YopJ Home Page
YopJ Activation by
IP6 (Inositol hexakisphosphate)
The identification of a cofactor for YopJ

In the previous page we showed how YopJ, a toxin secreted by Yersinia, is an enzyme that inactivates eukaryotic proteins necessary for the innate immune response, thereby suppressing the first line of defense against infection. Serine and threonine residues modified in the active sites of important enzymes are shown.

 Serine and threonine residues are acetylated by YopJ within the activation loops of MEK and IKK kinases.

In vitro acetylation reactions using purified YopJ and MEK are inefficient, yet in vivo there is a near complete inactivation of MEK (with levels of YopJ expression that are barely detectable), implying that in cells the enzyme is much more efficient. This prompted us to search for a possible cofactor for YopJ in mammalian cells.

 YopJ acetylates MEK efficiently in HeLa cells; search for a cofactor

Mammalian cell lysate (a few microlitres) gave a robust activation of YopJ acetyltransferase activity. Gel filtration showed the cofactor has a low molecular weight. Surprisingly, the cofactor activity was not lost, even after 18h dialysis (10kDa MWCO)

 Evidence for a cofactor in mammalian cell lysate for YopJ toxin from Yersinia

Fraction 52 from the gel filtration (which contained the cofactor) was heated for 30min and it still works, showing the cofactor is heat-stable.

 Heating at 95oC for 30min does not destroy cofactor activity

The YopJ cofactor is a:
low molecular weight
heat stable factor
not found in bacteria.
Inositol hexakisphosphate (phytic acid) fits these descriptions.

 Inositol phosphates are the likely activators of YopJ toxin

IP6 at low nanomolar concentrations stimulates YopJ activity. This result indicates that YopJ is selective in its requirement for phosphate groups and that three phosphates, as in IP3, are insufficient to stimulate the activity of YopJ.
IP6 stimulates YopJ toxin activity - but IP3 and IS6 do not
  An acid extract from HeLa cells shows cofactor activity for YopJ toxin

The cofactor from HeLa cells was purified by anion exchange chromatography and subjected to mass spectrometry...

 YopJ cofator activity as purified by anion exchange chromatography from HeLa cell lysate and subjected to mass spectrometry. Anaysis howed the presence of inositol hexakisphosphate (IP6).

The presence of the cofactor in yeast allowed us to test different deletion yeast strains. Acid extract from the impk(delta) strain of S.cerevisiae was deficient for the YopJ stimulatory cofactor. This strain accumulates IP2 and IP3, but has no IP4, IP5 or IP6.

 YopJ cofactor activity was also found in an acid extract of yeast cells.

Phytase degrades inositol phosphates and kills the cofactor activity, while a protein phosphatase has no effect.

 Phytase treatment destroys the YopJ cofactor activity of both IP6 and of HeLa acid extract; while alkaline phosphatase treatment on the other hand, does not.

HeLa Acid Extract (HAE) works like IP6 to activate YopJ acetyltransferase activity in vitro, leading to complete modification of MEK. MEK was detected using a discriminatory antibody (CST9122) directed against the activation loop of MEK. When the loop is acetylated then the antibody no longer recognizes MEK.

 Inclusion of IP6 into an invitro YopJ acetylation assay, leads to complete acetylation of MEK by the Yersinia toxin, YopJ.

IP6 binding to AvrA (the Salmonella equivalent of YopJ) leads to a change in tertiary structure. By tryptophan fluorescence YopJ also changes conformation on binding to IP6 (not shown).

 IP6 induces a conformational change in AvrA from Salmonella and in YopJ from Yersinia, both of which are acetyltransferases.

Our publications on YopJ
Mittal et al PNAS 2006
Mittal et al JBC 2010

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