PRMT6 increases the pool of HIV1 Tat in the cytosol

1
PRMT6 increases the poolof HIV-1 Tat in the
cytosol

• Haran Sivakumaran, Ann Apolloni, David Harrich
• HIV Molecular Virology Laboratory,Queensland
  Institute of Medical Research, Australia

2
Introduction Tat
Adapted from Barboric Peterlin. PLoS Biology
2005 3
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Introduction PRMT6

• PRMT6 is a type I methyltransferase
• Few known cellular substrates
• Directly methylates Tat at R52 and R53
• Residues part of nuclear localisation signal
• Does PRMT6 alter distribution of Tat in
  subcellular compartments?

4
Methods
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Tat-FLAG mutants
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Two-exon and one-exon Tat-FLAG
Cytosol
Nucleoplasm
Luciferase (103 CPS/µg)
5.1 8.5 8.3 0.1 0.3 0.2
PARP
Myc-PRMT6
Tat101-FLAG
W wildtype M mutant
- W M - W M
pMyc-PRMT6
Luciferase (103 CPS/µg)
5.2 6.6 5.3 0.2 0.1 0.2
PARP
Myc-PRMT6
Tat72-FLAG
Cytosol
Nucleoplasm
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R52K-R53K and K71Q mutants
Cytosol
Nucleoplasm
Luciferase (103 CPS/µg)
10.5 5.0 4.3 0.2 0.1 0.1
PARP
Myc-PRMT6
Tat101-R52K-R53K-FLAG
W wildtype M mutant
- W M - W M
pMyc-PRMT6
Luciferase (103 CPS/µg)
9.3 6.5 5.1 0.2 0.2 0.1
PARP
Myc-PRMT6
Tat101-K71Q-FLAG
Cytosol
Nucleoplasm
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Myc-PRMT6 similarly affectsin situ Tat in the
HIV-1 provirus
293T, whole cell lysates
Gag
FLAG-Tat
pLM-FLAG-Tat

pMyc-PRMT6
- W M
W wildtype M mutant
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Lactacystin increasesTat-FLAG abundance
Cytosol
N.p.
ß-galactosidase (U/g)
390 322 64 51
PARP
Tat101-FLAG
10 µM Lactacystin
- -
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Myc-PRMT6 doesnt inhibitdegradation of
phospho-I?Ba
HeLa, whole cell lysates
ß-tubulin
Myc-PRMT6
phospho-I?Ba
pMyc-PRMT6
W wildtype M mutant
5 µM Lactacystin
20 µg protein per lane
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Conclusions

• Myc-PRMT6 increases pools of Tat101-FLAG in
  cytosol and nucleoplasm
• Dependent on R52 and/or R53 residue(s)of Tat
• Tat72-FLAG is not increased in the nucleoplasm

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Conclusions

• Cytosolic increase isnt dependent on K71
• PRMT6 may rescue Tat from proteasome-mediated
  degradation
• Functions of Tat after transactivation may be
  facilitated by PRMT6

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Proposed Model
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Acknowledgements

• Monsef Benkirane (Institut de Génétique Humaine)
• For the Tat-FLAG construct
• Stéphane Richard (Lady Davis Institute)
• For the Myc-PRMT6 constructs
• Luke Meredith (QIMR)
• For the pLM construct

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Endogenous PRMT6observed in the cytosol
293T
HeLa
C N C N
kDa
C cytosol N nucleoplasm
130
PARP
95
55
PRMT6
43
Luciferase (103 CPS/µg)
- - 2.8 0.1
40 µg protein per lane
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No observed increase in Tat transactivation
function
n 3