1. 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

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

3. 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

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. 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.

6. 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.

8. EXAMPLE #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. 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 stainMass spectrometry

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

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

19. 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]

20. 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

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

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). PNAS 100, 1723 -1728.

25. 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

26. 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.

27. 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.

28. 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

29. 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

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

31. 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

32. 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.

33. 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.

34. EXAMPLE #2:

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. 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

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 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.