Activity of substituted thiophene sulfonamides against malarial and mammalian cyclin dependent protein kinases

Cyclin dependent protein kinases (CDKs) are pursued as drug targets for several eukaryotic pathogens. In this study, we identified thiophene and benzene sulfonamides as potent inhibitors of Pfmrk, a Plasmodium falciparum CDK with sequence homology to human CDK7. Several of the compounds demonstrated inhibitor selectivity for CDK7 over CDK1, CDK2, and CDK6. The compounds are moderate antimalarial agents against drug resistant parasites and possess encouraging in vitro therapeutic indices as determined against human cell lines. One particular sub-class of compounds, bromohydrosulfonylacetamides, was specific for Pfmrk with IC₅₀ values in the sub-micromolar range. These compounds represent the most potent Pfmrk inhibitors reported and provide support for further characterization and derivation as potential antimalarial agents.     

Inhibition of cytotoxic T lymphocyte-mediated lysis and cellular proliferation by isoquinoline sulfonamide protein kinase inhibitors. Evidence for the involvement of protein kinase C in lymphocyte function

The effects of the isoquinoline sulfonamides, a class of synthetic protein kinase inhibitors, namely 1-(5-isoquinoline sulfonyl)-2-methylpiperazine dihydrochloride (H7), N-[2-(methylamino)ethyl]-5-isoquinoline sulfonamide dihydrochloride (H8), N-(2-aminoethyl)-5-isoquinoline sulfonamide dihydrochloride (H9), and N-(2-guanidinoethyl)-5-isoquinoline sulfonamide hydrochloride (HA1004), on the lytic activity of in vivo-produced (H-2b anti-H-2d alloimmune) cytotoxic T lymphocytes (CTL) were investigated. The hierarchy of inhibition of lysis shown by these compounds resembled that of their inhibition of Ca2+/phospholipid-dependent enzyme (protein kinase C). H7 has the highest affinity for protein kinase C (Hidaka, H., Inagaki, M., Kawamoto, S., and Sasaki, Y. (1984) Biochemistry 23, 5036-5041) and gave the greatest inhibition of lysis by CTL. HA1004 has the weakest affinity for protein kinase C and gave very little inhibition of lysis, whereas H8 and H9 showed intermediate inhibition of lysis. In addition, the effect of the isoquinoline sulfonamides on cellular proliferation was examined. Interestingly, the pattern of inhibition observed for both lymphocytes and tumor cells closely mimicked the effects of these compounds on protein kinase C activity. These results demonstrate that modulation of an early biochemical signal affects both short-term (e.g. CTL-mediated lysis) and long-term (e.g. cellular proliferation) events. These data provide further evidence for the integral role of protein kinase C in the activation of the lytic signal in CTL. In addition, suggestive evidence is provided that protein kinase C, or some other enzyme with similar sensitivity to the isoquinoline sulfonamides, plays an important role in cellular proliferation.     

Mutants of protein kinase A that mimic the ATP-binding site of protein kinase B (AKT)

The mutation of well behaved enzymes in order to simulate less manageable cognates is the obvious approach to study specific features of the recalcitrant target. Accordingly, the prototypical protein kinase PKA serves as a model for many kinases, including the closely related PKB, an AGC family protein kinase now implicated as oncogenic in several cancers. Two residues that differ between the alpha isoforms of PKA and PKB at the adenine-binding site generate differing shapes of the binding surface and are likely to play a role in ligand selectivity. As the corresponding mutations in PKA, V123A would enlarge the adenine pocket, while L173M would alter both the shape and its electronic character of the adenine-binding surface. We have determined the structures of the corresponding double mutant (PKAB2: PKAalpha V123A, L173M) in apo and MgATP-bound states, and observed structural alterations of a residue not previously involved in ATP-binding interactions: the side-chain of Q181, which in native PKA points away from the ATP-binding site, adopts in apo double mutant protein a new rotamer conformation, which places the polar groups at the hinge region in the ATP pocket. MgATP binding forces Q181 back to the position seen in native PKA. The crystal structure shows that ATP binding geometry is identical with that in native PKA but in this case was determined under conditions with only a single Mg ion ligand. Surface plasmon resonance spectroscopy studies show that significant energy is required for this ligand-induced transition. An additional PKA/PKB mutation, Q181K, corrects the defect, as shown both by the crystal structure of triple mutant PKAB3 (PKAalpha V123A, L173M, Q181K) and by surface plasmon resonance spectroscopy binding studies with ATP and three isoquinoline inhibitors. Thus, the triple mutant serves well as an easily crystallizable model for PKB inhibitor interactions. Further, the phenomenon of Q181 shows how crystallographic analysis should accompany mutant studies to monitor possible spurious structural effects.     

Design and synthesis of pyridine-pyrazolopyridine-based inhibitors of protein kinase B/Akt

Thr-211 is one of three different amino acid residues in the kinase domain of protein kinase B/Akt as compared to protein kinase A (PKA), a closely related analog in the same AGC family. In an attempt to improve the potency and selectivity of our indazole-pyridine series of Akt inhibitors over PKA, efforts have focused on the incorporation of a chemical functionality to interact with the hydroxy group of Thr-211. Several substituents including an oxygen anion, amino, and nitro groups have been introduced at the C-6 position of the indazole scaffold, leading to a significant drop in Akt potency. Incorporation of a nitrogen atom into the phenyl ring at the same position (i.e., 9f) maintained the Akt activity and, in some cases, improved the selectivity over PKA. The structure-activity relationships of the new pyridine-pyrazolopyridine series of Akt inhibitors and their structural features when bound to PKA are also discussed.     

Targeting malaria with specific CDK inhibitors

Cyclin-dependent protein kinases (CDKs) are attractive targets for drug discovery and efforts have led to the identification of novel CDK selective inhibitors in the development of treatments for cancers, neurological disorders, and infectious diseases. More recently, they have become the focus of rational drug design programs for the development of new antimalarial agents. CDKs are valid targets as they function as essential regulators of cell growth and differentiation. To date, several CDKs have been characterized from the genome of the malaria-causing protozoan Plasmodium falciparum. Our approach employs experimental and virtual screening methodologies to identify and refine chemical inhibitors of the parasite CDK Pfmrk, a sequence homologue of human CDK7. Chemotypes of Pfmrk inhibitors include the purines, quinolinones, oxindoles, and chalcones, which have sub-micromolar IC50 values against the parasite enzyme, but not the human CDKs. Additionally, we have developed and validated a pharmacophore, based on Pfmrk inhibitors, which contains two hydrogen bond acceptor functions and two hydrophobic sites, including one aromatic ring hydrophobic site. This pharmacophore has been exploited to identify additional compounds that demonstrate significant inhibitory activity against Pfmrk. A molecular model of Pfmrk designed using the crystal structure of human CDK7 highlights key amino acid substitutions in the ATP binding pocket. Molecular modeling and docking of the active site pocket with selective inhibitors has identified possible receptor-ligand interactions that may be responsible for inhibitor specificity. Overall, the unique biochemical characteristics associated with this protein, to include distinctive active site amino acid residues and variable inhibitor profiles, distinguishes the Pfmrk drug screen as a paradigm for CDK inhibitor analysis in the parasite.     

Use of pseudosubstrate affinity to measure active protein kinase A

Traditional cAMP-dependent protein kinase (also known as protein kinase A [PKA]) assays, which are based on substrate phosphorylation, often have high background activity from other kinases, thereby limiting sensitivity and making it difficult to detect low levels of active PKA in cell lysates. Therefore, a better technique that measures active PKA in crude cell lysates undoubtedly is necessary. We developed an efficient and sensitive assay to compare active PKA levels based on binding of the active PKA catalytic subunit to its pseudosubstrate domain inhibitor (PKI) fused with glutathione S-transferase (GST-PKI). This pseudosubstrate affinity assay can detect variations in the active PKA levels in the presence of common inducers of PKA activity such as forskolin and prostaglandins. It has resolution to detect a concentration-dependent curve of active PKA in a linear range, and it also has sensitivity to detect up to 2.5 ng of active enzyme. An observed change in the binding affinity between PKA and PKI in the presence of the PKA inhibitor N-[2-(p-bromocinnamylamino)ethyl]-5-isoquinolinesulfonamide (H89) shows that this assay can be successfully used to measure how active PKA is affected by specific inhibitors. We conclude that this method is a simple, inexpensive, and nonhazardous method to compare active PKA levels with high sensitivity and specificity with negligible background.     

Balanol analogues probe specificity determinants and the conformational malleability of the cyclic 3',5'-adenosine monophosphate-dependent protein kinase catalytic subunit

The protein kinase family is a prime target for therapeutic agents, since unregulated protein kinase activities are linked to myriad diseases. Balanol, a fungal metabolite consisting of four rings, potently inhibits Ser/Thr protein kinases and can be modified to yield potent inhibitors that are selective-characteristics of a desirable pharmaceutical compound. Here, we characterize three balanol analogues that inhibit cyclic 3',5'-adenosine monophosphate-dependent protein kinase (PKA) more specifically and potently than calcium- and phospholipid-dependent protein kinase (PKC). Correlation of thermostability and inhibition potency suggests that better inhibitors confer enhanced protection against thermal denaturation. Crystal structures of the PKA catalytic (C) subunit complexed to each analogue show the Gly-rich loop stabilized in an "intermediate" conformation, disengaged from important phosphoryl transfer residues. An analogue that perturbs the PKA C-terminal tail has slightly weaker inhibition potency. The malleability of the PKA C subunit is illustrated by active site residues that adopt alternate rotamers depending on the ligand bound. On the basis of sequence homology to PKA, a preliminary model of the PKC active site is described. The balanol analogues serve to test the model and to highlight differences in the active site local environment of PKA and PKC. The PKA C subunit appears to tolerate balanol analogues with D-ring modifications; PKC does not. We attribute this difference in preference to the variable B helix and C-terminal tail. By understanding the details of ligand binding, more specific and potent inhibitors may be designed that differentiate among closely related AGC protein kinase family members.     

Selectivity of protein kinase inhibitors in human intact platelets

The specificity of commonly used protein kinase inhibitors has been evaluated in the intact human platelet. Protein kinase C (PKC) and cyclic AMP-dependent protein kinase (PKA) were activated selectively by treating platelets with phorbol dibutyrate (PDBu) or prostacyclin (PGl2). PKC activity was quantitated by measuring PDBu-specific phosphorylation of a 47,000 molecular weight protein, and PKA activity monitored by measuring prostacyclin-dependent phosphorylation of a 22,000 molecular weight protein. Staurosporine and 1-(5-isoquinolinylsulphonyl)-2-methyl-piperazine (H-7) were found to be non-specific inhibitors in the intact platelet, consistent with their effects on the isolated enzymes. Tamoxifen inhibited PKC activity (IC50 = 80 microM) but increased PKA-dependent protein phosphorylation. These results support the use of human platelets for measuring the specificity of protein kinase inhibitors and indicate that tamoxifen might have value for experimental purposes as a relatively selective PKC inhibitor.     

Proteolytic regulation of the mitochondrial cAMP-dependent protein kinase

The mitochondrial cAMP-dependent protein kinase (PKA) is activatable in a cAMP-independent fashion. The regulatory (R) subunits of the PKA holoenzyme (R(2)C(2)), but not the catalytic (C) subunits, suffer proteolysis upon exposure of bovine heart mitochondria to digitonin, Ca(2+), and a myriad of electron transport inhibitors. Selective loss of both the RI- and RII-type subunits was demonstrated via Western blot analysis, and activation of the C subunit was revealed by phosphorylation of a validated PKA peptide substrate. Selective proteolysis transpires in a calpain-dependent fashion as demonstrated by exposure of the R and C subunits of PKA to calpain and by attenuation of R and C subunit proteolysis in the presence of calpain inhibitor I. By contrast, exposure of mitochondria to cAMP fails to promote R subunit degradation, although it does result in enhanced C subunit catalytic activity. Treatment of mitochondria with electron transport chain inhibitors rotenone, antimycin A, sodium azide, and oligomycin, as well as an uncoupler of oxidative phosphorylation, also elicits enhanced C subunit activity. These results are consistent with the notion that signals, originating from cAMP-independent sources, elicit enhanced mitochondrial PKA activity.     

Disruption of the A-kinase anchoring domain in flagellar radial spoke protein 3 results in unregulated axonemal cAMP-dependent protein kinase activity and abnormal flagellar motility

Biochemical studies of Chlamydomonas flagellar axonemes revealed that radial spoke protein (RSP) 3 is an A-kinase anchoring protein (AKAP). To determine the physiological role of PKA anchoring in the axoneme, an RSP3 mutant, pf14, was transformed with an RSP3 gene containing a mutation in the PKA-binding domain. Analysis of several independent transformants revealed that the transformed cells exhibit an unusual phenotype: a fraction of the cells swim normally; the remainder of the cells twitch feebly or are paralyzed. The abnormal/paralyzed motility is not due to an obvious deficiency of radial spoke assembly, and the phenotype cosegregates with the mutant RSP3. We postulated that paralysis was due to failure in targeting and regulation of axonemal cAMP-dependent protein kinase (PKA). To test this, reactivation experiments of demembranated cells were performed in the absence or presence of PKA inhibitors. Importantly, motility in reactivated cell models mimicked the live cell phenotype with nearly equal fractions of motile and paralyzed cells. PKA inhibitors resulted in a twofold increase in the number of motile cells, rescuing paralysis. These results confirm that flagellar RSP3 is an AKAP and reveal that a mutation in the PKA binding domain results in unregulated axonemal PKA activity and inhibition of normal motility.     

Regulation of releasable vesicle pool sizes by protein kinase A-dependent phosphorylation of SNAP-25

Protein kinase A (PKA) is a key regulator of neurosecretion, but the molecular targets remain elusive. We combined pharmacological manipulations of kinase and phosphatase activities with mutational studies on the exocytotic machinery driving fusion of catecholamine-containing vesicles from chromaffin cells. We found that constitutive PKA activity was necessary to maintain a large number of vesicles in the release-ready, so-called primed, state, whereas calcineurin (protein phosphatase 2B) activity antagonized this effect. Overexpression of the SNARE protein SNAP-25a mutated in a PKA phosphorylation site (Thr-138) eliminated the effect of PKA inhibitors on the vesicle priming process. Another, unidentified, PKA target regulated the relative size of two different primed vesicle pools that are distinguished by their release kinetics. Overexpression of the SNAP-25b isoform increased the size of both primed vesicle pools by a factor of two, and mutations in the conserved Thr-138 site had similar effects as in the a isoform.     

Contribution of protein kinase A and protein kinase C pathways in ultraviolet B-induced IL-8 expression by human keratinocytes

We have previously demonstrated that treatment of the human keratinocyte cell line NCTC 2544 with a UVB dose equivalent to 1h exposure (100 mJ/cm2) results in a significant increase of IL-8 production. In this study, we use specific inhibitors to investigate the role of both PKA- and PKC-mediated pathways in the regulation of UVB-induced IL-8 expression in NCTC 2544 cell line. We show here that the treatment of irradiated human keratinocytes with PKA inhibitors [H89 and PKA inhibitor (PKAi)] induced a significant decrease of IL-8 production at both mRNA and protein levels. However, the regulation of IL-8 production seems to be mediated via a cAMP-independent PKA pathway, since drugs known to enhance cAMP concentrations [PGE2, cholera toxin and dibutyryl cAMP] decrease IL-8 production in irradiated cells by down-regulating NF-kappa B activation in response to UVB radiation. Using PMA (a potent pharmacological activator of PKC) and calphostin C (a specific PKC inhibitor), we demonstrated an up-regulation of IL-8 in NCTC 2544 cells and a down-regulation of the cytokine in UVB-irradiated cells, respectively. We also observed that in our experimental conditions, staurosporine, an inhibitor of both PKC and PMA-stimulated cellular responses, does not involve PKC inhibition in irradiated cells and significantly decreased NF-kappa B activity in response to UVB radiation. Finally, we concluded that a cAMP-independent PKA activation and a PKC-associated pathway are probably involved in the regulation of UVB-induced IL-8 synthesis in human keratinocytes.     

Characterisation of cAMP-dependent protein kinase isoforms in the brain of the crab Chasmagnathus

In the crab Chasmagnathus learning model, systemic administration of cAMP analogues that are specific activators or inhibitors of cAMP-dependent protein kinase (PKA) proved to respectively facilitate or impair long-term retention. The aims of the present work were to analyse PKA activity distribution in the crab brain and to characterise PKA isoforms. The neuropils from the eyestalk showed higher levels of induced PKA activity when compared with other neuropils of the central nervous system. Two PKA isoforms, homologous to mammalian PKA I and PKA II, were detected from central brain protein extracts using DEAE chromatography. Only PKA II was found in lateral protocerebrum extracts, suggesting a role of this isoform in the processing of visual inputs and in the integration of this information with other sensory inputs. PKA I was observed to be ten-fold more sensitive to cAMP than PKA II. cGMP induced a high activation of both PKA isoforms, similar to that obtained with cAMP. PKA I showed a two-fold greater sensitivity for cGMP than PKA II. An autophosphorylation assay was performed and a protein of 55 kDa, corresponding to phosphorylated R II regulatory subunit, was detected. The presence of a PKA I isoform with high sensitivity for cAMP in the central brain suggests a role of this subtype in long-term memory. Accepted: 29 August 2000     

Probes of the mitochondrial cAMP-dependent protein kinase

The development of a fluorescent assay to detect activity of the mitochondrial cAMP-dependent protein kinase (PKA) is described. A peptide-based sensor was utilized to quantify the relative amount of PKA activity present in each compartment of the mitochondria (the outer membrane, the intermembrane space, and the matrix). In the process of validating this assay, we discovered that PKA activity is regulated by the protease calpain. Upon exposure of bovine heart mitochondria to digitonin, Ca^2 +, and a variety of electron transport chain inhibitors, the regulatory subunits of the PKA holoenzyme (R₂C₂) are digested, releasing active catalytic subunits. This proteolysis is attenuated by calpain inhibitor I (ALLN). This article is part of a Special Issue entitled: Inhibitors of Protein Kinases (2012).     

Somatostatin stimulates menin gene expression by inhibiting protein kinase A

Somatostatin is a potent inhibitor of gastrin secretion and gene expression. Menin is a 67-kDa protein product of the multiple endocrine neoplasia type 1 (MEN1) gene that when mutated leads to duodenal gastrinomas, a tumor that overproduces the hormone gastrin. These observations suggest that menin might normally inhibit gastrin gene expression in its role as a tumor suppressor. Since somatostatin and ostensibly menin are both inhibitors of gastrin, we hypothesized that somatostatin signaling directly induces menin. Menin protein expression was significantly lower in somatostatin-null mice, which are hypergastrinemic. We found by immunohistochemistry that somatostatin receptor-positive cells (SSTR2A) express menin. Mice were treated with the somatostatin analog octreotide to determine whether activation of somatostatin signaling induced menin. We found that octreotide increased the number of menin-expressing cells, menin mRNA, and menin protein expression. Moreover, the induction by octreotide was greater in the duodenum than in the antrum. The increase in menin observed in vivo was recapitulated by treating AGS and STC cell lines with octreotide, demonstrating that the regulation was direct. The induction required suppression of protein kinase A (PKA) since forskolin treatment suppressed menin protein levels and octreotide inhibited PKA enzyme activity. Small-interfering RNA-mediated suppression of PKA levels raised basal levels of menin protein and prevented further induction by octreotide. Using AGS cells, we also showed for the first time that menin directly inhibits endogenous gastrin gene expression. In conclusion, somatostatin receptor activation induces menin expression by suppressing PKA activation.     

Autoregulation of kinase dephosphorylation by ATP binding to AGC protein kinases

AGC kinases, including the three Akt (protein kinase B) isoforms, protein kinase A (PKA) and all protein kinase C (PKC) isoforms, require activation loop phosphorylation (threonine 308 in Akt1) as well as phosphorylation of a C-terminal residue (serine 473 in Akt1) for catalytic activity and phosphorylation of downstream targets. Conversely, phosphatases reverse these phosphorylations. Virtually all cellular processes are affected by AGC kinases, a circumstance that has led to intense scrutiny of the molecular mechanisms that regulate phosphorylation of these kinases. Here, we review a new layer of control of phosphorylation in Akt, PKA and PKC pointing to ATP binding pocket occupancy as a means to decelerate dephosphorylation of these and, potentially, other kinases. This additional level of kinase regulation opens the door to search for new functional motifs for the rational design of non-ATP-competitive kinase inhibitors that discriminate within and between protein kinase families.Key words: inhibitors hijacking kinase activation, activation loop phosphorylation, dephosphorylation, phosphatase resistance, PKA, PKB, PKC     

Immune complex-induced integrin activation and L-plastin phosphorylation require protein kinase A.

Integrins in resting leukocytes are poorly adhesive, and cell activation is required to induce integrin-mediated adhesion. We recently demonstrated a close correlation between phosphorylation of Ser(5) in L-plastin (LPL), a leukocyte-specific 67-kDa actin bundling protein, and activation of alpha(M)beta(2)-mediated adhesion in polymorphonuclear neutrophils (PMN) (Jones, S. L., Wang, J., Turck, C. W., and Brown, E. J. (1998) Proc. Natl. Acad. Sci. U. S. A. 95, 9331-9336). However, the kinase that phosphorylates LPL Ser(5) has not been identified. We found that cAMP-dependent protein kinase (PKA), but not a variety of other serine kinases, can specifically phosphorylate LPL and LPL-derived peptides on Ser(5) in vitro. The cell-permeable cAMP analog 8-bromo-cAMP and the adenylate cyclase activator forskolin both induce LPL phosphorylation in cells. Two PKA inhibitors, H89 and KT5720, inhibited immune complex (IC)-stimulated LPL phosphorylation as well as IC-induced activation of alpha(M)beta(2)-mediated adhesion in PMN. The dose response of H89 inhibition of PMN adhesion correlated with its inhibition of LPL phosphorylation in response to IC. IC stimulation also transiently increased intracellular cAMP concentration in PMN. Thus, PKA functions in an integrin activation pathway initiated by IC binding to Fcgamma receptors in addition to its better known role as a negative regulator of cell activation by G protein-coupled receptors. In contrast, LPL Ser(5) phosphorylation and PMN adhesion induced by formylmethionyl-leucylphenylalanine or phorbol myristate acetate were not affected by PKA inhibitors, suggesting that a different kinase(s) is responsible for LPL phosphorylation in response to these agonists. Phosphoinositidyl 3-kinase also is required for FcgammaR but not formylmethionyl-leucylphenylalanine- or phorbol myristate acetate-induced LPL phosphorylation and activation of alpha(M)beta(2). Two phosphoinositidyl 3-kinase inhibitors blocked FcgammaR-induced cAMP accumulation, demonstrating that this kinase acts upstream of PKA. These data demonstrate a necessary role for PKA in IC-induced integrin activation and LPL phosphorylation.     

Effect of parathyroid hormone on rat renal cAMP-dependent protein kinase and protein kinase C activity measured using synthetic peptide substrates

The actions of parathyroid hormone (PTH) on the renal cortex are thought to be mediated primarily by cAMP-dependent protein kinase (PKA) with some suggestion of a role for protein kinase C (PKC). However, present methods for assaying PKA and PKC in subcellular fractions are insensitive and require large amounts of protein. Recently, a sensitive method for measuring the activity of protein kinases has been reported. This method uses synthetic peptides as substrates and a tandem chromatographic procedure for isolating the phosphorylated peptides. We have adapted this method to study the effect of PTH on PKA and PKC activity using thin slices of rat renal cortex. PTH (250 nM) stimulated cytosolic PKA activity four- to fivefold within 30 s, and PKA activity was sustained for at least 5 min. PTH also rapidly stimulated PKC activity in the membrane fraction and decreased PKC activity in the cytosol. These changes were maximal at 30 s, but unlike changes in PKA, they declined rapidly thereafter. PTH significantly activated PKC only at concentrations of 10 nM or greater. This study demonstrates that PTH does activate PKC in renal tissue, although the duration of activation is much less than for PKA. It also demonstrates that a combination of synthetic peptides with tandem chromatography can be used as a sensitive assay procedure for protein kinase activity in biological samples.     

Effects of protein phosphatase and kinase inhibitors on the cardiac L- type Ca current suggest two sites are phosphorylated by protein kinase A and another protein kinase

We previously showed (Frace, A.M. and H.C. Hartzell. 1993. Journal of Physiology. 472:305-326) that internal perfusion of frog atrial myocytes with the nonselective protein phosphatase inhibitors microcystin or okadaic acid produced an increase in the L-type Ca current (ICa) and a decrease in the delayed rectifier K current (IK). We hypothesized that microcystin revealed the activity of a protein kinase (PKX) that was basally active in the cardiac myocyte that could phosphorylate the Ca and K channels or regulators of the channels. The present studies were aimed at determining the nature of PKX and its phosphorylation target. The effect of internal perfusion with microcystin on ICa or IK was not attenuated by inhibitors of protein kinase A (PKA). However, the effect of microcystin on ICa was largely blocked by the nonselective protein kinase inhibitors staurosporine (10- 30 nM), K252a (250 nM), and H-7 (10 microM). Staurosporine and H-7 also decreased the stimulation of ICa by isoproterenol, but K252a was more selective and blocked the ability of microcystin to stimulate ICa without significantly reducing isoproterenol-stimulated current. Internal perfusion with selective inhibitors of protein kinase C (PKC), including the autoinhibitory pseudosubstrate PKC peptide (PKC(19-31)) and a myristoylated derivative of this peptide had no effect. External application of several PKC inhibitors had negative side effects that prevented their use as selective PKC inhibitors. Nevertheless, we conclude that PKX is not PKC. PKA and PKX phosphorylate sites with different sensitivities to the phosphatase inhibitors calyculin A and microcystin. In contrast to the results with ICa, the effect of microcystin on IK was not blocked by any of the kinase inhibitors tested, suggesting that the effect of microcystin on IK may not be mediated by a protein kinase but may be due to a direct effect of microcystin on the IK channel.