Elucidation of signaling pathways by functional proteomics

1. Elucidation of signaling pathways by functional proteomics Metodi V. MetodievDepartment of Biological Sciences, University of Essex, United Kingdom

2. Today’s talk: • Functional Proteomics of Mitogen Activated Protein Kinases (MAPK) – regulated signal transduction • Clinical Proteomics: Identification novel protein biomarkers of breast cancer

3. Mitogen Activated Protein Kinases (MAPK) • Proline directed protein serine/threonine kinases • Tightly regulated by dual phosphorylation on TXY motif in its phosphorylation loop • Respond to external stimuli: growth factors, chemokines, stress etc • Phosphorylate diverse array of substrates that regulate proliferation, differentiation, immune response, cytoskeleton rearrangements etc. • Only small fraction of these substrates are identified

4. The mating pathway of Saccharomyces cerevisiae is the prototypical MAPK – regulated signaling cascade acell The cell cycle of the haploid a cell acell conjugation zygote a/adiploid cell

5. The “super-sensitive” and “hyper-adaptive” alleles of the pheromone- responsive Ga protein Wild Type - about 3.5 cm halo Super-sensitive, hyper-adaptive, larger but filled-in halo Super-sensitive more than 5 cm halo

6. The pheromone response pathway of Saccharomyces cerevisiae a g b GDP GDP GTP FUS3 STE5 P Adhesion, Fusion STE12 P Activation of transcription Dig1/2 G1 to S cell cycle progression G1 cyclins FAR1 aF STE20 STE11 STE7 FUS3

7. Beyond Genomics: The completion of the genome of S. cerevisiae allowed us to apply post-genomic approaches, such as the expression profiling, sometimes also called “General Proteomics” WT High-resolution 2-DE using IPG strips can resolve thousands of Proteins by charge and size differences Mutant

8. The functional proteomics approach as applied to signal transduction: • It goes beyond expression profiling to attempt a system-wide analysis of signal-regulated protein-protein interactions andprotein post-translational modifications (phosphorylation mostly). • It is a highly integrated approach and employs diverse arsenal of techniques: • Affinity techniques including arrays of proteins and peptides; • Advanced separation methods; • Mass spectrometry; • Bioinformatics; • Molecular genetics - to set up the model system for optimal performance;

9. Proteomic screen for signal regulated protein-protein interactions I. Bait construction: GST-Gpa1 fusion protein under CUP1 promoter on a 2m vector. The GST entity (blue) confers high solubility and allows for highly specific affinity capture under mild conditions. Gpa1 GST II. Affinity capture on GSH sepharose beads and high-resolution 2D PAGE analysis, which adds to the fidelity of the identification. Input no aF + aF 2.43 III. In gel trypsin digestion, MALDI MS and identification by peptide fragment mass fingerprints. Left panel - MAPK Fus3 identified by ProFound. The Z value of 2.43 is the highest possible. Right panel - the kinesin motor Kar3 identified by MASCOT search engines. Hits outside the green area are significant (red bar).

10. The pheromone response pathway of Saccharomyces cerevisiae a g b GTP FUS3 FUS3 FUS3 STE5 P P P Adhesion, Fusion STE12 P P P Activation of transcription Dig1/2 G1 to S cell cycle progression G1 cyclins FAR1 aF STE20 STE11 STE7

11. Gpa1 affinity beads precipitated Fus3-myc. GST-Gpa1 precipitated Fus3-myc. GST-Gpa1 precipitated a 40 kDa protein that is recognized by the Anti-active antibody The interaction was inhibited by phosphatase. Validation of the proteomics results by pull-down...

12. * Gpa1 PD Lysates *From Metodiev et al., Science, 2002 [K/R][K/R]x…xLxL 18-LQNKRANDVIEQSLQLGPA1 7-LQRRNLKGLNLNL STE7 97- KRGRVPAPLNLDIG1 72- KRGNIPKPLNLFAR1 207-N KKN CILPKLDLNLPTP2 55-NNKRNHQKAHSLDL MPT5 K21E R22E = DSD (Docking-Site-Disrupted) mutant of Gpa1

13. gpa1DSD interacts normally with the receptor and bg but confers defects in adaptation and mating* B A C A. Defect in Gpa1 mediated adaptation and recovery B. Defect in overall mating ability C. Defect in mating fidelity (chemotropism) *Metodiev et al., Science, 2002

14. gpa1DSD confers a defect in Ste4 phosphorylation* *Metodiev et al., Science, 2002

15. gpa1DSD/bud1D cells do not shmoo 0 hours 2 hours 8 hours GPA1WT gpa1DSD It is worth noting that fus3 mutants also have defect in polarization.

16. Yeast Kar3 PRTPx…TP.x...PSP Mammalian CENP-E PRSPx…SP.x...PSP Kar3 shares domain architecture and MAPK phosphorylation sites with mammalian orthologs known to be substrates of Erk2

17. gpa1DSD confers abnormal microtubule dynamics gpa1DSD GPA1WT 44 16 12 18 10 % 91 4 0 4 1 % DSD confers several classes of abnormal MT morphology. Previously similar effects were attributed to mutations in KAR3, KAR9, SPA2, BNI1 and other genes.

18. Ste4 Far1* ?! A model to explain the plethora of effects of DSD (chemotropism) Bni1* Kar9** (MT attachment) Fus3PP Gpa1GTP Pea2**/Spa2* Kar3* (nuclear migration/MT length) Fus1* (plasma membrane fusion) Fus2* (adaptation) Sst2* X1, X2….Xn (many other proteins at the membrane contain PXT/SP and could potentially be Fus3 substrates - ion channels, pumps etc.) * = PXT/SP

19. WT bni1D 84 10 6 FUS3Q93G 28 20 12 40 FUS3Q93G + 1-Na PP1 78 13 9 gpa1-EE 13 31 20 36 9 11 30 50 Bni1 is a substrate of Fus3. It regulates the localization of Kar9. Kar9 is not localized properly in gpa1DSD strains and when Fus3 is inactivated*. *Matheos, Metodiev, Stone, and Rose Journal of Cell Biology, 2004