Lecture 10. Proteomics II

**Lewis TS, Hunt JB, Aveline LD, Jonscher KR, Louie DF, Yeh JM, Nahreini TS, Resing KA, Ahn NG. 2000. Identification of novel MAP kinase pathway signaling targets by functional proteomics and mass spectrometry. Mol Cell 6:1343-1354

Zhu H, Bilgin M, Bangham R, Hall D, Casamayor A, Bertone P, Lan N, Jansen R, Bidlingmaier S, Houfek T, Mitchell T, Miller P, Dean RA, Gerstein M, Snyder M. 2001. Global analysis of protein activities using proteome chips. Science. 293:2101-2105.

Paper 1.

Two parts:

1. 2D-gels to Display the Proteomeand Potential Erk1/2 Targets

2. MS to Identify the Erk1/2 Targets

Lewis et al. 2000. Identification of novel MAP kinase pathway signaling targets by functional proteomics and mass spectrometry. Mol Cell 6:1343-1354

The Mitogen Activated Protein Kinase (MAPKK) Family

MAPKKK

MAPKK

MAPK

Raf MKK1 MKK4

MEK1/2 JEK1/2 MKK6

ERK1/2 JNK1/2 p38

GrowthDifferentiationApoptosisStress Responses

Phorbol Ester (PMA)

ras

raf

MEK1/2

Erk1/2

Protein Kinase C (PKC)

Differentiation K562 cellsTo Megakaryocytes(Platelets)

Part 1. 2D Gels to Compare the Proteome in PMA treated K562 cells

3500 proteins resolved: detection from 5000-107 copies per cell

91 proteins found different in PMS treated cells (30 down, 61 up):magnitude change >1.5-fold;

reproducible in 3 gels from at least 2 experiments

Kinetic Analysis of PMA-dependent changes in expression

Down

Up

MobilityAltered

Cluster Analysis of the 91 Changes Reveals4 General Classes

Phorbol Ester (PMA)

ras

raf

MEK1/2

Erk1/2

Protein Kinase C (PKC)

Differentiation K562 cellsTo Megakaryocytes(Platelets)

U0126

Question: Are these proteins regulated by Erks or PKC?

1. Pharmacological InhibitorShould Inhibit Changes

1. Pharmacological Inhibitor Should Inhibit Changes

No Phorbol Ester (PMA)

ras

raf

MEK1/2*

Erk1/2 Differentiation K562 cellsTo Megakaryocytes(Platelets)

MEK1 Dominant Positive Mutation

Question: Are these proteins regulated by Erks or PKC?

2. Dominant Signaling Mutations Should Cause Changes in Absence of PMA

2. Dominant Signaling Mutations Should Cause Changes in Absence of PMA

CorrespondenceBetween U0126Inhibition and MKK1* Stimulation

Conclusion:

66% of ChangesAre DIRECT Targetsof Erk1/2

Peptide Mixture

MALDI (Matrix Assisted Laser Desorption Ionization) Mass SpecUsed to Fingerprint the 91 proteins

HPLC coupled with nanospray MS Used to Confirm Identity of the Proteins

-Result: 41 proteins account for the 91 Changes-25 are Direct Targets of Erks-20/25 Represent newly Defined Targets

Product:Precursor Relationships

Paper 2. Protein Chips to Study Protein:Proteinor Protein:Lipid Interactions

Zhu H, et al 2001 Global analysis of protein activities using proteome chips. Science. 293:2101-2105.

Yeast Proteome on a Single Glass Slide:5800 GST Proteins Purified and Placed on the Chip

Probed with anti-GST as a CONTROL

Finding All Protein Partners for Calmodulin:14/39 Contain a Conserved Calmodulin Motif

Biotinylated Calmodulin Used to Probe Chip

Identification of Lipid Binding Proteins

PI3 Interacting Proteins

Specificfir PI3

Bind PI3and PC

Conventional Techniques ConfirmLipid Binding by Proteins Identified

with the CHIP