|
Burkhardt M, Glazova M, Gambaryan S, Vollkommer T, Butt E, Bader
B, Heermeier K, Lincoln TM, Walter U, Palmetshofer A. J Biol
Chem. 2000 Oct 27;275(43):33536-41.
KT5823 inhibits cGMP-dependent protein kinase activity in
vitro but not in intact human platelets and rat mesangial cells.
Institute of Clinical Biochemistry and Pathobiochemistry and
the Division of Nephrology, Medical University Clinic Wuerzburg,
97080 Wuerzburg, Germany.
Many signal transduction pathways are mediated by the second
messengers cGMP and cAMP, cGMP- and cAMP-dependent protein
kinases (cGK and PKA), phosphodiesterases, and ion channels. To
distinguish among the different cGMP effectors, inhibitors of
cGK and PKA have been developed including the K-252 compound
KT5823 and the isoquinolinesulfonamide H89. KT5823, an in vitro
inhibitor of cGK, has also been used in numerous studies with
intact cells to implicate or rule out the involvement of this
protein kinase in a given cellular response. However, the
efficacy and specificity of KT5823 as cGK inhibitor in intact
cells or tissues have never been demonstrated. Here, we analyzed
the effects of both KT5823 and H89 on cyclic-nucleotide-mediated
phosphorylation of vasodilator-stimulated phosphoprotein (VASP)
in intact human platelets and rat mesangial cells. These two
cell types both express high levels of cGK. KT5823 inhibited
purified cGK. However, with both intact human platelets and rat
mesangial cells, KT5823 failed to inhibit cGK-mediated serine
157 and serine 239 phosphorylation of VASP induced by nitric
oxide, atrial natriuretic peptide, or the membrane-permeant cGMP
analog, 8-pCPT-cGMP. KT5823 enhanced 8-pCPT-cGMP-stimulated VASP
phosphorylation in platelets and did not inhibit forskolin-stimulated
VASP phosphorylation in either platelets or mesangial cells. In
contrast H89, an inhibitor of both PKA and cGK, clearly
inhibited 8-pCPT-cGMP and forskolin-stimulated VASP
phosphorylation in the two cell types. The data indicate that
KT5823 inhibits purified cGK but does not affect a cGK-mediated
response in the two different cell types expressing cGK I. These
observations indicate that data that interpret the effects of
KT5823 in intact cells as the major or only criteria supporting
the involvement of cGK clearly need to be reconsidered.
PMID: 10922374 |
