Post-Translational Modifications:CrossTalk

Robert Chalkley

Chem 204

•A large variety of PTMs are used by the cell

•The different modifications do not act independent of each other

•Most studies analyze one PTM (or one site) at a time

•Can this reveal the biological control?

•Present examples why multiple PTM analysis important

•Show examples of how multiple PTM analysis can be performed

Introduction

Multi-Site Phosphorylation

Many examples where multiple phosphorylations required for protein activation / regulation

Growth Factor receptors:

•Autophosphorylate (pY) to cause receptor dimerization

•Contain multiple phosphorylation sites (pY, pS and pT)

•Different phosphorylation sites represent binding sites for different proteins to start signaling cascades

•May be that phosphorylation of any one of several sites causes activation, but dephosphorylation of all sites required for inactivation.

Cohen, P. Trends Biochem Sci (2000) 25 12 596-601

O-GlcNAcylation and Phosphorylation

•O-GlcNAc modified proteins are also potential phosphoproteins

•Many examples where the same or neighboring residues can be either GlcNAcylated or phosphorylated.

•Multiple experiments have shown the two modifications interact/effect each other

Zeidan, Q. and Hart, G. W. J Cell Sci (2010) 123 13-22

O-GlcNAcylation and Phosphorylation: Ying-Yang

Sites of Modification are sometimes the same

Mutually exclusive and opposing effects?

c-Myc: Proto-oncogene transcription factor

Thr58 is a mutational ‘hotspot’ in lymphomas.

Thr58 can be phosphorylated or O-GlcNAc modified

O-GlcNAc in growth inhibited / starved cells

Ser62 phosphorylation required for Thr58 phosphorylation

Mutation of Ser62 increases O-GlcNAcylation at Thr58

Mutate Thr58: how do you know what PTM causes the effect?

Chou, T-Y et al. J Biol Chem (1995) 270 32 18961-18965

Wang, Z. et al. Mol Cell Proteomics (2007) 6 8 1365-1379

Effect of Inhibiting GSK-3 on O-GlcNAcylation

•GSK-3 inhibited using Lithium

•Quantitative study of the effect on O-GlcNAc modification

Wang, Z. et al. Mol Cell Proteomics (2007) 6 8 1365-1379

O-GlcNAcylation Changes upon GSK-3 Inhibition

•Enriched for modified proteins by IP

•SILAC for quantifying changes

•Increase in O-GlcNAc: 10 proteins

•Decrease in O-GlcNAc: 19 proteins

•Is it safe to assume changes in protein levels after IP correspond to PTM level changes?

•Modifications play different roles

•Phosphorylation can lead to increases or decreases of O-GlcNAcylation

O-GlcNAc and Phosphorylation Co-Analysis

•Mouse Post-Synaptic Density

GlcNAc-Enriched Fraction

GlcNAc-Depleted Fraction

Phospho-Enriched Fraction

PTM Depleted Fraction

LWAC

TiO2High pH RPLC

High pH RPLC High pH RPLC

Digest

LC/MS/MS

LC/MS/MS LC/MS/MS

O-GlcNAcylation and Phosphorlyation Identification

•≈2200 phosphorylated peptides

•≈250 GlcNAcylated peptides

•≈ 250 GlcNAcylated peptides => ≈ 200 O-GlcNAcylation sites on 80 different proteins

•For half of the O-GlcNAc modified proteins, phosphopeptides were also identified.

•4 peptides both O-GlcNAcylated and phosphorylated.

To understand relationship need quantitative data.

Bassoon is Heavily O-GlcNAcylated and Phosphorylated

•Bassoon is a major component of the cytomatrix in the presynaptic active zone.

•Involved in spatial and temporal control of neurotransmitter release.

Phospho/GlcNAc sites (3)

•Why is this protein so heavily post-translationally modified?

•What are the modifications doing?

•Regulating protein-protein interactions?

OGT regulation by PTM

Yang et al. Nature (2008) 451 7181 964-969

Ubiquitination

•Reversible modification of lysine (or protein N-terminus)

•Can be addition of single Ub or chain of Ub can be built up

•i.e. Ubiquitin becomes ubiquitinated

•7 different lysines in ubiquitin

•Site of linkage during polyubiquitination determines biological effect

Kirkpatrick et al. Nat Cell Biol (2005) 7 8 750-757

Kirkpatrick et al. Nat Cell Biol (2005) 7 8 750-757

Linkage-Specific Measurement

•Linkage-specific antibodies have also been developed.

Histones

•Four histones per nucleosome (H2A, H2B, H3, H4) + linker histone (H1)

•Heavily post-translationally modified:

•Acetylation

•Methylation, Dimethylation, Trimethylation

•Phosphorylation

•Ubiquitination

•GlcNAcylation

•Different combinations of PTMs interact to control gene expression – ‘Histone Code’ hypothesis1

1Jenuwein et al. Science (2001) 2931074-1079

Abcam

Approaches for Analyzing Multiple PTMs on a Protein

Mass spectrometry is the only practical way of monitoring multiple PTMs at the same time.

Problem: Most proteomic analysis is of short peptides; e.g. tryptic

If modifications occur on different peptides, how do you know if PTMs occur on same protein species?

Solution: Analyze larger protein fragments

More likely multiple PTMs will be present on the same fragment

•AspN cleavage of Histone H4 produces 23 aa peptide.

•74 Different Modified Versions of Histone H4 detected in single preparation.

Phanstiel et al. PNAS (2008) 105 11 4093-4098

Combinations of Modifications

Challenges of Intact Protein Analysis

Dynamic range: Amount of protein with a given modification combination may be very low

Multiple species with same modifications but on different residues

Same mass – cannot differentiate at MS level

Can only differentiate based on distinct fragment ions

m/z

Re

lativ

e A

bund

anc

e

mass

13466 13480

13494

1350813522

13536

13550

13564

13578

Intact Protein AnalysisHistone H2B

Challenges of Intact Protein Analysis

Dynamic range: Amount of protein with a given modification combination may be very low

Multiple species with same modifications but on different residues

Same mass – cannot differentiate at MS level

Can only differentiate based on distinct fragment ions

Differently modified versions observed at similar m/z

May not have sufficient resolution to isolate a single m/z for MSMS analysis

16+ Charge Envelope of Histone H3

Challenges of Intact Protein Analysis

Dynamic range: Amount of protein with a given modification combination may be very lowMay be multiple species with same modifications but on different residues => same mass

Challenges of Intact Protein Analysis

Dynamic range: Amount of protein with a given modification combination may be very low

Multiple species with same modifications but on different residues

Same mass – cannot differentiate at MS level

Can only differentiate based on distinct fragment ions

Differently modified versions observed at similar m/z

May not have sufficient resolution to isolate a single m/z for MSMS analysis

Proteins fragment at multiple peptide bonds in ECD and ETD

+ Can identify sites of modification

- Precursor signal split between many peaks

Need more sample

The larger the protein, the more the problem

ECD Histones

m/z250 500 750 1000 1250 1500 1750 2000

[M+12H]12+

[M+12H]11+

m/z250 500 750 1000 1250 1500 1750 2000

[M+12H]12+

[M+12H]11+

m/z

ECD Fragmentation of Histone H2B

+ Me3

+Me2 and Me3

+ Me3

•Fragments from 90/134 bond cleavages observed:

• 67% Sequence Coverage on a 15kDa protein!

•3 different PTMs observed

•Information on relative stoichiometry of modifications

c

z

c

z

c

z

ARTRQTARKS TGGKAPRKQL ASKAARKSAP STGGVKKPHR YKPGTVALRE

IRRFQKSTEL LIRKLPFQRL VREIAQDFKT DLRFQSSAIG ALQESVEAYL

VSLFEDTNLA AIHAKRVTIQ KKDIKLARRL RGERS

Fragments observed by ECD Fragmentation of Histone H2B

Challenges of Intact Protein Analysis

Resolution of protein chromatography is much lower than peptides

Is it possible to resolve differentially modified proteins?

•Need to tailor your chromatography to the modification

•The bigger the protein, the more difficult to resolve

Young, N.L. et al. Mol Cell Proteomics (2009) 8 10 2266-2284

Young, N.L. et al. Mol Cell Proteomics (2009) 8 10 2266-2284

Resolving Isobaric Protein Modification States

PTM Cross-Talk Between Proteins

Modifications on one protein lead to modifications of different proteins

•Phosphorylation Cascades

•Histone PTM cross-talk:

•Histone H2B ubiquitination required for Histone H3 methylation of K4 and K79

Sin3 Transcriptional Regulation Complex

•Gene transcription regulated by complexes binding to gene promoter regions.

•Activator and repressor complexes

•PTMs on histones regulate binding of transcription complexes

•Acetylation => Activation

•De-Acetylation => Repression

Sin3A

OGT

HDAC1HDAC2

N-CoROGase

Summary

•Proteins bear multiple PTMs simultaneously

•Strategies that only study one PTM miss important biological information

•To understand the interactions between modifications, you need:

•Information about co-occurrence on same molecule

•Quantitative information about changes upon stimulation

•Need to be able to distinguish between increased protein vs PTM levels

•Knowledge of protein complex composition and PTM cross-talk within complex