1. Protein Traps in AfCS Cells Dianne DeCamp, Yan Ni, Hongjun Shu, Iain Fraser

2. 1. Objective 2. Strategy for WEHI Cells 3. Progress 4. Future Directions HELP

3. 1.Identify protein-protein interactions in mammalian cells 2.Accomplish this in the AfCS cell line of choice for other studies Objectives:

4. 1. Design of Bait proteins (Tags) Requirements 4.Identification of associated proteins by mass spectrometry 3. Isolation of Bait proteins with associated protein complexes 2. Expression of Bait proteins

5. A Tale of Two Tags A Strategy for WEHI Cells

6. FTM Tag for Bait Proteins N EQKLISEEDLDYKDDDDK FLAG c-myc TEV protease cleavage site ENLYFQ C attB IgG beads N CBP Protein A TEV protease cleavage site ENLYFQBait C attB Ca-CaM Beads TAP Tag for Bait Proteins (tandem affinity purification) Bait

7. Expression of Bait Protein 1.Transfection 2. Infection with virus

8. Retroviral Vectors Promoter 1: CMV or MSCV, expression level is similar. TAG: FTM or TAP tag at the N- or C- terminus. Selection marker: puromycin or CD4 surface marker; both work well. Tag Promoter 1Bait gene IRES or promoter2 Selection marker

9. Progress

10. A Two Week Cycle Week 1. Plate packaging cells: eight 100 mm dishes MonTuWedThrFri Transfection: 8 genes Harvest virus, store in 4  C Harvest virus, infection 6 million/virus Remove virus, Passage cells FACS analysis or puromycin select Week 2. Mon Tu WedThrFri Sorting by FACS or CD4 beads western Passage cells (~40 M) Potential Capacity of 8 viruses/week

11. >75 retroviral constructs tested ~60 ‘cell lines’ generated xxx(enriched by selection) 29 genes

12. Genes Tested in WEHI-231 Cells BLNK Btk Calbindin CaMKII delta Cellubrevin Clk3 Erk1 Erk2 Furin Grb2 RhoB RhoC RSK3 PI3Kp85  PI3Kp110  Sec22 Stat3 Syk Vimentin YFP Grk2 Jnk1 Lyn MAPKK7  2 MEK3 Nek4 PDE4B3 rgRGS RGS4 RhoA = used in pull-downs = poor expression = not yet used in pull-downs

13. Enrich Expression by FACS 28% before sorting 95% after sorting MSCV-FTM-Erk1 - Before sorting: ~10 ng/1E6 WEHI cells - After sorting: ~50 ng/1E6 WEHI cells (endogenous Erk1, 25-30 ng/1E6)

14. FTM-YFP Elution FTM-ERK2 Elution ERK2 YFP 114 49 37 26 83 63 kDa TEV protease FTM Pull-Down Limitation: Myc portion of the FTM tag does not work

15. TAP tag requires both affinity steps 2-Step Pull-Down ERK2-TAP IgG beads only – TEV cleavage CaM beads only – TEV cleavage TAP-YFP IgG leaching from the beads Only nonspecific binding Purified bait protein

16. TAP tagged baits can be isolated to essential homogeneity – but this is not the goal!

17. Colloidal Coomassie-stained Gels FTM-YFP Elution FTM-ERK2 Elution ERK2 YFP 114 49 37 26 83 63 kDa TEV protease FTM Pull-Down TAP Pull-Down 114 ERK2 RhoB ERK1-TAP Elution RhoB-TAP Elution 26 83 63 49 37 kDa

18. Partial Success with FTM Pulldowns Example: FTM-RhoC Can reproducibly pull down RhoGDI Binds to FTM-RhoC (but not FTM-RhoB) Soluble protein, 5-10  g/ml in WEHI lysate Represents 0.1-0.2% of total soluble cell protein K d ’s for Rho-related proteins are 2-30 nM

19. MS Analysis of Coll. Coomassie-stained Gel 1 2 3 5 6 7 8 4 YFP Elution ERK2 Elution ERK2 Identified Proteins YFP Elution ERK2 Elution YFP 1. HSP 90 beta 1x10 -11 2. Keratin 1x10 -26 3. keratin 1x10 -42 4. BSA 2x10 -7 7. IgG 2x10 -8 8. IgG 6x10 -20 5. tropomyosin 5 8x10 -12 6. YFP 2x10 -33 F-actin capping protein 3x10 -7 114 49 37 26 83 63 kDa Score TEV protease

20. MS Analysis of SDS/PAGE Gels 1 23 4 56 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 YFP Elution Rho B Elution Rho C Elution 22 23 24 25 26 27 28 29 30 YFP Elution Rho B Elution Rho C Elution Colloidal Coomassie SilverQuest

22. Erk2 Associated Proteins in 293 cells ATP-dependent RNA helicase A chromatin-specific transcription elongation factor splicing Factor 3b, subunit 1 & 2 ribosomal protein S6 kinase (Rsk2P90) methylosome protein 50 (MEP50) Kinesin-related Motor Protein EG5 protein methyltransferase rErk2 kiaa0122 gene product Others… Erk2

23. Conclusions regarding FTM-Tagged Baits Current protocol can detect a few interacting proteins (RhoGDI, filamen) Isolate mostly “nonspecific” proteins The abundance of known interacting proteins in WEHI lysate may be too low

24. 1. Design of Bait proteins (Tags) Summary -- Requirements 4.Identification of associated proteins by mass spectrometry 3. Isolation of Bait proteins with associated protein complexes 2. Expression of Bait proteins OK Not optimal

25. Future Directions Increased communication with investigators whom have similar paradigms working Alternative epitope tags What’s needed? 1.Better methods: efficient and reproducible 2. New Cells Solutions Choice of cells – (not limited to AfCS cells?)

26. She Chen Richard Davis Debalina Siddeeq Melissa Stalder Joelle Zavzavadjian