_at_vanderbilt.edu Phone
3-2080
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.
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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 usually inactive in response to
the appropriate signal, the enzyme is activated.
Protein modules for the assembly of signaling
complexes. Modular domains recognizing specific
sequences on their target acceptor protein are
depicted.
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Anchoring and scaffold proteins (molecular glue)
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Identification of A-kinase anchoring proteins
(AKAPs)

• 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
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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. Following electrotransfer, RII-binding
proteins were visualized by gel overlay (using
radiolabeled RII) and autoradiography. 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
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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
• - frequently exploited in the analysis of
  AKAP-mediated functions (exhibits nM affinity).

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Mechanism of RII binding to AKAPs
PKA holoenzyme

• PKA holoenzyme
• 3 catalytic subunits (a, b, g) which exhibit
  virtually identical properties.
• 2 different R subunits (RI and RII)
• - distinct cAMP binding affinities
• - different localization (RI mainly cytosolic,
  RII mainly particulate)
• RII binds AKAPs - explains distribution

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Schematic representation of a prototypic AKAP
signaling complex
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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.
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Targeting of AKAPs to subcellular structures

• Membrane targeting of AKAP79 signaling complex
• Potential 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).

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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.

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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).

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Targeting proteins for serine/threonine kinases
and phosphatases
Ht31 peptide
Peptides containing RVXF motif
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Scaffold proteins for mitogen-activated protein
kinases (MAPKs)
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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.

• Advantages of scaffolds in the control of cell
  signaling
• Tight regulation - signals pass quickly from one
  kinase to the next.
• Allows localized responses within the cell.
• Prevents crosstalk between pathways.

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b-Arrestin 2 a receptor-regulated MAPK scaffold
for JNK activation
McDonald, et al. (2000) Science 2901574-1577

• b-arrestins are involved in GPCR desensitization
  (bind GRK phosphorylated GPCR, facilitate
  clathrin-mediated GPCR endocytosis).
• Recently, c-Jun amino-terminal kinase 3 (JNK3)
  was identified as a binding partner for
  b-arrestin.
• Apoptosis signal-regulating kinase (ASK1) and
  MKK4 were 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,
  which facilitates the phosphorylation of JNK3 by
  MKK4.

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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).

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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.