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

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### 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)

-ATM/ATR, CHK1/CHK2

-CDK1/CDK2

-Cell Cycle Checkpoints

- Project objectives
- Recent findings&results
- Further tasks

DNA Damage Response (DDR)

Shiloh, Nature Review Cancer, 2003

DDR Signaling Pathway

Zhou et al, Nature, 2000

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

CHK1/CHK2

- Serine/threonine kinases
- take care of cell cycle control checkpoints
- CHK1/CHK2 targets

Zhou et al, Nature, 2000

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

Cell Cycle Checkpoints

Shiloh, Nature Review Cancer, 2003

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

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

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

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.

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:

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.

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

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

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

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