The Yeast Two-Hybrid System

Transcription

1 The Yeast Two-Hybrid System

2 What is the yeast two-hybrid system used for? Identifies novel protein-protein interactions Can identify protein cascades Identifies mutations that affect protein-protein binding Can identify interfering proteins in known interactions (Reverse Two-Hybrid System)

3 How does it work? Uses yeast as a model for eukaryotic protein interactions A library is screened or a protein is characterized using a bait construct Interactions are identified by the transcription of reporter genes Positives are selected using differential media

4 The Model Transcription Activating Region Bait Protein Prey Protein DNA-Binding Domain Reporter Gene DNA-Binding Site

5 Steps to Screen a Library Create the Bait Plasmid Construct from the gene of interest and the DNA binding domain of Gal4 or LexA or other suitable domain Transform with the bait construct a yeast strain lacking the promoter for the reporter genes and select for transformed yeast Transform the yeast again with the library plasmids Select for interaction

6 Sequence analysis Isolate plasmid from yeast and transform E. coli Purify plasmid from E. coli and sequence Blast sequence against database for known proteins or construct a possible protein sequence from the DNA sequence and compare to other proteins

7 Reporter Genes LacZ reporter - Blue/White Screening HIS3 reporter - Screen on His+ media (usually need to add 3AT to increase selectivity) LEU2 reporter - Screen on Leu+ media ADE2 reporter - Screen on Ade+ media URA3 reporter - Screen on Ura+ media (can do negative selection by adding FOA)

8 Plasmid Constructs Plasmids are constructed with the Gal4 DNA binding domain (or other suitable domain) in front of a Multiple Cloning Site (MCS) The plasmid contains genes that can be used for selection such as Amp, Leu2, Ura3, or Trp1

9 Sample Plasmid From Golemis Lab Homepage

10 False Positives False positives are the largest problem with the yeast two-hybrid system Can be caused by: Non-specific binding of the prey Ability to induce transcription without interaction with the bait (Majority of false positives)

11 Elimination of False Positives Sequence Analysis Plasmid Loss Assays Retransformation of both strain with bait plasmid and strain without bait plasmid Test for interaction with an unrelated protein as bait Two (or more) step selections

12 Advantages Immediate availability of the cloned gene of the interacting protein Only a single plasmid construction is required Interactions are detected in vivo Weak, transient interactions can be detected Can accumulate a weak signal over time

13 Examples of Uses of the Yeast Two- Hybrid System Identification of caspase substrates Interaction of Calmodulin and L-Isoaspartyl Methyltransferase Genetic characterization of mutations in E2F1 Peptide hormone-receptor interactions Pha-4 interactions in C. elegans

14 The Two-Hybrid System Bait! Protein! Binding! Domain! Activation! Domain! Prey! Protein! Reporter Gene

15 The Two-Hybrid System Interaction of bait and prey proteins localizes the activation domain to the reporter gene, thus activating transcription. Since the reporter gene typically codes for a survival factor, yeast colonies will grow only when an interaction occurs. Activation! Domain! Prey! Protein! Bait! Protein! Binding! Domain! Reporter Gene

16 Myriad s ProNet Two-Hybrid Process Industrial-scale application of the yeast two-hybrid system Roboticized bait creation process Custom activation domain libraries Efficient mating strategy Automated for quality and throughput Positive sample tracking Robots for media preparation, liquid handling, colony picking Statistical quality control

17 ProNet Process Flowchart 1 4! 3! 5! 7! 8! 0! 1! 2! 6! 9! 10! 23! 24! 2 22! 25! 29! 27! 26! 30! 28! 3 46! 40! 41! 44! 45! 48! 49! 42! 43! 47! 1 Bait Construction 2 Bait Verification 3 Two-Hybrid Screen 4 Identification and Confirmation of Interactions 12! 11! 34! 33! 31! 32! 50! 13! 35! 36! Seq! 51! 56! 14! 15! 16! 37! 52! 53! 57! 17! 38! 39! 54! 55! Seq! 18! 19! 20! 58! 59! 60! 21! 61! 62! 64! 4 63! Seq!

18 Bait Selection A number of criteria were taken into consideration in the selection of bait protein candidates: Proteins known to be expressed in B cells and/or cardiac myocytes. Proteins known or suspected to be involved in signaling pathways in B cells and/or cardiac myocytes. Proteins believed important in GPCR or BCR signaling leading to PIP3 generation. These were selected first in an attempt to define interactions within a focused network of signaling molecules. Preference was also given to proteins that had a higher likelihood of success in the yeast two-hybrid assay proteins with well-defined modular domains exhibiting secondary structure suggestive of involvement in protein-protein interactions.

19 Bait Design Fragments of proteins representing folded domains are often more effective than the full-length protein in identifying physiologically relevant interactions If the domain structure of a given bait protein was already established, the specific baits were designed to represent one or more folded domains. For cases in which domain structure was not available, a variety of secondary structure prediction algorithms were used to predict domains and thus direct bait design. were designed to cover the entire protein, with several overlapping fragments, as not all baits will work effectively. Phosphorylation sites Proline-rich SH3 binding BLNK Src homology 2 domain P! P! P! Pro! Pro! SH2! 110" 116" 134" 146" 350" 436" (P sites)" (linker)" (N-term)" (SH2)" (Pro regions)" (C-term)"

20 B Cell Receptor Pathway

21 B Cell Receptor Pathway

22 B Cell Receptor Pathway z Endophilin2 BAN-P PDE4B3 Nck Pak - Pix/Cool Ruk1 Git2 PP2C gamma

23 What can we learn from the larger data set? 170 interactions derived from just 16 bait proteins Limited interconnectivity We now have 473 interactions from 55 bait proteins How can the average biologist analyze this data set? With difficulty! All 473 interactions uploaded to Access database table Query: which prey IDs are correlated with more than one bait ID?

24 Interaction examples:1 AFCS ID Bait Protein Name Prey Nuc GI Prey ID Prey Protein Name A Grb A Blnk A Calcium-dependent protein kinase II alpha A Blnk A Grb A Blnk A Grb A Blnk A Grb A Blnk AFCS ID Bait Protein Name Prey Nuc GI Prey ID Prey Protein Name A cdc A Pak1 A PIX/COOL A Pak1 AFCS ID Bait Protein Name Prey Nuc GI Prey ID Prey Protein Name A Integrin beta A Nucleoside diphosphate kinase B A Integrin beta A Nucleoside diphosphate kinase B A Ryanodine receptor type I A Nucleoside diphosphate kinase B A PI3 kinase p85 regulatory subunit (alpha) A Nucleoside diphosphate kinase B A Dbl A Nucleoside diphosphate kinase B A Integrin beta A Nucleoside diphosphate kinase B AFCS ID Bait Protein Name Prey Nuc GI Prey ID Prey Protein Name A PI3 kinase p85 regulatory subunit (alpha) A Endophilin 2 A PI3K p85beta A Endophilin 2 A BLNK A Endophilin 2 AFCS ID Bait Protein Name Prey Nuc GI Prey ID Prey Protein Name A Nck A Bcap A Bcap A Bcap A Bcap A Bcap A Bcap A Bcap A Grb A Bcap A Grb A Bcap

25 Interaction examples: 2 AFCS ID Bait Protein Name Prey Nuc GI Prey ID A Lyn A CD AFCS ID Bait Protein Name Prey Nuc GI Prey ID A phospholipase C gamma A Rac AFCS ID Bait Bait Protein Name Prey Nuc GI Prey ID A phospholipase C gamma A phospholipase C gamma A phospholipase C gamma A phospholipase C gamma A phospholipase C gamma A Grb A Calcium-dependent protein kinase II alpha A Calcium-dependent protein kinase II alpha A phospholipase C gamma A phospholipase C gamma AFCS ID Bait Protein Name Prey Nuc GI Prey ID A vav A Grb A Grb A Grb A Nck AFCS ID Bait Protein Name Prey Nuc GI Prey ID A Wasp A Nck A Phosphatidyl-inositol 3'-kinase gamma A Nck A Wasp A Wasp

26

27 CamK II SOS2 CD19 CD22 Dbl Btk Fyn cdc42 CAP WISH NdkB PI3Kγ (p110) Actin Cytoskeleton PDK1

28 AIP Cbl-b CamK II SOS2 CD19 CD Dbl Btk Fyn cdc42 CAP WISH NdkB PI3Kγ (p110) Sam68 Actin Cytoskeleton PDK Protein 4.1G

29 Caveats! Y2H data requires validation by secondary assay Protein complementation Pull-downs Didn t observe source library in data analysis Didn t analyze all bait and prey coordinates to map sites of interaction Cant assume multi-protein complexes since proteins may be competing for same site of interaction This level of analysis requires more sophisticated computational approach

30 Bait Status Protein name Date Submitted AFCS ID Ordered Created Searched Status adenylyl cyclase V 11-Jul-01 A No Interactions N-Ras 12-Jul-02 A Screens in progress K-Ras 4-Apr-02 A Data Released p115rhogef 11-Jul-01 A Data Released Beta arrestin 1 11-Jul-01 A Data Released RGS Jul-02 A Screens in progress NFATc1 (AKA NFAT2) 8-Aug-01 A Data Released RGS 2 11-Jul-01 A No Interactions G alpha13 11-Jul-01 A No Interactions RGS Jul-02 A Screens in progress btk 25-Jul-01 A Data Released G alpha12 13-Jul-01 A No Interactions PKG 23-Jul-02 A No Interactions Protein kinase A RI alpha 10-Oct-02 A Screens in progress Protein kinase A RII beta 10-Oct-02 A No Interactions PAK1 11-Jul-01 A Data Released Acetylcholine (muscarinic) 10-Oct-02 A Screens in progress N-WASp 11-Jul-01 A Data Released RGS Sep-01 A Data Released I kappa B alpha 11-Jul-01 A No Interactions PDE4B3 11-Jul-01 A No Interactions G alpha i3 11-Jul-01 A Data Released Nck1 13-Sep-01 A Data Released Inositol (1,4,5,) P3 receptor 2 11-Jul-01 A No Interactions Wasp 4-Apr-02 A Data Released dock2 1-Feb-02 A Data Released RasGap p Oct-02 A Screens in progress Adrenergic receptor beta 1 10-Oct-02 A No Interactions Adrenergic receptor beta 3 10-Oct-02 A No Interactions AKAP12 11-Jul-01 A Data Released PKB-alpha (= Akt1) 8-Aug-01 A Data Released Akt2 1-Feb-02 A Data Released Akt3 10-Oct-02 A No Interactions AP-3 beta3a 10-Oct-02 A Screens in progress Bam32 13-Sep-01 A No Interactions Bcap 4-Apr-02 A Data Released Blk 12-Jul-02 A No Interactions BLNK 13-Jul-01 A Data Released calcineurin A-subunit 23-Jul-02 A No Interactions Calcineurin A beta 1-Feb-02 A No Interactions Calcium channel voltage- 1-Feb-02 A Data Released Calcium channel voltage- 8-Aug-01 A Screens in progress Calmodulin III 25-Jul-01 A No Interactions CaMKI 23-Jul-02 A No Interactions Calcium-dependent protein 1-Feb-02 A Data Released Calcium-dependent protein 1-Feb-02 A No Interactions caveolin3 13-Sep-01 A No Interactions Cbl 8-Aug-01 A No Interactions GRP-1 4-Apr-02 A No Interactions p70s6k 10-Oct-02 A No Interactions CD19 4-Apr-02 A Data Released CD21 12-Jul-02 A Screens in progress CD22 12-Jul-02 A Screens in progress CD32 4-Apr-02 A No Interactions cd44 1-Feb-02 A No Interactions Protein name Date Submitted AFCS ID Ordered Created Searched Status CD45 4-Apr-02 A No Interactions CD79a 4-Apr-02 A No Interactions CD79b 4-Apr-02 A Screens in progress CD81 12-Jul-02 A No Interactions cdc42 1-Feb-02 A Data Released CCR7 1-Feb-02 A No Interactions CXCR4 1-Feb-02 A No Interactions Chemokine receptor CXCR5 4-Apr-02 A Data Released PIX/COOL 13-Jul-01 A Data Released Dock1 12-Jul-02 A Screens in progress Dbl 13-Sep-01 A Data Released Dbs 10-Oct-02 A Screens in progress Dok2 12-Jul-02 A No Interactions CD150 4-Apr-02 A No Interactions Fyn 12-Jul-02 A Screens in progress G protein alpha i2 4-Apr-02 A No Interactions G protein beta 1 4-Apr-02 A No Interactions G protein beta 2 4-Apr-02 A No Interactions G protein gamma 2 4-Apr-02 A No Interactions GAB-1 23-Apr-02 A Data Released Emk 10-Oct-02 A Screens in progress Grb2 13-Sep-01 A Data Released GRK2 11-Jul-01 A No Interactions glycogen synthase kinase- 23-Jul-02 A No Interactions HDAC5 23-Jul-02 A No Interactions Integrin beta 2 4-Apr-02 A Data Released Integrin beta 1 4-Apr-02 A Data Released Kv Oct-02 A No Interactions Kv1.2 1-Feb-02 A Data Released Kv Oct-02 A No Interactions Kv Oct-02 A No Interactions Kv Oct-02 A No Interactions Lck 4-Apr-02 A Data Released Lyn 8-Aug-01 A Data Released Mcip-1 1-Feb-02 A Data Released MEF2A 23-Jul-02 A No Interactions MEF 2C 1-Feb-02 A Data Released MEF2D 23-Jul-02 A Screens in progress MEK5 23-Jul-02 A Screens in progress MKK6 23-Jul-02 A No Interactions Mitr 23-Jul-02 A Screens in progress Na/Ca exchanger 11-Jul-01 A No Interactions Nav Oct-02 A Screens in progress Neurofibromin 2 10-Oct-02 A Screens in progress NFAT2 8-Aug-01 A No Interactions NFAT3 23-Jul-02 A No Interactions NFkappaB p65 4-Apr-02 A Data Released P Jul-02 A Screens in progress p38 MAPK 23-Jul-02 A Screens in progress HDAC7 23-Jul-02 A Screens in progress G2A receptor 13-Sep-01 A No Interactions PDK1 25-Jul-01 A Data Released Phosphoinositide 3-kinase 4-Apr-02 A No Interactions Phosphatidyl-inositol 3'-kinase 8-Aug-01 A Data Released Protein name Date Submitted AFCS ID Ordered Created Searched Status p110 beta class Ia 8-Aug-01 A Data Released Phosphoinositide 3-kinase 12-Jul-02 A Screens in progress Phosphatidyl-inositol 3'-kinase 13-Jul-01 A Data Released PI3 kinase p85 regulatory 8-Aug-01 A Data Released PI3K p85beta 1-Feb-02 A Data Released Phosphoinositide 4-phosphate 10-Oct-02 A Screens in progress Phosphoinositide 4-phosphate 10-Oct-02 A No Interactions Phosphoinositide 4-phosphate 10-Oct-02 A No Interactions Phosphoinositide 5-phosphate 10-Oct-02 A Data Released Phospholamban 8-Aug-01 A No Interactions Phospholipase C beta 2 11-Jul-01 A Data Released phospholipase C gamma1 13-Jul-01 A Data Released phospholipase C gamma2 13-Jul-01 A Data Released PKA catalytic subunit alpha 8-Aug-01 A No Interactions Protein kinase A RI beta 10-Oct-02 A Screens in progress Protein kinase A RII alpha 10-Oct-02 A Screens in progress Protein kinase C beta1 11-Jul-01 A No Interactions PKC-epsilon 11-Jul-01 A No Interactions PKC zeta 25-Jul-01 A Data Released PTEN 13-Jul-01 A No Interactions Rac1 13-Sep-01 A Data Released Rac2 13-Jul-01 A No Interactions Rac3 12-Jul-02 A Screens in progress RasGEF1 10-Oct-02 A Screens in progress RasGEF2 10-Oct-02 A No Interactions RasGRF1 10-Oct-02 A Screens in progress RGS 1 11-Jul-01 A Data Released RGS Jul-02 A No Interactions RGS Jul-02 A No Interactions RGS 3 8-Aug-01 A No Interactions RhoA 1-Feb-02 A Data Released ROCK1 2-Aug-01 A No Interactions Rock2 10-Oct-02 A No Interactions p90rsk1 23-Apr-02 A Data Released Ryanodine receptor type I 13-Sep-01 A Data Released Ryanodine receptor Type II 1-Feb-02 A Data Released S100A1 1-Feb-02 A No Interactions SRF 23-Jul-02 A No Interactions Shc 4-Apr-02 A Data Released SHIP1 8-Aug-01 A No Interactions SHIP2 1-Feb-02 A No Interactions SHP-1 4-Apr-02 A Data Released Tec 23-Apr-02 A No Interactions Tumor necrosis factor 10-Oct-02 A Screens in progress Vav1 12-Jul-02 A Screens in progress vav2 1-Feb-02 A Data Released Vav3 12-Jul-02 A No Interactions RasGRF2 10-Oct-02 A Screens in progress Abi1 10-Oct-02 A Screens in progress Scar-2/WAVE-2 9-Aug-01 A Data Released HDAC4 23-Jul-02 A Screens in progress Gpr90 23-Jul-02 A No Interactions Fmr1 10-Oct-02 A Screens in progress PRex1 10-Oct-02 A No Interactions

31 Bait Status Protein name Date Submitted AFCS ID Ordered Created Searched Status K-Ras 4-Apr-02 A Data Released p115rhogef 11-Jul-01 A Data Released Beta arrestin 1 11-Jul-01 A Data Released NFATc1 (AKA NFAT2) 8-Aug-01 A Data Released btk 25-Jul-01 A Data Released PAK1 11-Jul-01 A Data Released N-WASp 11-Jul-01 A Data Released RGS Sep-01 A Data Released G alpha i3 11-Jul-01 A Data Released Nck1 13-Sep-01 A Data Released Wasp 4-Apr-02 A Data Released dock2 1-Feb-02 A Data Released AKAP12 11-Jul-01 A Data Released PKB-alpha (= Akt1) 8-Aug-01 A Data Released Akt2 1-Feb-02 A Data Released Bcap 4-Apr-02 A Data Released BLNK 13-Jul-01 A Data Released Calcium channel voltage- 1-Feb-02 A Data Released Calcium-dependent protein 1-Feb-02 A Data Released CD19 4-Apr-02 A Data Released cdc42 1-Feb-02 A Data Released Chemokine receptor CXCR5 4-Apr-02 A Data Released PIX/COOL 13-Jul-01 A Data Released Dbl 13-Sep-01 A Data Released GAB-1 23-Apr-02 A Data Released Grb2 13-Sep-01 A Data Released Integrin beta 2 4-Apr-02 A Data Released Integrin beta 1 4-Apr-02 A Data Released Kv1.2 1-Feb-02 A Data Released Lck 4-Apr-02 A Data Released Lyn 8-Aug-01 A Data Released Mcip-1 1-Feb-02 A Data Released MEF 2C 1-Feb-02 A Data Released NFkappaB p65 4-Apr-02 A Data Released PDK1 25-Jul-01 A Data Released Phosphatidyl-inositol 3'-kinase 8-Aug-01 A Data Released Phosphatidyl-inositol 3'-kinase 8-Aug-01 A Data Released Phosphatidyl-inositol 3'-kinase 13-Jul-01 A Data Released PI3 kinase p85 regulatory 8-Aug-01 A Data Released PI3K p85beta 1-Feb-02 A Data Released Phosphoinositide 5-phosphate 10-Oct-02 A Data Released Phospholipase C beta 2 11-Jul-01 A Data Released phospholipase C gamma1 13-Jul-01 A Data Released phospholipase C gamma2 13-Jul-01 A Data Released PKC zeta 25-Jul-01 A Data Released Rac1 13-Sep-01 A Data Released RGS 1 11-Jul-01 A Data Released RhoA 1-Feb-02 A Data Released p90rsk1 23-Apr-02 A Data Released Ryanodine receptor type I 13-Sep-01 A Data Released Ryanodine receptor Type II 1-Feb-02 A Data Released Shc 4-Apr-02 A Data Released SHP-1 4-Apr-02 A Data Released vav2 1-Feb-02 A Data Released Scar-2/WAVE-2 9-Aug-01 A Data Released Protein name Date Submitted AFCS ID Ordered Created Searched Status Abi1 10-Oct-02 A Screens in progress Acetylcholine (muscarinic) 10-Oct-02 A Screens in progress AP-3 beta3a 10-Oct-02 A Screens in progress Calcium channel voltage- 8-Aug-01 A Screens in progress CD21 12-Jul-02 A Screens in progress CD22 12-Jul-02 A Screens in progress CD79b 4-Apr-02 A Screens in progress Dbs 10-Oct-02 A Screens in progress Dock1 12-Jul-02 A Screens in progress Emk 10-Oct-02 A Screens in progress Fmr1 10-Oct-02 A Screens in progress Fyn 12-Jul-02 A Screens in progress HDAC4 23-Jul-02 A Screens in progress HDAC7 23-Jul-02 A Screens in progress MEF2D 23-Jul-02 A Screens in progress MEK5 23-Jul-02 A Screens in progress Mitr 23-Jul-02 A Screens in progress Nav Oct-02 A Screens in progress Neurofibromin 2 10-Oct-02 A Screens in progress N-Ras 12-Jul-02 A Screens in progress P Jul-02 A Screens in progress p38 MAPK 23-Jul-02 A Screens in progress Phosphoinositide 3-kinase 12-Jul-02 A Screens in progress Phosphoinositide 4-phosphate 10-Oct-02 A Screens in progress Protein kinase A RI alpha 10-Oct-02 A Screens in progress Protein kinase A RI beta 10-Oct-02 A Screens in progress Protein kinase A RII alpha 10-Oct-02 A Screens in progress Rac3 12-Jul-02 A Screens in progress RasGap p Oct-02 A Screens in progress RasGEF1 10-Oct-02 A Screens in progress RasGRF1 10-Oct-02 A Screens in progress RasGRF2 10-Oct-02 A Screens in progress RGS Jul-02 A Screens in progress RGS Jul-02 A Screens in progress Tumor necrosis factor 10-Oct-02 A Screens in progress Vav1 12-Jul-02 A Screens in progress adenylyl cyclase V 11-Jul-01 A No Interactions Adrenergic receptor beta 1 10-Oct-02 A No Interactions Adrenergic receptor beta 3 10-Oct-02 A No Interactions Akt3 10-Oct-02 A No Interactions Bam32 13-Sep-01 A No Interactions Blk 12-Jul-02 A No Interactions Calcineurin A beta 1-Feb-02 A No Interactions calcineurin A-subunit 23-Jul-02 A No Interactions Calcium-dependent protein 1-Feb-02 A No Interactions Calmodulin III 25-Jul-01 A No Interactions CaMKI 23-Jul-02 A No Interactions caveolin3 13-Sep-01 A No Interactions Cbl 8-Aug-01 A No Interactions CCR7 1-Feb-02 A No Interactions CD150 4-Apr-02 A No Interactions CD32 4-Apr-02 A No Interactions cd44 1-Feb-02 A No Interactions CD45 4-Apr-02 A No Interactions Protein name Date Submitted AFCS ID Ordered Created Searched Status CD79a 4-Apr-02 A No Interactions CD81 12-Jul-02 A No Interactions CXCR4 1-Feb-02 A No Interactions Dok2 12-Jul-02 A No Interactions G alpha12 13-Jul-01 A No Interactions G alpha13 11-Jul-01 A No Interactions G protein alpha i2 4-Apr-02 A No Interactions G protein beta 1 4-Apr-02 A No Interactions G protein beta 2 4-Apr-02 A No Interactions G protein gamma 2 4-Apr-02 A No Interactions G2A receptor 13-Sep-01 A No Interactions glycogen synthase kinase- 23-Jul-02 A No Interactions Gpr90 23-Jul-02 A No Interactions GRK2 11-Jul-01 A No Interactions GRP-1 4-Apr-02 A No Interactions HDAC5 23-Jul-02 A No Interactions I kappa B alpha 11-Jul-01 A No Interactions Inositol (1,4,5,) P3 receptor 2 11-Jul-01 A No Interactions Kv Oct-02 A No Interactions Kv Oct-02 A No Interactions Kv Oct-02 A No Interactions Kv Oct-02 A No Interactions MEF2A 23-Jul-02 A No Interactions MKK6 23-Jul-02 A No Interactions Na/Ca exchanger 11-Jul-01 A No Interactions NFAT2 8-Aug-01 A No Interactions NFAT3 23-Jul-02 A No Interactions p70s6k 10-Oct-02 A No Interactions PDE4B3 11-Jul-01 A No Interactions Phosphoinositide 3-kinase 4-Apr-02 A No Interactions Phosphoinositide 4-phosphate 10-Oct-02 A No Interactions Phosphoinositide 4-phosphate 10-Oct-02 A No Interactions Phospholamban 8-Aug-01 A No Interactions PKA catalytic subunit alpha 8-Aug-01 A No Interactions PKC-epsilon 11-Jul-01 A No Interactions PKG 23-Jul-02 A No Interactions PRex1 10-Oct-02 A No Interactions Protein kinase A RII beta 10-Oct-02 A No Interactions Protein kinase C beta1 11-Jul-01 A No Interactions PTEN 13-Jul-01 A No Interactions Rac2 13-Jul-01 A No Interactions RasGEF2 10-Oct-02 A No Interactions RGS Jul-02 A No Interactions RGS 2 11-Jul-01 A No Interactions RGS Jul-02 A No Interactions RGS 3 8-Aug-01 A No Interactions ROCK1 2-Aug-01 A No Interactions Rock2 10-Oct-02 A No Interactions S100A1 1-Feb-02 A No Interactions SHIP1 8-Aug-01 A No Interactions SHIP2 1-Feb-02 A No Interactions SRF 23-Jul-02 A No Interactions Tec 23-Apr-02 A No Interactions Vav3 12-Jul-02 A No Interactions

32 Pattern Among Negative? Of 72 negative baits, 45 are either membrane proteins (receptors, cell-surface antigens, channels) or are membrane associated (G proteins, GEFs, RGS etc.) May be overcome by generous and creative bait design but these proteins will always give lower return in Y2H assay Also several proteins that could be expected to do better (cytosolic protein kinases and some adaptors) Further baits advisable Future baits sets should also include some of novel preys identified in primary screen

33 References Bartel, Paul, C. Chien, R. Sternglanz, S. Fields. Elimination of False Positives that Arise in Using the Two- Hybrid System. Biotechniques (1993) Vol. 14, no. 6, p Chien, Cheng-ting, P. Bartel, R. Sternglanz, S. Fields. The two-hybrid system: A method to identify and clone genes for proteins that interact with a protein of interest. Proc. Natl. Acad. Sci. USA (1991) Vol. 88, p Fields, Stanley, O. Song. "A novel genetic system to detect protein-protein interactions." Nature (1989) Vol. 340, p James, Philip, J. Halladay, E. Craig. "Genomic Libraries and a Host Strain Designed for Highly Efficient Two-Hybrid Selection in Yeast." Genetics (1996) Vol. 144, p Kamada, S, H. Kusano, H. Fujita, M. Ohtsu, R. Koya, N. Kuzumaki, Y. Tsujimoto. "A cloning method for caspase substrates that uses the yeast two-hybrid system: Cloning of the antiapoptotic gene gelsolin." Proc. Natl. Acad. Sci. USA (1998) Vol 95, p O'Connor, Mirriam, C. O'Connor. "Complex Interactions of the Protein L-Isoaspartyl Methyltransferase and Calmodulin Revealed with the Yeast Two-hybrid System." The Journal of Biological Chemistry (1998) Vol. 273, p Staudinger, Jeff, J. Zhou, R. Burgess, S. Elledge, E. Olson. "PICK1: A Perinuclear Binding Protein and Substrate for Protein Kinase C Isolated by the Yeast Two-Hybrid System." The Journal of Cell Biology (1995) Vol. 128, p

34 References continued Vidal, Marc, P. Braun, E. Chen, J. Boeke, E. Harlow. "Genetic Characterization of a mammalian proteinprotein interaction domain by using a yeast reverse two-hybrid system." Proc. Natl. Acad. Sci. USA (1996) Vol. 93, p White, Michael. "The yeast two-hybrid system: Forward and reverse." Proc. Natl. Acad. Sci. USA (1996) Vol 93, p Zhu, Jianwei, C. Kahn. "Analysis of a peptide hormone-receptor interaction in the yeast two-hybrid system." Proc. Natl. Acad. Sci. USA (1997) Vol. 94, p Lab of Erica Golemis Special thanks to Dr. Susan Mango and the University of Utah