I did my PhD on one of the most studied enzyme-systems of all time, the cAMP-dependent protein kinase (PKA). One would think that the tools we used were accurate when the enzyme was as thoroughly characterized as it was. But alas!, now we learn they had major flaws. The endless kinase assay’s lasting until the wee hours, were they all in vain ?
“….nonspecific effects are
widespread; they include actions on other protein kinases and signaling
molecules and also on basic cellular functions, such as transcription.”
We had two teams in my group, one was the gene characterizing team – we did cloning, sequencing, expression and transcription analyzes mostly. The other was the protein activity and interaction team who did a lot of enzyme activity (kinase) assays, protein-protein interaction and immunoblots. Members frequently crossed team borders to learn the necessary methods. I was in the gene-group, but I did my occasional kinase assay and cell-culture experiment as well. As a rule, the kinase assays (as well as cell culture assays) always included inhibitors of the kinase and very often those inhibitors were H89 and/or KT5720. These inhibitors were thought to be specific so that any decrease in enzyme activity one would see upon adding them, was attributed too a loss of PKA activity.
A recent review by Andrew J. Murray in Science puts a serious dent in that assumption. These inhibitors seems to act on a range of other signaling systems. Their targets seems to be very diverse indeed. Specific they most certainly are not. That means that a lot of the inhibition we would see and base our conclusions on, was wrong.
body of evidence has now accumulated
that indicates that both H89 and KT 5720
can have effects independent of PKA inhibition.
These actions are extremely varied;
some of the most worrisome actions are the
substantial effects on the MAPK and calcium
signaling pathways, which interact with
the PKA pathway and mediate multiple
and even more worrying:
“Furthermore, many of
these non–PKA-based actions of H89 and
KT 5720 occur at concentrations that have
been widely used to investigate PKA function.”
The final conclusions and the cellular mechanisms we unraveled, one can only hope were not wrong, which would be fortunate for me and others with a history in the field. But, one can never be sure and reevaluation may be in place for some of the past studies
“the molecular bases of some
cellular processes attributed to PKA solely
through the use of these compounds may
have to be reevaluated.”
Any experiment with H89 in the future needs to take this new information into account. And in the review, the author outlines a number of alternative approaches to inhibit PKA activity in a specific manner.
The above outlined studies indicate that neither
KT 5720 nor H89 should be used alone
to study the function of PKA. As these compounds
are so commonly used, it will therefore
be necessary to devise strategies that
can overcome their shortcomings.
Although annoying and possibly detrimental to papers I have published in the past, I cannot help having this proud feeling. Because, this is the beauty of the scientific method: not only do we admit we’re wrong (and publish it in high impact journals), we have efficient methods to prevent the wrongdoings from continuing. Any responsible and updated reviewer will now dispute all conclusions based on experiments involving these inhibitors. Then scientific results in the field will become more accurate. In addition, new conclusions and insight may come from this increased understanding of the limitations on previous results.
Science involves unrestricted sharing, regardless of the nature of the information. Add peer-review and you have the scientific method working at its best.