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J. Biol. Chem., Vol. 275, Issue 43, 33536-33541, October 27, 2000
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From the 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.
KT5823 Inhibits cGMP-dependent Protein Kinase
Activity in Vitro but Not in Intact Human Platelets and Rat
Mesangial Cells*
§,§¶,¶,,,,
,, and
Institute of Clinical Biochemistry and
Pathobiochemistry and the Division of Nephrology, Medical
University Clinic Wuerzburg, 97080 Wuerzburg, Germany, the
¶ Sechenov Institute of Evolutionary Physiology and Biochemistry,
Academy of Sciences, 194223 St. Petersburg, Russia, and the ** Division
of Molecular and Cellular Pathology, University of Alabama, Birmingham,
Alabama 35294-0019.
*
This work was supported by the Deutsche
Forschungsgemeinschaft (SFB 355) and by the Wilhelm Sander Stiftung.The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
To whom correspondence should be addressed: Institut für
Klinische Biochemie und Pathobiochemie, Medizinische
Universitaetsklinik, Josef-Schneider Str. 2, D-97080 Wuerzburg,
Germany. Tel.: 49-931-201-2782; Fax: 49-931-201-3137; E-mail:
palme@klin-biochem.uni-wuerzburg.de.
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