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Predicting New Substrates for Kinases in the Pathway Network Of DNA Damage Response (DDR)

SYMBIO 2009 Oral Presentation. Predicting New Substrates for Kinases in the Pathway Network Of DNA Damage Response (DDR). Chengcheng LIU CSB,SMA Singapore Advisor: Christopher Hogue MIT Advisor: Michael Yaffe. Outline. Biological significance -DNA Damage Response (DDR)

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Predicting New Substrates for Kinases in the Pathway Network Of DNA Damage Response (DDR)

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  1. SYMBIO 2009 Oral Presentation Predicting New Substrates for Kinases in the Pathway Network Of DNA Damage Response (DDR) Chengcheng LIU CSB,SMA Singapore Advisor: Christopher Hogue MIT Advisor: Michael Yaffe

  2. Outline • Biological significance -DNA Damage Response (DDR) -ATM/ATR, CHK1/CHK2 -CDK1/CDK2 -Cell Cycle Checkpoints • Project objectives • Recent findings&results • Further tasks

  3. DNA Damage Response (DDR) Shiloh, Nature Review Cancer, 2003

  4. DDR Signaling Pathway Zhou et al, Nature, 2000

  5. ATM/ATR • ATM (ataxia telangiectasia mutated)and ATR (ATM and Rad3-ralated )are central components of DDR. • Double Stranded Breaks (DSB)- deadly DNA lesion-activate ATM • ATM/ATR effectors • More in Matsuoka et al, Science 2007

  6. CHK1/CHK2 • Serine/threonine kinases • take care of cell cycle control checkpoints • CHK1/CHK2 targets Zhou et al, Nature, 2000

  7. CDK1/CDK2 • Cyclin-dependent kinases Cdk1/Cdk2 • ATM/ATR activate CHK1/CHK2 which inactivate CDC25C/CDC25A. • CDC25C/CDC25A activate CDK1/CDK2 • CDK2: drives G1-S transition and S phase • CDK1: drives G2 to M phase • More in Blethrow et al, PNAS 2008

  8. Cell Cycle Checkpoints Shiloh, Nature Review Cancer, 2003

  9. Project objectives • ATM/ATR, CDK1/CDK2 have many substrates that function in DNA Damage Response • To study the sequence properties of ATM/ATR binding peptides • To predict new substrates of ATM/ATR and the downstream target kinases (CDK1/CDK2) based on the obtained sequence properties

  10. Phosphorylation/Substrate Motifs • ATM/ATR: ‘SQ/TQ’ motif • Cdk consensus sequence: [ST]Px[KR] (while many of the newly discovered Cdk targets lack this consensus sequence) Blethrow, PNAS 2008

  11. Peptide sequences

  12. Flowchart FASTA File 50aa long peptides with pS/pT at position 0 and Q at position +1 for ATM/ATR substrate peptides Sequence Logo Sequence logo by WebLogo Weblogo.berkeley.edu Choose positions based on sequence logo. Get matrices for occurrences of pairs of amino acids as well as one position matrix. Pairwise Matrices Significant tests for ENRICHMENT and REDUCTION on di-amino-acid pair in each pairwise matrix Statistical Significance Tests

  13. Sequence Logo A sequence logo in bioinformatics is a graphical representation of the sequence conservation of nucleotides or amino acids. The fewer the number of residues, the higher the letters will be, because the better the conservation is at that position.

  14. 861 phospho peptides in Matsuoka paper 3.189 0.528

  15. Example: Position Matrix

  16. Example: Pairwise Matrix for Positions -2, -1

  17. Example: Pairwise Matrix for Positions 0,+2

  18. Hypergeometric Test

  19. Null hypothesis: amino acid A at position i and amino acid B at position j are independent. • Alternative hypothesis: they are dependent. i.e. amino acid A at position i influences amino acid B at position j, either positively (ENRICHMENT) or negatively (REDUCTION). • ENRICHMENT: • REDUCTION:

  20. Example: hypothesis testing • In our case: N: the number of peptide sequences=861 n: the number of amino acid A at position i m: the number of amino acid B at position j k: the number of observed amino acid pair AB S at position 0: 739 (n) G at position +2: 111 (m) SG:106 (k) Therefore, null hypothesis might be rejected. i.e. S at position 0 influences G at position +2.

  21. Positions 0,+2 ENRICHED Total Sequences: 861 (0:S): 739 (2:G): 111 (SG): 106 Sig: 0.000493308536540346 REDUCED Total Sequences: 861 (0:T): 122 (2:G): 111 (TG): 5 Sig: 0.000493308536540392 ENRICHED Total Sequences: 861 (0:T): 122 (2:T): 34 (TT): 11 Sig: 0.00472844685500321 REDUCED Total Sequences: 861 (0:S): 739 (2:T): 34 (ST): 23 Sig: 0.00472844685500322 ENRICHED Total Sequences: 861 (0:S): 739 (2:K): 42 (SK): 40 Sig: 0.0473251226050667 REDUCED Total Sequences: 861 (0:T): 122 (2:K): 42 (TK): 2 Sig: 0.0473251226050678

  22. Positions -2,-1 ENRICHED Total Sequences: 861 (-2:H): 16 (-1:F): 16 (HF): 3 Sig: 0.00254946945599526 REDUCED Total Sequences: 861 (-2:D): 52 (-1:A): 82 (DA): 0 Sig: 0.0046374261048227 ENRICHED Total Sequences: 861 (-2:E): 87 (-1:D): 61 (ED): 13 Sig: 0.00526369190934148 ENRICHED Total Sequences: 861 (-2:T): 56 (-1:A): 82 (TA): 11 Sig: 0.0124000515535599 ENRICHED Total Sequences: 861 (-2:F): 20 (-1:K): 8 (FK): 2 Sig: 0.0132088855455469 REDUCED Total Sequences: 861 (-2:A): 68 (-1:E): 67 (AE): 1 Sig: 0.0232455022362195 ENRICHED Total Sequences: 861 (-2:Q): 63 (-1:D): 61 (QD): 9 Sig: 0.0274924230194965 ENRICHED Total Sequences: 861 (-2:D): 52 (-1:V): 30 (DV): 5 Sig: 0.0296924668645824 ENRICHED Total Sequences: 861 (-2:S): 142 (-1:Y): 12 (SY): 5 Sig: 0.0338288610340189 ENRICHED Total Sequences: 861 (-2:D): 52 (-1:G): 116 (DG): 12 Sig: 0.0361073735008351 REDUCED Total Sequences: 861 (-2:E): 87 (-1:V): 30 (EV): 0 Sig: 0.038624197238983 REDUCED Total Sequences: 861 (-2:D): 52 (-1:P): 49 (DP): 0 Sig: 0.0431171204415983 ENRICHED Total Sequences: 861 (-2:Y): 20 (-1:Q): 35 (YQ): 3 Sig: 0.043638245462674 ENRICHED Total Sequences: 861 (-2:V): 27 (-1:M): 11 (VM): 2 Sig: 0.0437649300063072 REDUCED Total Sequences: 861 (-2:P): 65 (-1:D): 61 (PD): 1 Sig: 0.0443151307382988 REDUCED Total Sequences: 861 (-2:S): 142 (-1:I): 27 (SI): 1 Sig: 0.0463337184998628 REDUCED Total Sequences: 861 (-2:N): 30 (-1:A): 82 (NA): 0 Sig: 0.0470285778499934 ENRICHED Total Sequences: 861 (-2:G): 54 (-1:G): 116 (GG): 12 Sig: 0.047248899303214 ENRICHED Total Sequences: 861 (-2:V): 27 (-1:I): 27 (VI): 3 Sig: 0.0486891538521751 ENRICHED Total Sequences: 861 (-2:A): 68 (-1:L): 113 (AL): 14 Sig: 0.0490508987497569

  23. 112 Previously identified phospho SQ/TQ peptides in literature

  24. 71 Identified phospho peptides in Blethrow paper

  25. Further tasks • Certain types of amino acids are preferred at neighbor positions of pSQ/TQ according to the obtained sequence logo • Particular pair of amino acids frequently appear around pSQ/TQ sites • Try to build a model based on the obtained information about pairs of amino acids and their specific positions (PSSM) • Predict new substrates of ATM/ATR or downstream kinases

  26. Acknowledgement A/P Chris Hogue Prof. Michael Yaffe Brian Joughin

  27. Thank You

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