I treat cells with forskolin and measure CRE-luc reporter gene activity (which is elevated 30 to 50 fold by 10 µM forskolin). Rp-cAMPS and 8Br-Rp-cAMPS (both 1 mM) inhibit this nicely. Myr-PKI should also inhibit this CRE-luc activity. Can some recommend a concentration?
Yes, it works very well. We have used it in primary cells (HUVECs) as well as LNCaP prostate epithelial cells. Using forskolin-stimulated CREB phosphorylation on Ser133 as a readout, a 1 hr pre-incubation with 10 nM myrPKI pretty much abolishes CREB phosphorylation.
I used H89 and it works well with C2C12 cells (and also MC3T3). I heard that KT 5720 work better but never tried it !
Dear Mauricio and Timothy, thanks for the hints! Do you remember which solvent you used? Water? Or a buffered solution?
Dear Chafik, H89 is not at all PKA-specific and KT5720 is not much better (see papers on protein kinase inhibitor specificity by Sir Philipp Cohen in the Biochemical journal).
Soluble in water, we store as 1000x concentrated stock, i.e. 10 microM, in -20 freezer.
Another way to go would be to use a PKIalpha adenovirus (natural inhibitory peptide). See: "Meja 2004, Adenovirus-Mediated Delivery and Expression of a cAMP-Dependent Protein Kinase Inhibitor Gene to BEAS-2B Epithelial Cells Abolishes the Anti-Inflammatory Effects of Rolipram, Salbutamol, and Prostaglandin E2: A Comparison with H-89, JPET 309(2) 833-44". While we have had some success with H89 and KT, these are not particularly specific as Erik Maronde has pointed out.
Dear Erik,
I agree with you, H89 is not specific to PKA (H89 inhibits other kinases) but you should use it in combination with PKI-myr (or siRNA knockdown of PKA, catalytic subunit of PKA).
Thanks for all the good comments! In our 2007 paper we used transfected PKI, but this time I didn`t want to transfect too many different things, so I appreciate the comments on the use of myr-PKI as a reagent a lot.
I used PKI from BioSource (Camarillo, CA, USA). Cells were pretreated for 30 min with 1 mM IBMX and then treated with 10 μM forskolin and REAGENTS for 72 h or with 1.5 μM PKI for 90 min and then incubated with REGANTS for 72 h.
Dear Ewa, could you please tell me what your end-point (the measured parameter) was? In my assay myr-PKI-14-22amide from 0.1 to 30 µM does not inhibit 1 or 10 µM Forskolin-or 100 µM Sp-cDBIMPS (a cAMP agonist) induced CRE-luciferase reporter gene activity. Rp-8-Br-cAMPS (1 mM) however inhibits nicely. And finally the EPAC-specific agonist does nothing.
Dear Erik,
in my research I used primary cultures of ovarian cells or breast cancer cell line MCF 7. As an end point- active PKA levels, cell proliferation or steroids secretion.
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