Post-Translational Modifications: CrossTalk

1. Post-Translational Modifications:CrossTalk Robert Chalkley Chem 204

2. Introduction • 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

3. 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

4. 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

5. 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

6. 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

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

8. O-GlcNAc and Phosphorylation Co-Analysis • Mouse Post-Synaptic Density Digest LWAC GlcNAc-Enriched Fraction GlcNAc-Depleted Fraction High pH RPLC TiO2 Phospho-Enriched Fraction PTM Depleted Fraction High pH RPLC High pH RPLC LC/MS/MS LC/MS/MS LC/MS/MS

9. 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.

10. 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?

11. OGT regulation by PTM Yang et al. Nature (2008) 451 7181 964-969

12. 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

13. Linkage-Specific Measurement • Linkage-specific antibodies have also been developed. Kirkpatrick et al. Nat Cell Biol (2005) 7 8 750-757

14. 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

15. Abcam

16. 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

17. Combinations of Modifications • 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

18. 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

19. Intact Protein Analysis Histone H2B 13536 13578 13494 13550 13522 13466 13480 13508 13564

20. 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

21. Challenges of Intact Protein Analysis 16+ Charge Envelope of Histone H3 Dynamic range: Amount of protein with a given modification combination may be very low May be multiple species with same modifications but on different residues => same mass

22. 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

23. ECD Fragmentation of Histone H2B ECD Histones

24. Fragments observed by ECD Fragmentation of Histone H2B c z c z c z + 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

25. 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

26. Resolving Isobaric Protein Modification States Young, N.L. et al. Mol Cell Proteomics (2009) 8 10 2266-2284

27. 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

28. 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 OGT N-CoR OGase Sin3A HDAC1 HDAC2

29. 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