Post on 14-Jan-2016
description
Elucidation of signaling pathways by functional proteomics
Metodi V. MetodievDepartment 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-sensitivemore than 5 cm halo
Super-sensitive, hyper-adaptive, larger but filled-in halo
F
STE20
STE11
STE7
STE5
FUS3
P
P
FUS3
FAR1G1 cyclins
G1 to Scell cycleprogression
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 ofProteins 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 proteinunder 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
STE20
STE11
STE7
STE5
P
P
FUS3
FAR1G1 cyclins
G1 to Scell cycleprogression
The pheromone response pathway of Saccharomyces cerevisiae
GTP
Activation of transcription
STE12
Adhesion, Fusion
Dig1/2
P
P
FUS3 P
P
FUS3
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-LQNKRANDVIEQSLQL GPA1 7-LQRRNLKGLNLNL STE7 97- KRGRVPAPLNL DIG1 72- KRGNIPKPLNL FAR1207-N KKN CILPKLDLNL PTP2 55-NNKRNHQKAHSLDL MPT5
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
gpa1DSD 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
gpa1DSD confers a defect in Ste4 phosphorylation*
*Metodiev et al., Science, 2002
GPA1WT
0 hours 2 hours 8 hours
gpa1DSD
It is worth noting that fus3 mutants also have defect in polarization.
gpa1DSD/bud1 cells do not shmoo
PRTPx…TP.x...PSP Yeast Kar3
PRSPx…SP.x...PSP
Mammalian CENP-E
Kar3 shares domain architecture and MAPK phosphorylationsites with mammalian orthologs known to be substrates of Erk2
GPA1WT gpa1DSD
91 4 0 4 1 % 44 16 12 18 10 %
DSD confers several classes of abnormal MT morphology.Previously similar effects were attributed to mutations in KAR3, KAR9, SPA2, BNI1 and other genes.
gpa1DSD confers abnormal microtubule dynamics
Gpa1GTP Fus3PP
Ste4Far1*
Kar3*
Kar9**
Fus1*Fus2*
Bni1*
(chemotropism)
(MT attachment)
(plasma membrane fusion)
(nuclear migration/MT length)
X1, X2….Xn (many other proteins at the membranecontain 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
bni1FUS3Q93G
FUS3Q93G
+ 1-Na PP1
WT
gpa1-EE
78
13
84
9
28
13
31
10
11
20
20
30
12
9
36
6
50
40
*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 gpa1DSD strains and when Fus3 is inactivated*.