AID Phosphorylation

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AID Phosphorylation

• By ?? ???
• 2008.7.6

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Background Introduction

• B cells undergo two types of genomic alterations
  to increase antibody diversity
• somatic hypermutation (SHM)
• class switch recombination (CSR)
• Activation-induced cytidine deaminase(AID)
  initiates SHM and CSR

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Background Introduction

• A Brief Mechanism
• Deamination by AID
• Deaminated DNA is subsequently replicated or
  repaired by different cellular repair mechanisms
  (BER, MMR, DSBs etc)

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Background Introduction

• Substrate of AID
• Linear ssDNA, but not linear dsDNA, serves as an
  effective AID substrate in vitro. (principal
  substrate)
• How can AID gain efficient access to dsDNA in
  vivo?
• Transcription is required
• R-loop mechanism
• RPA/transcription dependent mechanism (RGYW).

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AID Phosphorylation

• RPA/transcription dependent mechanism
• Native AID isolated from stimulated primary
  B-cell nuclei is phosphorylated on Ser38 and
  Tyr184 residues.
• Protein kinase A (PKA) was found to interact with
  AID and appears to be a primary kinase
  responsible for phosphorylation at Ser38 and
  possibly at Thr27 residues in vivo and in vitro.

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AID Phosphorylation

• RPA/transcription dependent mechanism
• phosphorylated AID from activated B cells can
  interact with RPA, a single strand DNA binding
  protein.
• RPA can target AID to transcribed switch
  regions. Then the complex can deaminate the
  non-template strand of RGYW-containing
  substrates.
• The higher density of RGYW sequences in
  transcribed Ig loci (compared to random regions
  of the genome) might help facilitate access of
  AID to these sequences

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AID Phosphorylation

• Experiment
• Inhibition of PKA in B cells decrease CSR
  activity
• Conditional deletion of the regulatory subunit of
  PKA increase CSR activity
• Mutation of the AID Serine38 to Alanine(the S38A
  mutation) had no effect on overall AID enzymatic
  activity on ssDNA in vitro,
• But such mutation markedly reduces RPA-dependent
  dsDNA deamination activity and severely impairs
  the ability of AID to effect CSR in vivo.

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AID Phosphorylation

• What do these indicate?
• PKA(and phosphorylation) is needed for AID
  activity in vivo.
• The mutaton on Ser38 did not adversely affect the
  catalytic site
• The residual CSR activity reflects the R-Loop
  mechanism (phosphorylation-independent) But
  still phosphorylation-dependent mechanism is more
  dominant
• AID phosphorylation probably has other functions.

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AID Phosphorylation

• Other Implications of AID Phosphorylation
• Phosphorylated AID appears to be preferentially
  present in the nucleus of B cells as opposed to
  the cytoplasm.
• AID exists in an inactive state in cytoplasm,
  until activation via the B-cell receptor leads to
  PKA-dependent AID activation. AID is
  phosphorylated, and then transported into the
  nucleus, where it binds to a co-factor RPA to
  effect specific transcriptional events.

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Function of AID
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Thank You For Attention?

• By ?? ???
• 2008.7.6