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Organization of Macromolecular Complexes

Organization of Macromolecular Complexes. Role of Scaffold Proteins. Maria Diverse-Pierluissi, Ph.D. Department of Pharmacology and Biological Chemistry Mount Sinai School of Medicine. April 12, 2005. What is a scaffold protein? What roles do they play in signal transduction?.

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Organization of Macromolecular Complexes

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  1. Organization of Macromolecular Complexes Role of Scaffold Proteins Maria Diverse-Pierluissi, Ph.D. Department of Pharmacology and Biological Chemistry Mount Sinai School of Medicine April 12, 2005

  2. What is a scaffold protein? What roles do they play in signal transduction?

  3. Roles of scaffold proteins • Spatial localization or targeting - create microenvironment in close proximity to effectors. • Substrate specificity • Signal integration - scaffold proteins bring together signal initiators and terminators.

  4. Targeting of type 1 phosphatase Yotiao-green PKA-blue PP1-red Kinase- and phosphatase-anchoring proteins: harnessing the dynamic duo. Bauman AL and Scott JD (2002). Nature Cell Biol. 4, 203 - 206.

  5. Targeting M110/MBS: 1) selectivity of phosphatase activity towards a subgroup of muscle proteins, 2) assembles complex with Rho and PKG. Kinase- and phosphatase-anchoring proteins: harnessing the dynamic duo. Bauman AL and Scott JD (2002). Nature Cell Biol. 4, 203 - 206.

  6. RACK can bring together protein kinase C with several signaling molecules RACK-receptor for activated C kinase Kinase- and phosphatase-anchoring proteins: harnessing the dynamic duo. Bauman AL and Scott JD (2002). Nature Cell Biol. 4, 203 - 206.

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

  8. EXAMPLE #1: WAVE-1

  9. 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.

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

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

  12. 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.

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

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

  15. 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.

  16. 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.

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

  18. 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.

  19. 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.

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

  21. 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.

  22. Mutant WAVE does not interact with WRP The WRP component of the WAVE-1 complex attenuates Rac-mediated signalling. Soderling et al. (2002). Nature Cell Biol.4, 970 - 975.

  23. 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.

  24. Loss of WAVE-1 causes sensorimotor retardation and reduced learning and memory in mice. Soderling et al. (2003). PNAS100, 1723 -1728.

  25. 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). PNAS100, 1723 – 1728.

  26. 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). PNAS100, 1723 – 1728.

  27. 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). PNAS100, 1723 – 1728.

  28. Learning and memory deficits Spatial learning: Morris water maze Traces indicating swim-paths Loss of WAVE-1 causes sensorimotor retardation and reduced learning and memory in mice. Soderling et al. (2003). PNAS100, 1723 – 1728.

  29. Quantification of learning and memory deficits Loss of WAVE-1 causes sensorimotor retardation and reduced learning and memory in mice. Soderling et al. (2003). PNAS100, 1723 – 1728.

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

  31. 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.

  32. 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.

  33. WAVE-2 mutant lacking phosphoinositide-binding activity inhibits proper lamellipodia formation. PtdIns(3,4,5)P3 binding is necessary for WAVE2-induced formation of lamellipodia. Oikawa et al. (2004). Nature Cell Biol. 6, 421 – 426.

  34. EXAMPLE #2: PDZ domains in neurons

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

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

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

  38. Colocalization of Mint1 and CASK with N-type channels in hippocampal neurons Synaptic targeting of N-type calcium channels in hippocampal neurons. Maximov A and Bezprozvanny I. (2002). J Neurosci. 22, 6939 – 6952.

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

  40. Summary • Scaffold proteins can create substrate specificity (i.e. PP1 phosphatase). • Scaffold proteins bring together signaling molecules and cytoskeleton components to control structural and mechanical signal induced modifications. • 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.

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