KT5823 Inhibits cGMP-dependent Protein Kinase Activity in Vitro but Not in Intact Human Platelets and Rat Mesangial Cells*

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.

[1]  D. Wray,et al.  Regulation of Cloned Cardiac L-type Calcium Channels by cGMP-dependent Protein Kinase* , 2000, The Journal of Biological Chemistry.

[2]  S. Fukumoto,et al.  Distinct role of cAMP and cGMP in the cell cycle control of vascular smooth muscle cells: cGMP delays cell cycle transition through suppression of cyclin D1 and cyclin-dependent kinase 4 activation. , 1999, Circulation research.

[3]  T. Lincoln,et al.  Activation of Mitogen-activated Protein Kinase Pathways by Cyclic GMP and Cyclic GMP-dependent Protein Kinase in Contractile Vascular Smooth Muscle Cells* , 1999, The Journal of Biological Chemistry.

[4]  E. Schlatter,et al.  A novel cGMP‐regulated K+ channel in immortalized human kidney epithelial cells (IHKE‐1) , 1999, The Journal of physiology.

[5]  K. Knobeloch,et al.  Megakaryocyte hyperplasia and enhanced agonist-induced platelet activation in vasodilator-stimulated phosphoprotein knockout mice. , 1999, Proceedings of the National Academy of Sciences of the United States of America.

[6]  P. Klatt,et al.  Increased Adhesion and Aggregation of Platelets Lacking Cyclic Guanosine 3′,5′-Monophosphate Kinase I , 1999, The Journal of experimental medicine.

[7]  B. Horowitz,et al.  Cyclic GMP-dependent Protein Kinase Activates Cloned BKCa Channels Expressed in Mammalian Cells by Direct Phosphorylation at Serine 1072* , 1999, The Journal of Biological Chemistry.

[8]  A. Aszódi,et al.  The vasodilator‐stimulated phosphoprotein (VASP) is involved in cGMP‐ and cAMP‐mediated inhibition of agonist‐induced platelet aggregation, but is dispensable for smooth muscle function , 1999, The EMBO journal.

[9]  R. Ye,et al.  Activation of p38 Mitogen-activated Protein Kinase by Lipopolysaccharide in Human Neutrophils Requires Nitric Oxide-dependent cGMP Accumulation* , 1999, The Journal of Biological Chemistry.

[10]  Jesse D. Roberts,et al.  Adenovirus-mediated Gene Transfer of cGMP-dependent Protein Kinase Increases the Sensitivity of Cultured Vascular Smooth Muscle Cells to the Antiproliferative and Pro-apoptotic Effects of Nitric Oxide/cGMP* , 1998, The Journal of Biological Chemistry.

[11]  F. Hofmann,et al.  The Large Conductance, Voltage-dependent, and Calcium-sensitive K+ Channel, Hslo, Is a Target of cGMP-dependent Protein Kinase Phosphorylation in Vivo * , 1998, The Journal of Biological Chemistry.

[12]  T. Lincoln,et al.  Nitric oxide--cyclic GMP pathway regulates vascular smooth muscle cell phenotypic modulation: implications in vascular diseases. , 1998, Acta physiologica Scandinavica.

[13]  D. Bredt,et al.  Regulation of Sensory Neuron Precursor Proliferation by Cyclic GMP‐Dependent Protein Kinase , 1998, Journal of neurochemistry.

[14]  U. Walter,et al.  Analysis and Regulation of Vasodilator-stimulated Phosphoprotein Serine 239 Phosphorylation in Vitro and in Intact Cells Using a Phosphospecific Monoclonal Antibody* , 1998, The Journal of Biological Chemistry.

[15]  U. Walter,et al.  Functional analysis of cGMP-dependent protein kinases I and II as mediators of NO/cGMP effects , 1998, Naunyn-Schmiedeberg's Archives of Pharmacology.

[16]  J. Elferink,et al.  The involvement of protein kinase G in stimulation of neutrophil migration by endothelins. , 1998, European journal of pharmacology.

[17]  P. Klatt,et al.  Defective smooth muscle regulation in cGMP kinase I‐deficient mice , 1998, The EMBO journal.

[18]  U. Walter,et al.  Distinct and specific functions of cGMP-dependent protein kinases. , 1997, Trends in biochemical sciences.

[19]  D. Cohen,et al.  Mitogen-activated protein kinase cascades and the signaling of hyperosmotic stress to immediate early genes. , 1997, Comparative biochemistry and physiology. Part A, Physiology.

[20]  T. Simmet,et al.  Plasmin is a potent and specific chemoattractant for human peripheral monocytes acting via a cyclic guanosine monophosphate-dependent pathway. , 1997, Blood.

[21]  L. Hung,et al.  cGMP-elevating agents suppress proliferation of vascular smooth muscle cells by inhibiting the activation of epidermal growth factor signaling pathway. , 1997, Circulation.

[22]  W. Müller-Esterl,et al.  Two distinct Ca2+ influx pathways activated by the bradykinin B2 receptor. , 1996, European journal of biochemistry.

[23]  T. Lincoln,et al.  Phosphorylation of the Inositol 1,4,5-Trisphosphate Receptor , 1996, The Journal of Biological Chemistry.

[24]  C. Wanner,et al.  Interaction of native and oxidized lipoprotein(a) with human mesangial cells and matrix. , 1996, Kidney international.

[25]  J. P. Huggins,et al.  Inhibition of cyclic GMP‐dependent protein kinase‐mediated effects by (Rp)‐8‐bromo‐PET‐cyclic GMPS , 1995, British journal of pharmacology.

[26]  U. Walter,et al.  Expression of cGMP-dependent protein kinase I and phosphorylation of its substrate, vasodilator-stimulated phosphoprotein, in human endothelial cells of different origin. , 1995, Circulation research.

[27]  U. Walter,et al.  Endogenous expression of type II cGMP-dependent protein kinase mRNA and protein in rat intestine. Implications for cystic fibrosis transmembrane conductance regulator. , 1995, The Journal of clinical investigation.

[28]  R. Solaro,et al.  Troponin I isoforms and differential effects of acidic pH on soleus and cardiac myofilaments. , 1995, The American journal of physiology.

[29]  A. Miettinen,et al.  The glomerular mesangium: studies of its developmental origin and markers in vivo and in vitro , 1995, APMIS : acta pathologica, microbiologica, et immunologica Scandinavica.

[30]  E. Arnold,et al.  Inhibition of smooth muscle cell growth by nitric oxide and activation of cAMP-dependent protein kinase by cGMP. , 1994, The American journal of physiology.

[31]  H. Genieser,et al.  (Rp)-8-pCPT-cGMPS, a novel cGMP-dependent protein kinase inhibitor. , 1994, European journal of pharmacology.

[32]  M. Krieger,et al.  cAMP- and cGMP-dependent protein kinase phosphorylation sites of the focal adhesion vasodilator-stimulated phosphoprotein (VASP) in vitro and in intact human platelets. , 1994, The Journal of biological chemistry.

[33]  M. Kaetzel,et al.  Activation of the cystic fibrosis transmembrane conductance regulator by cGMP in the human colonic cancer cell line, Caco-2. , 1994, The Journal of biological chemistry.

[34]  U. Walter,et al.  Defective nitrovasodilator-stimulated protein phosphorylation and calcium regulation in cGMP-dependent protein kinase-deficient human platelets of chronic myelocytic leukemia. , 1993, The Journal of biological chemistry.

[35]  J. Beavo,et al.  Analysis of the functional role of cGMP-dependent protein kinase in intact human platelets using a specific activator 8-para-chlorophenylthio-cGMP. , 1992, Biochemical pharmacology.

[36]  U. Walter,et al.  Concentration and regulation of cyclic nucleotides, cyclic-nucleotide-dependent protein kinases and one of their major substrates in human platelets. Estimating the rate of cAMP-regulated and cGMP-regulated protein phosphorylation in intact cells. , 1992, European journal of biochemistry.

[37]  T. Lincoln,et al.  Vimentin is transiently co-localized with and phosphorylated by cyclic GMP-dependent protein kinase in formyl-peptide-stimulated neutrophils. , 1991, The Journal of biological chemistry.

[38]  B. Jastorff,et al.  Inhibition of cGMP‐dependent protein kinase by (Rp)‐guanosine 3',5'‐monophosphorothioates , 1990, FEBS letters.

[39]  M. Hagiwara,et al.  Inhibition of forskolin-induced neurite outgrowth and protein phosphorylation by a newly synthesized selective inhibitor of cyclic AMP-dependent protein kinase, N-[2-(p-bromocinnamylamino)ethyl]-5-isoquinolinesulfonamide (H-89), of PC12D pheochromocytoma cells. , 1990, The Journal of biological chemistry.

[40]  H. Cheng,et al.  Differential and common recognition of the catalytic sites of the cGMP-dependent and cAMP-dependent protein kinases by inhibitory peptides derived from the heat-stable inhibitor protein. , 1986, The Journal of biological chemistry.

[41]  J. Kreisberg,et al.  Glomerular cells in culture. , 1983, Kidney international.

[42]  P. Greengard,et al.  Microinjection of catalytic subunit of cyclic AMP-dependent protein kinase enhances calcium action potentials of bag cell neurons in cell culture. , 1980, Proceedings of the National Academy of Sciences of the United States of America.

[43]  P. Greengard,et al.  Immunological distinction between guanosine 3':5'-monophosphate-dependent and adenosine 3':5'-monophosphate-dependent protein kinases. , 1980, The Journal of biological chemistry.

[44]  P. Butterworth.  The use of Dixon plots to study enzyme inhibition. , 1972, Biochimica et biophysica acta.

[45]  M. Dixon.  The graphical determination of Km and Ki , 1972 .

[46]  P. Klatt,et al.  Structure and function of cGMP-dependent protein kinases. , 1999, Reviews of physiology, biochemistry and pharmacology.

[47]  A. Friebe,et al.  Soluble guanylyl cyclase: structure and regulation. , 1999, Reviews of physiology, biochemistry and pharmacology.

[48]  S. Robinson,et al.  Mechanisms of regulation and functions of guanylyl cyclases. , 1999, Reviews of physiology, biochemistry and pharmacology.

[49]  S. Dollinger,et al.  Nitric oxide donor SIN-1 inhibits mammalian cardiac calcium current through cGMP-dependent protein kinase. , 1995, The American journal of physiology.

[50]  T. Lincoln,et al.  cGMP signaling through cAMP- and cGMP-dependent protein kinases. , 1995, Advances in pharmacology.

[51]  K. Sekizawa,et al.  Interferon-gamma increases cytoplasmic motility of alveolar macrophages via nitric oxide-dependent signaling pathways. , 1994, American journal of respiratory cell and molecular biology.

[52]  H. Hidaka,et al.  Pharmacology of protein kinase inhibitors. , 1992, Annual review of pharmacology and toxicology.

[53]  U Walter,et al.  Physiological role of cGMP and cGMP-dependent protein kinase in the cardiovascular system. , 1989, Reviews of physiology, biochemistry and pharmacology.

[54]  H. Cheng,et al.  Peptide inhibitors of cAMP-dependent protein kinase. , 1988, Methods in enzymology.

[55]  R. Roskoski.  [1] Assays of protein kinase , 1983 .