April 12, 2005

Maria Diverse-Pierluissi, Ph.D.Department of Pharmacology and Biological Chemistry

Mount Sinai School of Medicine

Organization of Macromolecular Complexes

Role of Scaffold Proteins

What is a scaffold protein?

What roles do they play in signal transduction?

a. Spatial localization or targeting - create microenvironment in close proximity to effectors.

b. Substrate specificity

c. Signal integration - scaffold proteins bring together signal initiators and terminators.

Roles of scaffold proteins

Targeting of type 1 phosphatase

Yotiao-greenPKA-bluePP1-red

Kinase- and phosphatase-anchoring proteins: harnessing the dynamic duo. Bauman AL and Scott JD (2002). Nature Cell Biol. 4, 203 - 206.

1) selectivity of phosphatase activity towards a subgroup of muscle proteins, 2) assembles complex with Rho and PKG.

Targeting M110/MBS:

Kinase- and phosphatase-anchoring proteins: harnessing the dynamic duo. Bauman AL and Scott JD (2002). Nature Cell Biol. 4, 203 - 206.

RACK-receptor for activated C kinase

RACK can bring together protein kinase C with several signaling molecules

Kinase- and phosphatase-anchoring proteins: harnessing the dynamic duo. Bauman AL and Scott JD (2002). Nature Cell Biol. 4, 203 - 206.

Kinase- and phosphatase-anchoring proteins: harnessing the dynamic duo. Bauman AL and Scott JD (2002). Nature Cell Biol. 4, 203 - 206.

EXAMPLE #1:

WAVE-1

Member of the Wiskott-Aldrich syndrome protein family of scaffolding proteins.

Coordinates actin reorganization by coupling Rho GTPases to the mobilization of the Arp 2/3 complex.

Identified in a screen for AKAPs that bind to the SH3 domain of Abelson tyrosine kinase.

Kinase- and phosphatase-anchoring proteins: harnessing the dynamic duo. Bauman AL and Scott JD (2002). Nature Cell Biol. 4, 203 - 206.

The WRP component of the WAVE-1 complex attenuates Rac-mediated signalling.

Soderling et al. (2002). Nature Cell Biol. 4, 970 - 975.

The WRP component of the WAVE-1 complex attenuates Rac-mediated signalling. Soderling et al. (2002). Nature Cell Biol. 4, 970 - 975.

Immunoprecipitation of WAVE-binding proteins from rat brain.

Silver stain Mass spectrometry

The WRP component of the WAVE-1 complex attenuates Rac-mediated signalling. Soderling et al. (2002). Nature Cell Biol. 4, 970 - 975.

The WRP component of the WAVE-1 complex attenuates Rac-mediated signalling. Soderling et al. (2002). Nature Cell Biol. 4, 970 - 975.

Amino-acid sequence of WRP

EST clone-Probe for Northern blot

The WRP component of the WAVE-1 complex attenuates Rac-mediated signalling. Soderling et al. (2002). Nature Cell Biol. 4, 970 - 975.

Tissue expression of mRNA of WRP determined by Northern blot

The WRP component of the WAVE-1 complex attenuates Rac-mediated signalling. Soderling et al. (2002). Nature Cell Biol. 4, 970 - 975.

Northern blot

The WRP component of the WAVE-1 complex attenuates Rac-mediated signalling. Soderling et al. (2002). Nature Cell Biol. 4, 970 - 975.

The WRP component of the WAVE-1 complex attenuates Rac-mediated signalling. Soderling et al. (2002). Nature Cell Biol. 4, 970 - 975.

Confirmation by reciprocal co-immunoprecipitation experiments of the WRP-WAVE interaction

The WRP component of the WAVE-1 complex attenuates Rac-mediated signalling. Soderling et al. (2002). Nature Cell Biol. 4, 970 - 975.

Immunoprecipitation of [WAVE with WRP] and [WRP with WAVE]

The WRP component of the WAVE-1 complex attenuates Rac-mediated signalling. Soderling et al. (2002). Nature Cell Biol. 4, 970 - 975.

WRP in rat brain extracts

Screening of a solid-phase peptide array spanning the polyproline region of WAVE-1 using GST-WRP SH3

The WRP component of the WAVE-1 complex attenuates Rac-mediated signalling. Soderling et al. (2002). Nature Cell Biol. 4, 970 - 975.

The WRP component of the WAVE-1 complex attenuates Rac-mediated signalling. Soderling et al. (2002). Nature Cell Biol. 4, 970 - 975.

Mutant WAVE does not interact with WRP

WRP stimulates the intrinsic GTPase activity of Rac

The WRP component of the WAVE-1 complex attenuates Rac-mediated signalling. Soderling et al. (2002). Nature Cell Biol. 4, 970 - 975.

Loss of WAVE-1 causes sensorimotor retardation and reduced learning and memory in mice.

Soderling et al. (2003). PNAS 100, 1723 -1728.

Loss of WAVE-1 causes sensorimotor retardation and reduced learning and memory in mice. Soderling et al. (2003). PNAS 100, 1723 – 1728.

WAVE-1 knockout mice exhibit decreased body size

Loss of WAVE-1 causes sensorimotor retardation and reduced learning and memory in mice. Soderling et al. (2003). PNAS 100, 1723 – 1728.

Expression pattern of WAVE-1

WAVE-1 is the brain-specific isoform of the WAVE protein family.

Loss of WAVE-1 causes sensorimotor retardation and reduced learning and memory in mice. Soderling et al. (2003). PNAS 100, 1723 – 1728.

WAVE-1 knockout mice show sensorimotor deficits and reduced anxiety levels.

Loss of WAVE-1 causes sensorimotor retardation and reduced learning and memory in mice. Soderling et al. (2003). PNAS 100, 1723 – 1728.

Spatial learning: Morris water maze

Traces indicating swim-paths

Learning and memory deficits

Loss of WAVE-1 causes sensorimotor retardation and reduced learning and memory in mice. Soderling et al. (2003). PNAS 100, 1723 – 1728.

Quantification of learning and

memory deficits

Oikawa et al. (2004) Nature Cell Biol. 6, 421 - 426.

PtdIns(3,4,5)P3 binding is necessary for WAVE2-induced formation of lamellipodia. Oikawa et al. (2004). Nature Cell Biol. 6, 421 – 426.

WAVE-2 binds to PtdIns(3,4,5)P3

PtdIns(3,4,5)P3 binding is necessary for WAVE2-induced formation of lamellipodia. Oikawa et al. (2004). Nature Cell Biol. 6, 421 – 426.

Basic 1 region (aa 171-183) of WAVE-2 is sufficient for lipid binding, GFP-WAVE2-N, which has the wild-type Basic1 region, was localized along the leading edges.

Wortmannin decreases the degree of co-localization of WAVE2-N with Myr-p110a.

PtdIns(3,4,5)P3 binding is necessary for WAVE2-induced formation of lamellipodia. Oikawa et al. (2004). Nature Cell Biol. 6, 421 – 426.

WAVE-2 mutant lacking phosphoinositide-binding activity inhibits proper lamellipodia formation.

EXAMPLE #2:

PDZ domains in synapse assembly and signalling. Garner et al. (2000). Trends in Cell Biol. 10, 274 - 280.

PDZ domains in synapse assembly and signalling. Garner et al. (2000). Trends in Cell Biol. 10, 274 - 280.

PDZ domains in synapse assembly and signalling. Garner et al. (2000). Trends in Cell Biol. 10, 274 - 280.

Synaptic targeting of N-type calcium channels in hippocampal neurons.Maximov A and Bezprozvanny I. (2002). J Neurosci. 22, 6939 – 6952.

Colocalization of Mint1 and CASK with N-type channels in hippocampal neurons

A role for Mints in transmitter release: Mint 1 knockout mice exhibit impaired GABAergic synaptic transmission. Ho et al. (2003). PNAS. 100, 1409 -1414.

Summary

a. Scaffold proteins can create substrate specificity (i.e. PP1 phosphatase).

b. Scaffold proteins bring together signaling molecules and cytoskeleton components to control structural and mechanical signal induced modifications.

c. Conserved protein-protein interactions binding motifs such as PDZ domains help scaffolding proteins to organize multi-signaling complexes as seen in postsynaptic densities and presynaptic active zones.