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

Today’s talk: 1.Functional Proteomics of Mitogen Activated Protein Kinases (MAPK) – regulated signal transduction 2.Clinical Proteomics: Identification novel protein biomarkers of breast cancer

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

The mating pathway of Saccharomyces cerevisiae is the prototypical MAPK – regulated signaling cascade The cell cycle of the haploid a cell  cell a cell a  diploid cell zygote conjugation

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

FF   STE20 STE11 STE7 STE5 FUS3 P P FAR1 G1 cyclins G1 to S cell cycle progression The pheromone response pathway of Saccharomyces cerevisiae GDP GTP  Activation of transcription STE12 Adhesion, Fusion Dig1/2 GDP

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 Mutant High-resolution 2-DE using IPG strips can resolve thousands of Proteins by charge and size differences

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 and protein 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;

Input no  F +  F II. Affinity capture on GSH sepharose beads and high-resolution 2D PAGE analysis, which adds to the fidelity of the identification. Proteomic screen for signal regulated protein-protein interactions I. Bait construction: GST-Gpa1 fusion protein under CUP1 promoter on a 2  vector. The GST entity (blue) confers high solubility and allows for highly specific affinity capture under mild conditions. Gpa1 GST 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). 2.43

FF   STE20 STE11 STE7 STE5 P P FUS3 FAR1 G1 cyclins G1 to S cell cycle progression The pheromone response pathway of Saccharomyces cerevisiae GTP  Activation of transcription STE12 Adhesion, Fusion Dig1/2 P P FUS3 P P

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

K21E R22E = DSD (Docking-Site-Disrupted) mutant of Gpa1 [K/R][K/R]x…xLxL 18-LQNKRANDVIEQSLQLGPA1 7-LQRRNLKGLNLNLSTE7 97- KRGRVPAPLNLDIG1 72- KRGNIPKPLNLFAR1 207-N KKN CILPKLDLNLPTP2 55-NNKRNHQKAHSLDLMPT5 Gpa1 PD Lysates *From Metodiev et al., Science, 2002 *

A. Defect in Gpa1 mediated adaptation and recovery A B. Defect in overall mating ability B gpa1 DSD interacts normally with the receptor and  but confers defects in adaptation and mating* C. Defect in mating fidelity (chemotropism) C *Metodiev et al., Science, 2002

gpa1 DSD confers a defect in Ste4 phosphorylation* *Metodiev et al., Science, 2002

G PA1 WT 0 hours2 hours8 hours gpa1 DSD It is worth noting that fus3 mutants also have defect in polarization. gpa1 DSD /bud1  cells do not shmoo

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

GPA1 WT gpa1 DSD % % DSD confers several classes of abnormal MT morphology. Previously similar effects were attributed to mutations in KAR3, KAR9, SPA2, BNI1 and other genes. gpa1 DSD confers abnormal microtubule dynamics

Gpa1 GTP Fus3 PP Ste4 Far1* Kar3* Kar9** Fus1* Fus2* Bni1* (chemotropism) (MT attachment) (plasma membrane fusion) (nuclear migration/MT length) X 1, X 2 ….X n ( many other proteins at the membrane contain PXT/SP and could potentially be Fus3 substrates - ion channels, pumps etc.) ?! Sst2* (adaptation) Pea2**/Spa2* * = PXT/SP A model to explain the plethora of effects of DSD

bni1  FUS3 Q93G + 1-Na PP1 WT gpa1-EE *Matheos, Metodiev, Stone, and Rose Journal of Cell Biology, 2004 Bni1 is a substrate of Fus3. It regulates the localization of Kar9. Kar9 is not localized properly in gpa1 DSD strains and when Fus3 is inactivated*.