Identification of Protein-Protein Interactions by the Yeast 2-Hybrid System Alliance for Cell...

Identification of Protein-Protein Interactions by the Yeast 2-Hybrid

System

Alliance for Cell SignalingMyriad Genetics Inc.

Reporter Gene

BaitProtein

BindingDomain

Prey Protein

ActivationDomain

• Two hybrid proteins are generated with transcription factor domains

• Both fusions are expressed in a yeast cell that carries a reporter gene whose expression is under the control of binding sites for the DNA-binding domain

The Two-Hybrid System

Reporter Gene

BaitProtein

BindingDomain

Prey Protein

ActivationDomain

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.

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

ProNet Process Flowchart

1

0

2

6

10

11

13

17

9

1615

5

19

20

21

25

29

24

35 36

38

18

41

27

43

45

49

50

54

62

4846

5352

5958

47

63

61

60

30

7 8

3

12

14

34

39

57

56

32

37

51

22

4

42

33

44

40

28

23

64

55

31

26

Seq

Seq

Seq

1

2

3

4

1 Bait Construction

2 Bait Verification

3 Two-Hybrid Screen4 Identification and Confirmation of Interactions

AfCS Cell Type-Specific Libraries

• Screening of an activation domain library that is representative of a specific cell type may permit the identification of cell type–specific protein-protein interactions.

• Since a number of proteins that play a central role in B-cell signaling pathways may be expressed at low levels in resting cells, mRNA was isolated from B cells stimulated with a cocktail of ligands selected to activate the major B-cell signaling pathways.

B-Cell mRNA Sources Cardiac Myocyte mRNA Sources

Murine primary resting B cells Murine primary cardiac myocytes

Murine primary B cells stimulated with a ligand cocktail of anti-IgM, anti-CD40, and IL-4 for 4 hr

Nk-TAg murine cardiac myocyte cell line

WEHI-231 murine B-cell line

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.

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.

• Baits were designed to cover the entire protein, with several overlapping fragments, as not all baits will work effectively.

1-100 (P sites)

100-200 (Pro regions)

1-240 (N-term)

150-350 (linker)

348-457 (SH2)

210-457 (C-term)

110 116 134 146 350 436

P P P SH2Pro Pro

Phosphorylation sites

Proline-rich SH3 binding Src homology 2 domainBLNK

Project Status-One Year Later

May 2002 May 2003

Bait Proteins Approved

98 163

Screens Initiated 302 706

Bait Fragments in Preparation

119 -

Bait Proteins with Released Data

16 (26) 55 (90)

Total Interactions 170 473

B Cell Receptor Pathway

B Cell Receptor Pathway

Pak

Nck

Pix/Cool

Git2

14-3-3z

Endophilin2

BAN-PPDE4B3

Ruk1

-

PP2C gamma


May 2002 May 2003


98 163



119 -


16 (26) 55 (90)


What can we learn from the larger data set?

• Last year, 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?

Interaction examples:1AFCS ID Bait Protein Name Prey Nuc GI Prey ID Prey Protein NameA001088 Grb2 3406752 A000381 BlnkA000456 Calcium-dependent protein kinase II alpha 3406752 A000381 BlnkA001088 Grb2 3406752 A000381 BlnkA001088 Grb2 3406752 A000381 BlnkA001088 Grb2 3406752 A000381 Blnk

AFCS ID Bait Protein Name Prey Nuc GI Prey ID Prey Protein NameA000605 cdc42 6754977 A000070 Pak1A000676 PIX/COOL 6754977 A000070 Pak1

AFCS ID Bait Protein Name Prey Nuc GI Prey ID Prey Protein NameA001220 Integrin beta 1 53353 A001678 Nucleoside diphosphate kinase BA001220 Integrin beta 1 53353 A001678 Nucleoside diphosphate kinase BA002108 Ryanodine receptor type I 53353 A001678 Nucleoside diphosphate kinase BA001775 PI3 kinase p85 regulatory subunit (alpha) 53353 A001678 Nucleoside diphosphate kinase BA000754 Dbl 53353 A001678 Nucleoside diphosphate kinase BA001220 Integrin beta 1 53353 A001678 Nucleoside diphosphate kinase B

AFCS ID Bait Protein Name Prey Nuc GI Prey ID Prey Protein NameA001775 PI3 kinase p85 regulatory subunit (alpha) 7305484 A000834 Endophilin 2A001776 PI3K p85beta 7305484 A000834 Endophilin 2A000381 BLNK 7305484 A000834 Endophilin 2

AFCS ID Bait Protein Name Prey Nuc GI Prey ID Prey Protein NameA000113 Nck1 13878194 A000365 BcapA000365 Bcap 13878194 A000365 BcapA000365 Bcap 13878194 A000365 BcapA000365 Bcap 13878194 A000365 BcapA001088 Grb2 13878194 A000365 BcapA001088 Grb2 13878194 A000365 Bcap

Interaction examples: 2AFCS ID Bait Protein Name Prey Nuc GI Prey IDA001441 Lyn 19070197A000538 CD19 19070197

AFCS ID Bait Protein Name Prey Nuc GI Prey IDA001810 phospholipase C gamma2 3064262A002001 Rac1 3064262

AFCS ID Bait Bait Protein Name Prey Nuc GI Prey IDA001810 phospholipase C gamma2 4633514A001810 phospholipase C gamma2 4633514A001810 phospholipase C gamma2 4633514A001810 phospholipase C gamma2 4633514A001810 phospholipase C gamma2 4633514A001088 Grb2 4633514A000456 Calcium-dependent protein kinase II alpha 4633514A000456 Calcium-dependent protein kinase II alpha 4633514A001810 phospholipase C gamma2 4633514A001810 phospholipase C gamma2 4633514

AFCS ID Bait Protein Name Prey Nuc GI Prey IDA002361 vav2 20894430A001088 Grb2 20894430A001088 Grb2 20894430A001088 Grb2 20894430A000113 Nck1 20894430

AFCS ID Bait Protein Name Prey Nuc GI Prey IDA000118 Wasp 26326968A000113 Nck1 26326968A001771 Phosphatidyl-inositol 3'-kinase gamma 26326968A000113 Nck1 26326968A000118 Wasp 26326968A000118 Wasp 26326968

CD19CD19

Actin Cytoskeleton

PDK1PDK1

PI3K (p110)PI3K (p110)

BtkBtk

WISHWISH

CAPCAP

DblDbl

CamK IICamK II

NdkBNdkB

cdc42cdc42

CD22CD22

FynFyn

SOS2SOS2

CD19CD19

BtkBtk

Actin Cytoskeleton

SOS2SOS2

WISHWISH

CAPCAP

DblDbl

CamK IICamK II

NdkBNdkB

cdc42cdc42

PDK1PDK1

CD22CD22

FynFyn

PI3K (p110)PI3K (p110)

1907019719070197

67553996755399

66715386671538

46335144633514

85673258567325

13542677135426772089443020894430

2632696826326968

30642623064262

Sam68

Protein 4.1G

AIP

Cbl-b

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


May 2002 May 2003


98 163



119 -


16 (26) 55 (90)


Bait Status

Protein nameDate

Submitted AFCS ID

Baits Ordered

Baits Created

Baits Searched Status

adenylyl cyclase V 11-Jul-01 A000001 9 5 5 No InteractionsN-Ras 12-Jul-02 A000004 4 4 3 Screens in progressK-Ras 4-Apr-02 A000005 1 1 1 Data Releasedp115RhoGEF 11-Jul-01 A000008 9 7 7 Data ReleasedBeta arrestin 1 11-Jul-01 A000019 5 5 5 Data ReleasedRGS 13 12-Jul-02 A000020 5 5 4 Screens in progressNFATc1 (AKA NFAT2) 8-Aug-01 A000024 11 5 3 Data ReleasedRGS 2 11-Jul-01 A000028 5 5 4 No InteractionsG alpha13 11-Jul-01 A000029 4 3 3 No InteractionsRGS 18 12-Jul-02 A000034 4 4 3 Screens in progressbtk 25-Jul-01 A000038 9 9 9 Data ReleasedG alpha12 13-Jul-01 A000039 4 1 1 No InteractionsPKG 23-Jul-02 A000043 5 5 3 No InteractionsProtein kinase A RI alpha 10-Oct-02 A000049 5 4 3 Screens in progressProtein kinase A RII beta 10-Oct-02 A000050 1 0 0 No InteractionsPAK1 11-Jul-01 A000070 6 5 5 Data ReleasedAcetylcholine (muscarinic) 10-Oct-02 A000080 6 6 6 Screens in progressN-WASp 11-Jul-01 A000091 8 4 3 Data ReleasedRGS 16 13-Sep-01 A000093 5 5 5 Data ReleasedI kappa B alpha 11-Jul-01 A000097 4 4 1 No InteractionsPDE4B3 11-Jul-01 A000105 6 6 3 No InteractionsG alpha i3 11-Jul-01 A000112 3 3 3 Data ReleasedNck1 13-Sep-01 A000113 6 6 6 Data ReleasedInositol (1,4,5,) P3 receptor 2 11-Jul-01 A000116 4 2 2 No InteractionsWasp 4-Apr-02 A000118 5 3 2 Data Releaseddock2 1-Feb-02 A000129 14 14 14 Data ReleasedRasGap p120 10-Oct-02 A000133 9 5 5 Screens in progressAdrenergic receptor beta 1 10-Oct-02 A000219 9 1 1 No InteractionsAdrenergic receptor beta 3 10-Oct-02 A000221 6 5 5 No InteractionsAKAP12 11-Jul-01 A000239 13 8 8 Data ReleasedPKB-alpha (= Akt1) 8-Aug-01 A000249 4 3 3 Data ReleasedAkt2 1-Feb-02 A000250 6 6 6 Data ReleasedAkt3 10-Oct-02 A000251 6 6 6 No InteractionsAP-3 beta3A 10-Oct-02 A000295 10 10 10 Screens in progressBam32 13-Sep-01 A000361 6 6 5 No InteractionsBcap 4-Apr-02 A000365 5 2 1 Data ReleasedBlk 12-Jul-02 A000380 6 6 4 No InteractionsBLNK 13-Jul-01 A000381 6 6 6 Data Releasedcalcineurin A-subunit 23-Jul-02 A000420 4 4 1 No InteractionsCalcineurin A beta 1-Feb-02 A000421 6 4 4 No InteractionsCalcium channel voltage- 1-Feb-02 A000431 6 6 6 Data ReleasedCalcium channel voltage- 8-Aug-01 A000440 9 8 6 Screens in progressCalmodulin III 25-Jul-01 A000454 6 6 3 No InteractionsCaMKI 23-Jul-02 A000455 4 3 1 No InteractionsCalcium-dependent protein 1-Feb-02 A000456 3 3 3 Data ReleasedCalcium-dependent protein 1-Feb-02 A000461 5 5 5 No Interactionscaveolin3 13-Sep-01 A000522 3 3 3 No InteractionsCbl 8-Aug-01 A000525 10 6 4 No InteractionsGRP-1 4-Apr-02 A000529 1 0 0 No Interactionsp70s6k 10-Oct-02 A000530 5 0 0 No InteractionsCD19 4-Apr-02 A000538 4 3 3 Data ReleasedCD21 12-Jul-02 A000541 5 3 1 Screens in progressCD22 12-Jul-02 A000542 7 7 5 Screens in progressCD32 4-Apr-02 A000556 4 3 3 No Interactionscd44 1-Feb-02 A000566 7 3 3 No Interactions

Protein nameDate

Submitted AFCS ID

Baits Ordered

Baits Created


CD45 4-Apr-02 A000567 7 5 5 No InteractionsCD79a 4-Apr-02 A000586 3 3 2 No InteractionsCD79b 4-Apr-02 A000587 3 3 3 Screens in progressCD81 12-Jul-02 A000591 4 4 4 No Interactionscdc42 1-Feb-02 A000605 3 3 3 Data ReleasedCCR7 1-Feb-02 A000630 4 4 4 No InteractionsCXCR4 1-Feb-02 A000636 4 4 4 No InteractionsChemokine receptor CXCR5 4-Apr-02 A000637 3 3 2 Data ReleasedPIX/COOL 13-Jul-01 A000676 7 6 6 Data ReleasedDock1 12-Jul-02 A000746 12 11 11 Screens in progressDbl 13-Sep-01 A000754 13 12 8 Data ReleasedDbs 10-Oct-02 A000755 11 11 8 Screens in progressDok2 12-Jul-02 A000779 5 5 3 No InteractionsCD150 4-Apr-02 A000905 3 3 3 No InteractionsFyn 12-Jul-02 A000969 5 5 3 Screens in progressG protein alpha i2 4-Apr-02 A000975 3 3 3 No InteractionsG protein beta 1 4-Apr-02 A000982 3 3 3 No InteractionsG protein beta 2 4-Apr-02 A000983 3 3 3 No InteractionsG protein gamma 2 4-Apr-02 A000991 1 1 1 No InteractionsGAB-1 23-Apr-02 A000997 5 5 2 Data ReleasedEmk 10-Oct-02 A001086 9 8 8 Screens in progressGrb2 13-Sep-01 A001088 4 4 4 Data ReleasedGRK2 11-Jul-01 A001094 5 5 5 No Interactionsglycogen synthase kinase- 23-Jul-02 A001106 4 4 3 No InteractionsHDAC5 23-Jul-02 A001113 6 3 3 No InteractionsIntegrin beta 2 4-Apr-02 A001213 5 5 5 Data ReleasedIntegrin beta 1 4-Apr-02 A001220 7 6 4 Data ReleasedKv1.1 10-Oct-02 A001352 6 5 5 No InteractionsKv1.2 1-Feb-02 A001353 5 5 4 Data ReleasedKv1.4 10-Oct-02 A001355 8 8 7 No InteractionsKv4.2 10-Oct-02 A001366 8 7 7 No InteractionsKv4.3 10-Oct-02 A001367 8 7 6 No InteractionsLck 4-Apr-02 A001394 4 3 2 Data ReleasedLyn 8-Aug-01 A001441 5 5 5 Data ReleasedMcip-1 1-Feb-02 A001481 3 3 3 Data ReleasedMEF2A 23-Jul-02 A001499 6 6 0 No InteractionsMEF 2C 1-Feb-02 A001503 5 5 5 Data ReleasedMEF2D 23-Jul-02 A001504 7 5 0 Screens in progressMEK5 23-Jul-02 A001509 6 5 0 Screens in progressMKK6 23-Jul-02 A001510 9 0 0 No InteractionsMitr 23-Jul-02 A001543 7 7 7 Screens in progressNa/Ca exchanger 11-Jul-01 A001590 6 6 6 No InteractionsNav1.1 10-Oct-02 A001610 11 3 3 Screens in progressNeurofibromin 2 10-Oct-02 A001631 6 6 3 Screens in progressNFAT2 8-Aug-01 A001640 11 8 6 No InteractionsNFAT3 23-Jul-02 A001641 17 0 0 No InteractionsNFkappaB p65 4-Apr-02 A001645 5 5 2 Data ReleasedP300 23-Jul-02 A001716 14 9 8 Screens in progressp38 MAPK 23-Jul-02 A001717 7 5 0 Screens in progressHDAC7 23-Jul-02 A001725 11 7 0 Screens in progressG2A receptor 13-Sep-01 A001726 4 4 4 No InteractionsPDK1 25-Jul-01 A001740 6 6 5 Data ReleasedPhosphoinositide 3-kinase 4-Apr-02 A001767 7 7 7 No InteractionsPhosphatidyl-inositol 3'-kinase 8-Aug-01 A001768 10 9 8 Data Released

Protein nameDate

Submitted AFCS ID

Baits Ordered

Baits Created

Baits Searched StatusPhosphatidyl-inositol 3'-kinase

p110 beta class Ia 8-Aug-01 A001769 9 7 6 Data ReleasedPhosphoinositide 3-kinase 12-Jul-02 A001770 9 9 4 Screens in progressPhosphatidyl-inositol 3'-kinase 13-Jul-01 A001771 8 8 7 Data ReleasedPI3 kinase p85 regulatory 8-Aug-01 A001775 8 6 5 Data ReleasedPI3K p85beta 1-Feb-02 A001776 9 9 8 Data ReleasedPhosphoinositide 4-phosphate 10-Oct-02 A001780 6 6 5 Screens in progressPhosphoinositide 4-phosphate 10-Oct-02 A001781 6 6 6 No InteractionsPhosphoinositide 4-phosphate 10-Oct-02 A001782 7 6 6 No InteractionsPhosphoinositide 5-phosphate 10-Oct-02 A001783 5 5 5 Data ReleasedPhospholamban 8-Aug-01 A001786 2 2 2 No InteractionsPhospholipase C beta 2 11-Jul-01 A001804 11 7 7 Data Releasedphospholipase C gamma1 13-Jul-01 A001809 9 7 7 Data Releasedphospholipase C gamma2 13-Jul-01 A001810 8 8 8 Data ReleasedPKA catalytic subunit alpha 8-Aug-01 A001914 4 4 4 No InteractionsProtein kinase A RI beta 10-Oct-02 A001917 5 5 4 Screens in progressProtein kinase A RII alpha 10-Oct-02 A001918 6 6 5 Screens in progressProtein kinase C beta1 11-Jul-01 A001920 10 2 2 No InteractionsPKC-epsilon 11-Jul-01 A001925 7 5 5 No InteractionsPKC zeta 25-Jul-01 A001934 6 3 3 Data ReleasedPTEN 13-Jul-01 A001941 5 3 3 No InteractionsRac1 13-Sep-01 A002001 3 3 3 Data ReleasedRac2 13-Jul-01 A002002 5 5 5 No InteractionsRac3 12-Jul-02 A002003 4 4 3 Screens in progressRasGEF1 10-Oct-02 A002030 10 6 6 Screens in progressRasGEF2 10-Oct-02 A002031 9 8 7 No InteractionsRasGRF1 10-Oct-02 A002032 11 8 8 Screens in progressRGS 1 11-Jul-01 A002040 4 4 4 Data ReleasedRGS 14 12-Jul-02 A002044 4 4 2 No InteractionsRGS 20 12-Jul-02 A002046 4 1 0 No InteractionsRGS 3 8-Aug-01 A002048 5 4 4 No InteractionsRhoA 1-Feb-02 A002062 3 3 3 Data ReleasedROCK1 2-Aug-01 A002088 11 11 10 No InteractionsRock2 10-Oct-02 A002089 9 9 8 No Interactionsp90rsk1 23-Apr-02 A002099 7 7 7 Data ReleasedRyanodine receptor type I 13-Sep-01 A002108 29 6 6 Data ReleasedRyanodine receptor Type II 1-Feb-02 A002109 18 18 17 Data ReleasedS100A1 1-Feb-02 A002111 3 3 1 No InteractionsSRF 23-Jul-02 A002147 4 2 0 No InteractionsShc 4-Apr-02 A002150 6 5 4 Data ReleasedSHIP1 8-Aug-01 A002154 12 11 11 No InteractionsSHIP2 1-Feb-02 A002155 9 9 7 No InteractionsSHP-1 4-Apr-02 A002156 6 6 6 Data ReleasedTec 23-Apr-02 A002264 5 5 5 No InteractionsTumor necrosis factor 10-Oct-02 A002346 6 6 5 Screens in progressVav1 12-Jul-02 A002360 7 5 2 Screens in progressvav2 1-Feb-02 A002361 11 10 9 Data ReleasedVav3 12-Jul-02 A002362 5 5 0 No InteractionsRasGRF2 10-Oct-02 A002441 9 8 8 Screens in progressAbi1 10-Oct-02 A002545 6 5 5 Screens in progressScar-2/WAVE-2 9-Aug-01 A002549 4 3 3 Data ReleasedHDAC4 23-Jul-02 A002996 8 8 8 Screens in progressGpr90 23-Jul-02 A003212 7 0 0 No InteractionsFmr1 10-Oct-02 A003252 8 7 6 Screens in progressPRex1 10-Oct-02 A003270 9 0 0 No Interactions

Bait Status

Protein nameDate

Submitted AFCS ID

Baits Ordered

Baits Created


K-Ras 4-Apr-02 A000005 1 1 1 Data Releasedp115RhoGEF 11-Jul-01 A000008 9 7 7 Data ReleasedBeta arrestin 1 11-Jul-01 A000019 5 5 5 Data ReleasedNFATc1 (AKA NFAT2) 8-Aug-01 A000024 11 5 3 Data Releasedbtk 25-Jul-01 A000038 9 9 9 Data ReleasedPAK1 11-Jul-01 A000070 6 5 5 Data ReleasedN-WASp 11-Jul-01 A000091 8 4 3 Data ReleasedRGS 16 13-Sep-01 A000093 5 5 5 Data ReleasedG alpha i3 11-Jul-01 A000112 3 3 3 Data ReleasedNck1 13-Sep-01 A000113 6 6 6 Data ReleasedWasp 4-Apr-02 A000118 5 3 2 Data Releaseddock2 1-Feb-02 A000129 14 14 14 Data ReleasedAKAP12 11-Jul-01 A000239 13 8 8 Data ReleasedPKB-alpha (= Akt1) 8-Aug-01 A000249 4 3 3 Data ReleasedAkt2 1-Feb-02 A000250 6 6 6 Data ReleasedBcap 4-Apr-02 A000365 5 2 1 Data ReleasedBLNK 13-Jul-01 A000381 6 6 6 Data ReleasedCalcium channel voltage- 1-Feb-02 A000431 6 6 6 Data ReleasedCalcium-dependent protein 1-Feb-02 A000456 3 3 3 Data ReleasedCD19 4-Apr-02 A000538 4 3 3 Data Releasedcdc42 1-Feb-02 A000605 3 3 3 Data ReleasedChemokine receptor CXCR5 4-Apr-02 A000637 3 3 2 Data ReleasedPIX/COOL 13-Jul-01 A000676 7 6 6 Data ReleasedDbl 13-Sep-01 A000754 13 12 8 Data ReleasedGAB-1 23-Apr-02 A000997 5 5 2 Data ReleasedGrb2 13-Sep-01 A001088 4 4 4 Data ReleasedIntegrin beta 2 4-Apr-02 A001213 5 5 5 Data ReleasedIntegrin beta 1 4-Apr-02 A001220 7 6 4 Data ReleasedKv1.2 1-Feb-02 A001353 5 5 4 Data ReleasedLck 4-Apr-02 A001394 4 3 2 Data ReleasedLyn 8-Aug-01 A001441 5 5 5 Data ReleasedMcip-1 1-Feb-02 A001481 3 3 3 Data ReleasedMEF 2C 1-Feb-02 A001503 5 5 5 Data ReleasedNFkappaB p65 4-Apr-02 A001645 5 5 2 Data ReleasedPDK1 25-Jul-01 A001740 6 6 5 Data ReleasedPhosphatidyl-inositol 3'-kinase 8-Aug-01 A001768 10 9 8 Data ReleasedPhosphatidyl-inositol 3'-kinase 8-Aug-01 A001769 9 7 6 Data ReleasedPhosphatidyl-inositol 3'-kinase 13-Jul-01 A001771 8 8 7 Data ReleasedPI3 kinase p85 regulatory 8-Aug-01 A001775 8 6 5 Data ReleasedPI3K p85beta 1-Feb-02 A001776 9 9 8 Data ReleasedPhosphoinositide 5-phosphate 10-Oct-02 A001783 5 5 5 Data ReleasedPhospholipase C beta 2 11-Jul-01 A001804 11 7 7 Data Releasedphospholipase C gamma1 13-Jul-01 A001809 9 7 7 Data Releasedphospholipase C gamma2 13-Jul-01 A001810 8 8 8 Data ReleasedPKC zeta 25-Jul-01 A001934 6 3 3 Data ReleasedRac1 13-Sep-01 A002001 3 3 3 Data ReleasedRGS 1 11-Jul-01 A002040 4 4 4 Data ReleasedRhoA 1-Feb-02 A002062 3 3 3 Data Releasedp90rsk1 23-Apr-02 A002099 7 7 7 Data ReleasedRyanodine receptor type I 13-Sep-01 A002108 29 6 6 Data ReleasedRyanodine receptor Type II 1-Feb-02 A002109 18 18 17 Data ReleasedShc 4-Apr-02 A002150 6 5 4 Data ReleasedSHP-1 4-Apr-02 A002156 6 6 6 Data Releasedvav2 1-Feb-02 A002361 11 10 9 Data ReleasedScar-2/WAVE-2 9-Aug-01 A002549 4 3 3 Data Released

Protein nameDate

Submitted AFCS ID

Baits Ordered

Baits Created


Abi1 10-Oct-02 A002545 6 5 5 Screens in progressAcetylcholine (muscarinic) 10-Oct-02 A000080 6 6 6 Screens in progressAP-3 beta3A 10-Oct-02 A000295 10 10 10 Screens in progressCalcium channel voltage- 8-Aug-01 A000440 9 8 6 Screens in progressCD21 12-Jul-02 A000541 5 3 1 Screens in progressCD22 12-Jul-02 A000542 7 7 5 Screens in progressCD79b 4-Apr-02 A000587 3 3 3 Screens in progressDbs 10-Oct-02 A000755 11 11 8 Screens in progressDock1 12-Jul-02 A000746 12 11 11 Screens in progressEmk 10-Oct-02 A001086 9 8 8 Screens in progressFmr1 10-Oct-02 A003252 8 7 6 Screens in progressFyn 12-Jul-02 A000969 5 5 3 Screens in progressHDAC4 23-Jul-02 A002996 8 8 8 Screens in progressHDAC7 23-Jul-02 A001725 11 7 0 Screens in progressMEF2D 23-Jul-02 A001504 7 5 0 Screens in progressMEK5 23-Jul-02 A001509 6 5 0 Screens in progressMitr 23-Jul-02 A001543 7 7 7 Screens in progressNav1.1 10-Oct-02 A001610 11 3 3 Screens in progressNeurofibromin 2 10-Oct-02 A001631 6 6 3 Screens in progressN-Ras 12-Jul-02 A000004 4 4 3 Screens in progressP300 23-Jul-02 A001716 14 9 8 Screens in progressp38 MAPK 23-Jul-02 A001717 7 5 0 Screens in progressPhosphoinositide 3-kinase 12-Jul-02 A001770 9 9 4 Screens in progressPhosphoinositide 4-phosphate 10-Oct-02 A001780 6 6 5 Screens in progressProtein kinase A RI alpha 10-Oct-02 A000049 5 4 3 Screens in progressProtein kinase A RI beta 10-Oct-02 A001917 5 5 4 Screens in progressProtein kinase A RII alpha 10-Oct-02 A001918 6 6 5 Screens in progressRac3 12-Jul-02 A002003 4 4 3 Screens in progressRasGap p120 10-Oct-02 A000133 9 5 5 Screens in progressRasGEF1 10-Oct-02 A002030 10 6 6 Screens in progressRasGRF1 10-Oct-02 A002032 11 8 8 Screens in progressRasGRF2 10-Oct-02 A002441 9 8 8 Screens in progressRGS 13 12-Jul-02 A000020 5 5 4 Screens in progressRGS 18 12-Jul-02 A000034 4 4 3 Screens in progressTumor necrosis factor 10-Oct-02 A002346 6 6 5 Screens in progressVav1 12-Jul-02 A002360 7 5 2 Screens in progressadenylyl cyclase V 11-Jul-01 A000001 9 5 5 No InteractionsAdrenergic receptor beta 1 10-Oct-02 A000219 9 1 1 No InteractionsAdrenergic receptor beta 3 10-Oct-02 A000221 6 5 5 No InteractionsAkt3 10-Oct-02 A000251 6 6 6 No InteractionsBam32 13-Sep-01 A000361 6 6 5 No InteractionsBlk 12-Jul-02 A000380 6 6 4 No InteractionsCalcineurin A beta 1-Feb-02 A000421 6 4 4 No Interactionscalcineurin A-subunit 23-Jul-02 A000420 4 4 1 No InteractionsCalcium-dependent protein 1-Feb-02 A000461 5 5 5 No InteractionsCalmodulin III 25-Jul-01 A000454 6 6 3 No InteractionsCaMKI 23-Jul-02 A000455 4 3 1 No Interactionscaveolin3 13-Sep-01 A000522 3 3 3 No InteractionsCbl 8-Aug-01 A000525 10 6 4 No InteractionsCCR7 1-Feb-02 A000630 4 4 4 No InteractionsCD150 4-Apr-02 A000905 3 3 3 No InteractionsCD32 4-Apr-02 A000556 4 3 3 No Interactionscd44 1-Feb-02 A000566 7 3 3 No InteractionsCD45 4-Apr-02 A000567 7 5 5 No Interactions

Protein nameDate

Submitted AFCS ID

Baits Ordered

Baits Created


CD79a 4-Apr-02 A000586 3 3 2 No InteractionsCD81 12-Jul-02 A000591 4 4 4 No InteractionsCXCR4 1-Feb-02 A000636 4 4 4 No InteractionsDok2 12-Jul-02 A000779 5 5 3 No InteractionsG alpha12 13-Jul-01 A000039 4 1 1 No InteractionsG alpha13 11-Jul-01 A000029 4 3 3 No InteractionsG protein alpha i2 4-Apr-02 A000975 3 3 3 No InteractionsG protein beta 1 4-Apr-02 A000982 3 3 3 No InteractionsG protein beta 2 4-Apr-02 A000983 3 3 3 No InteractionsG protein gamma 2 4-Apr-02 A000991 1 1 1 No InteractionsG2A receptor 13-Sep-01 A001726 4 4 4 No Interactionsglycogen synthase kinase- 23-Jul-02 A001106 4 4 3 No InteractionsGpr90 23-Jul-02 A003212 7 0 0 No InteractionsGRK2 11-Jul-01 A001094 5 5 5 No InteractionsGRP-1 4-Apr-02 A000529 1 0 0 No InteractionsHDAC5 23-Jul-02 A001113 6 3 3 No InteractionsI kappa B alpha 11-Jul-01 A000097 4 4 1 No InteractionsInositol (1,4,5,) P3 receptor 2 11-Jul-01 A000116 4 2 2 No InteractionsKv1.1 10-Oct-02 A001352 6 5 5 No InteractionsKv1.4 10-Oct-02 A001355 8 8 7 No InteractionsKv4.2 10-Oct-02 A001366 8 7 7 No InteractionsKv4.3 10-Oct-02 A001367 8 7 6 No InteractionsMEF2A 23-Jul-02 A001499 6 6 0 No InteractionsMKK6 23-Jul-02 A001510 9 0 0 No InteractionsNa/Ca exchanger 11-Jul-01 A001590 6 6 6 No InteractionsNFAT2 8-Aug-01 A001640 11 8 6 No InteractionsNFAT3 23-Jul-02 A001641 17 0 0 No Interactionsp70s6k 10-Oct-02 A000530 5 0 0 No InteractionsPDE4B3 11-Jul-01 A000105 6 6 3 No InteractionsPhosphoinositide 3-kinase 4-Apr-02 A001767 7 7 7 No InteractionsPhosphoinositide 4-phosphate 10-Oct-02 A001781 6 6 6 No InteractionsPhosphoinositide 4-phosphate 10-Oct-02 A001782 7 6 6 No InteractionsPhospholamban 8-Aug-01 A001786 2 2 2 No InteractionsPKA catalytic subunit alpha 8-Aug-01 A001914 4 4 4 No InteractionsPKC-epsilon 11-Jul-01 A001925 7 5 5 No InteractionsPKG 23-Jul-02 A000043 5 5 3 No InteractionsPRex1 10-Oct-02 A003270 9 0 0 No InteractionsProtein kinase A RII beta 10-Oct-02 A000050 1 0 0 No InteractionsProtein kinase C beta1 11-Jul-01 A001920 10 2 2 No InteractionsPTEN 13-Jul-01 A001941 5 3 3 No InteractionsRac2 13-Jul-01 A002002 5 5 5 No InteractionsRasGEF2 10-Oct-02 A002031 9 8 7 No InteractionsRGS 14 12-Jul-02 A002044 4 4 2 No InteractionsRGS 2 11-Jul-01 A000028 5 5 4 No InteractionsRGS 20 12-Jul-02 A002046 4 1 0 No InteractionsRGS 3 8-Aug-01 A002048 5 4 4 No InteractionsROCK1 2-Aug-01 A002088 11 11 10 No InteractionsRock2 10-Oct-02 A002089 9 9 8 No InteractionsS100A1 1-Feb-02 A002111 3 3 1 No InteractionsSHIP1 8-Aug-01 A002154 12 11 11 No InteractionsSHIP2 1-Feb-02 A002155 9 9 7 No InteractionsSRF 23-Jul-02 A002147 4 2 0 No InteractionsTec 23-Apr-02 A002264 5 5 5 No InteractionsVav3 12-Jul-02 A002362 5 5 0 No Interactions

Pattern Among Negative Baits?

• 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

Project Status-The Year Ahead

May 2003 May 2004


163 ?


55 ?

Bait Proteins in Screening

36 ?

Bait Proteins with No Interactions

72 ?

Total Interactions 473 ?

Acknowledgements

Myriad

Terrece Pearman

Brandi Williams

Karen HeichmanPaul Bartel

AfCS

Data analysis and displayGil SambranoBob SinkovitsJoshua Li

RNA preparationKeng-Mean LinRobert HsuehZhen YanJoella GrossoehmeRead PierceJason PolasekJody Girouard

Identification of Protein-Protein Interactions by the Yeast 2-Hybrid System Alliance for Cell...

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