Gas Damping in Capacitive MEMS Transducers in the Free Molecular Flow Regime.

We present a novel analysis of gas damping in capacitive MEMS transducers that is based on a simple analytical model, assisted by Monte-Carlo simulations performed in Molflow+ to obtain an estimate for the geometry dependent gas diffusion time. This combination provides results with minimal computational expense and through freely available software, as well as insight into how the gas damping depends on the transducer geometry in the molecular flow regime. The results can be used to predict damping for arbitrary gas mixtures. The analysis was verified by experimental results for both air and helium atmospheres and matches these data to within 15% over a wide range of pressures.

In vitro reconstructed human epithelia reveal contributions of Candida albicans EFG1 and CPH1 to adhesion and invasion.

The individual and synergistic contributions of two transcription factors, EFG1 and CPH1, have been characterized with regard to adhesion to, and invasion of, human epithelia by Candida albicans. For this purpose two in vitro reconstructed tissue models were developed. A multi-layered model of human epidermis was used to simulate superficial infections of the skin, whereas a reconstructed human intestinal model was used to mimic the first steps of systemic infections. It was shown that C. albicans deleted for both transcription factors CPH1 and EFG1, in contrast to the congenic clinical isolate Sc5314, was neither able to adhere to, nor to penetrate, either of the model systems. A strain deleted for EFG1 alone showed significant reduction in adhesion and was not able to penetrate through the stratum corneum. However, strains deleted for CPH1 showed phenotypes paralleling the phenotypes of the clinical isolate Sc5314. Using different types of multi-layered human tissues reconstructed in vitro the individual contributions of Efg1p and Cph1p to two important virulence factors of C. albicans, namely adhesion and invasion, could be defined.

Protein kinase C mu selectively activates the mitogen-activated protein kinase (MAPK) p42 pathway.

Here we show that human protein kinase C mu (PKC mu) activates the mitogen-activated protein kinase (MAPK). Transient expression of constitutive active PKC mu leads to an activation of Raf-1 kinase as demonstrated by in vitro phosphorylation of MAPK. PKC mu enhances transcriptional activity of a basal thymidine kinase promotor containing serum response elements (SREs) as shown by luciferase reporter gene assays. SRE driven gene activation by PKC mu is triggered by the Elk-1 ternary complex factor. PKC mu-mediated activation of SRE driven transcription can be inhibited by the MEK1 inhibitor PD98059. In contrast to the activation of the p42/ERK1 MAPK cascade, transient expression of constitutive active PKC mu does neither affect c-jun N-terminal kinase nor p38 MAPK.

Intracellular action of the cytokine MIF to modulate AP-1 activity and the cell cycle through Jab1.

Cytokines are multifunctional mediators that classically modulate immune activity by receptor-mediated pathways. Macrophage migration inhibitory factor (MIF) is a cytokine that has a critical role in several inflammatory conditions but that also has endocrine and enzymatic functions. The molecular targets of MIF action have so far remained unclear. Here we show that MIF specifically interacts with an intracellular protein, Jab1, which is a coactivator of AP-1 transcription that also promotes degradation of the cyclin-dependent kinase inhibitor p27Kip1 (ref. 10). MIF colocalizes with Jab1 in the cytosol, and both endogenous and exogenously added MIF following endocytosis bind Jab1. MIF inhibits Jab1- and stimulus-enhanced AP-1 activity, but does not interfere with the induction of the transcription factor NFkappaB. Jab1 activates c-Jun amino-terminal kinase (JNK) activity and enhances endogenous phospho-c-Jun levels, and MIF inhibits these effects. MIF also antagonizes Jab1-dependent cell-cycle regulation by increasing p27Kip1 expression through stabilization of p27Kip1 protein. Consequently, Jab1-mediated rescue of fibroblasts from growth arrest is blocked by MIF. Amino acids 50-65 and Cys 60 of MIF are important for Jab1 binding and modulation. We conclude that MIF may act broadly to negatively regulate Jab1-controlled pathways and that the MIF-Jab1 interaction may provide a molecular basis for key activities of MIF.

Protein kinase C [micro] is regulated by the multifunctional chaperon protein p32.

We identified the multifunctional chaperon protein p32 as a protein kinase C (PKC)-binding protein interacting with PKCalpha, PKCzeta, PKCdelta, and PKC mu. We have analyzed the interaction of PKC mu with p32 in detail, and we show here in vivo association of PKC mu, as revealed from yeast two-hybrid analysis, precipitation assays using glutathione S-transferase fusion proteins, and reciprocal coimmunoprecipitation. In SKW 6.4 cells, PKC mu is constitutively associated with p32 at mitochondrial membranes, evident from colocalization with cytochrome c. p32 interacts with PKC mu in a compartment-specific manner, as it can be coimmunoprecipitated mainly from the particulate and not from the soluble fraction, despite the presence of p32 in both fractions. Although p32 binds to the kinase domain of PKC mu, it does not serve as a substrate. Interestingly, PKC mu-p32 immunocomplexes precipitated from the particulate fraction of two distinct cell lines, SKW 6.4 and 293T, show no detectable substrate phosphorylation. In support of a kinase regulatory function of p32, addition of p32 to in vitro kinase assays blocked, in a dose-dependent manner, aldolase but not autophosphorylation of PKC mu, suggesting a steric hindrance of substrate within the kinase domain. Together, these findings identify p32 as a novel, compartment-specific regulator of PKC mu kinase activity.

Proteolytic cleavage and activation of protein kinase C [micro] by caspase-3 in the apoptotic response of cells to 1-beta -D-arabinofuranosylcytosine and other genotoxic agents.

Protein kinase C (PKC) mu is a novel member of the PKC family that differs from the other isozymes in structural and biochemical properties. The precise function of PKCmu is not known. The present studies demonstrate that PKCmu is cleaved during apoptosis induced by 1-beta-d-arabinofuranosylcytosine (ara-C) and other genotoxic agents. PKCmu cleavage is blocked in cells that overexpress the anti-apoptotic Bcl-x(L) protein or the baculovirus p35 protein. Our results demonstrate that PKCmu is cleaved by caspase-3 at the CQND(378)S site. Cleavage of PKCmu is associated with release of the catalytic domain and activation of its kinase function. We also show that, unlike the cleaved fragments of PKCdelta and theta, overexpression of the PKCmu catalytic domain is not lethal. Cells stably expressing the catalytic fragment of PKCmu, however, are more sensitive to apoptosis induced by genotoxic stress. In addition, expression of the caspase-resistant PKCmu mutant partially inhibits DNA damage-induced apoptosis. These findings demonstrate that PKCmu is cleaved by caspase-3 and that expression of the catalytic domain sensitizes cells to the cytotoxic effects of ara-C and other anticancer agents.

Bruton's tyrosine kinase (Btk) associates with protein kinase C mu.

Bruton's tyrosine kinase (Btk) is considered an essential signal transducer in B-cells. Mutational defects are associated with a severe immunodeficiency syndrome, X-chromosome linked agammaglobulinemia (XLA). Here we show by coimmunoprecipitation that a member of the protein kinase C (PKC) family, PKCmu, is constitutively associated with Btk. Neither antigen receptor (Ig) crosslinking nor stimulation of B-cells with phorbol ester or H(2)O(2) affected Btk/PKCmu interaction. GST precipitation analysis revealed association of the Btk pleckstrin/Tec homology domain with PKCmu. Transient overexpression of PKCmu deletion mutants as well as expression of selected PKCmu domains in 293T cells revealed that both the kinase domain and the regulatory C1 region are independently capable of binding to the Btk PH-TH domain. These data show the existence of a PKCmu/Btk complex in vivo and identify two PKCmu domains that participate in Btk interaction.

Protein kinase C mu is negatively regulated by 14-3-3 signal transduction proteins.

Recent studies have documented direct interaction between 14-3-3 proteins and key molecules in signal transduction pathways like Ras, Cbl, and protein kinases. In T cells, the 14-3-3tau isoform has been shown to associate with protein kinase C theta and to negatively regulate interleukin-2 secretion. Here we present data that 14-3-3tau interacts with protein kinase C mu (PKCmu), a subtype that differs from other PKC members in structure and activation mechanisms. Specific interaction of PKCmu and 14-3-3tau can be shown in the T cell line Jurkat by immunocoprecipitiation and by pulldown assays of either endogenous or overexpressed proteins using PKCmu-specific antibodies and GST-14-3-3 fusion proteins, respectively. Using PKCmu deletion mutants, the 14-3-3tau binding region is mapped within the regulatory C1 domain. Binding of 14-3-3tau to PKCmu is significantly enhanced upon phorbol ester stimulation of PKCmu kinase activity in Jurkat cells and occurs via a Cbl-like serine containing consensus motif. However, 14-3-3tau is not a substrate of PKCmu. In contrast 14-3-3tau strongly down-regulates PKCmu kinase activity in vitro. Moreover, overexpression of 14-3-3tau significantly reduced phorbol ester induced activation of PKCmu kinase activity in intact cells. We therefore conclude that 14-3-3tau is a negative regulator of PKCmu in T cells.

Protein-kinase-Cmu expression correlates with enhanced keratinocyte proliferation in normal and neoplastic mouse epidermis and in cell culture.

In order to gain insight into the biological function of a PKC iso-enzyme, the protein kinase Cmu, we analyzed the expression pattern of this protein in mouse epidermis and keratinocytes in culture. Daily analysis of neonatal mouse epidermis immediately after birth showed a time-dependent reduction in the PKCmu content. Expression of the proliferating-cell nuclear antigen (PCNA), indicative of the proliferative state of cells, was reduced synchronously with PKCmu as the hyperplastic state of the neonatal tissue declined. In epidermal mouse keratinocytes, fractionated according to their maturation state, PKCmu expression was restricted to PCNA-positive basal-cell fractions. In primary cultures of those cells, growth arrest and induction of terminal differentiation by Ca2+ resulted in strongly reduced PKCmu expression, concomitantly with the loss of PCNA expression. Treatment of PMK-R1 keratinocytes with 100 nM of the mitogen 12-O-tetradecanoylphorbol-13-acetate (TPA) resulted in activation of PKCmu, reflected by translocation from the cytosolic to the particulate fraction and by shifts in electrophoretic mobility. DNA synthesis was significantly inhibited by the PKCmu inhibitor Goedecke 6976, while Goedecke 6983 did not inhibit PKCmu. Carcinomas generated according to the 2-stage carcinogenesis protocol in mouse skin consistently exhibited high levels of PKCmu. These data correlate PKCmu expression with the proliferative state of murine keratinocytes and point to a role of PKCmu in growth stimulation. A correlation between PKCmu expression and enhanced cell proliferation was also observed for NIH3T3 fibroblasts transfected with and overexpressing human PKCmu.

Proteolytic cleavage of protein kinase Cmu upon induction of apoptosis in U937 cells.

Treatment of U937 cells with various apoptosis-inducing agents, such as TNFalpha and beta-D-arabinofuranosylcytosine (ara-C) alone or in combination with the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA), bryostatin 1 or cycloheximide, causes proteolytic cleavage of protein kinase Cmu (PKCmu) between the regulatory and catalytic domain, generating a 62 kDa catalytic fragment of the kinase. The formation of this fragment is effectively suppressed by the caspase-3 inhibitor Z-DEVD-FMK. In accordance with these in vivo data, treatment of recombinant PKCmu with caspase-3 in vitro results also in the generation of a 62 kDa fragment (p62). Treatment of several aspartic acid to alanine mutants of PKCmu with caspase-3 resulted in an unexpected finding. PKCmu is not cleaved at one of the typical cleavage sites containing the motif DXXD but at the atypical site CQND378/S379. The respective fragment (amino acids 379-912) was expressed in bacteria as a GST fusion protein (GST-p62) and partially purified. In contrast to the intact kinase, the fragment does not respond to the activating cofactors TPA and phosphatidylserine and is thus unable to phosphorylate substrates effectively.

Protein kinase Cmu downregulation of tumor-necrosis-factor-induced apoptosis correlates with enhanced expression of nuclear-factor-kappaB-dependent protective genes.

Protein kinase Cmu (PKCmu) represents a new subtype of the PKC family characterized by the presence of a pleckstrin homology (PH) domain and an amino-terminal hydrophobic region. In order to analyse the potential role of PKCmu in signal-transduction pathways, stable PKCmu transfectants were established with human and murine cell lines. All transfectants showed a reduced sensitivity to tumor-necrosis-factor (TNF)-induced apoptosis, which correlated with the amount of transgene expressed and with an enhanced basal transcription rate of NF-kappaB-driven genes including the inhibitor of apoptosis protein 2 (cIAP2) and TNF-receptor-associated protein 1 (TRAF1). Sensitivity to apoptosis induced by the lipid mediator ceramide was unchanged in PKCmu transfectants. In support of a PKCmu action on NF-kappaB, we show enhancement and downregulation of TNF-induced expression of a NF-kappaB-dependent reporter gene by transient overexpression of wild-type and kinase-negative mutants of PKCmu, respectively. Interestingly, no significant changes were found in an electrophoretic mobility shift assay, indicative of PKCmu action downstream of IkappaB degradation, probably by modulation of the transactivation capacity of NF-kappaB. The dominant negative action of the kinase-negative mutant further suggest a regulatory role of PKCmu for NF-kappaB-dependent gene expression.

Association of protein kinase Cmu with type II phosphatidylinositol 4-kinase and type I phosphatidylinositol-4-phosphate 5-kinase.

Protein kinase Cmu (PKCmu), also named protein kinase D, is an unusual member of the PKC family that has a putative transmembrane domain and pleckstrin homology domain. This enzyme has a substrate specificity distinct from other PKC isoforms (Nishikawa, K., Toker, A., Johannes, F. J., Songyang, Z., and Cantley, L. C. (1997) J. Biol. Chem. 272, 952-960), and its mechanism of regulation is not yet clear. Here we show that PKCmu forms a complex in vivo with a phosphatidylinositol 4-kinase and a phosphatidylinositol-4-phosphate 5-kinase. A region of PKCmu between the amino-terminal transmembrane domain and the pleckstrin homology domain is shown to be involved in the association with the lipid kinases. Interestingly, a kinase-dead point mutant of PKCmu failed to associate with either lipid kinase activity, indicating that autophosphorylation may be required to expose the lipid kinase interaction domain. Furthermore, the subcellular distribution of the PKCmu-associated lipid kinases to the particulate fraction depends on the presence of the amino-terminal region of PKCmu including the predicted transmembrane region. These results suggest a novel model in which the non-catalytic region of PKCmu acts as a scaffold for assembly of enzymes involved in phosphoinositide synthesis at specific membrane locations.

Dominant-negative FADD inhibits TNFR60-, Fas/Apo1- and TRAIL-R/Apo2-mediated cell death but not gene induction.

Fas/Apo1 and other cytotoxic receptors of the tumor necrosis factor receptor (TNFR) family contain a cytoplasmic death domain (DD) [1] [2] [3] [4] [5] [6] [7] [8] [9] [10] [11] that activates the apoptotic process by interacting with the DD-containing adaptor proteins TNFR-associated DD protein (TRADD) [12] [13] and Fas-associated DD protein (FADD/MORT1) [14] [15], leading to the activation of cysteine proteases of the caspase family [16]. Stimulation of Fas/Apo1 leads to the formation of a receptor-bound death-inducing signaling complex (DISC), consisting of FADD and two different forms of caspase-8 [17] [18] [19]. Transient expression of a dominant-negative mutant of FADD impairs TNFR60-mediated and Fas/Apo1-mediated apoptosis [13] [20], but has no effect on TNF-related apoptosis-inducing ligand (TRAIL/Apo2L)-induced cell death [7] [8] [9] [10] [21]. To study the function of FADD in DD-receptor signaling in more detail, we established HeLa cells that stably expressed a green fluorescent protein (GFP)-tagged dominant-negative mutant of FADD, GFP-DeltaFADD. Interestingly, expression of this mutant inhibited cell death induced by TNFR60, Fas/Apo1 and TRAIL-R/Apo2. In addition, GFP-DeltaFADD did not interfere with TNF-mediated gene induction or with activation of NF-kappaB or Jun N-terminal kinase (JNK), demonstrating that FADD is part of the TNFR60-initiated apoptotic pathway but does not play a role in TNFR60-mediated gene induction. Fas/Apo1-mediated activation of JNK was unaffected by the expression of GFP-DeltaFADD, suggesting that in Fas/Apo1 signaling the apoptotic pathway and the activation of JNK diverge at a level proximal to the receptor, upstream of or parallel to FADD.

Differential effects of suramin on protein kinase C isoenzymes. A novel tool for discriminating protein kinase C activities.

Suramin, a hexasulfonated naphthylurea, is known to induce differentiation and inhibit proliferation, angiogenesis, and development of tumors. It has also been shown to suppress the activity of the protein kinase C (PKC) isoenzymes alpha, beta, and gamma. Here we report on a differential effect of suramin on PKCmu and various PKC isoforms representing the cPKC, nPKC, and aPKC group of the PKC family. In the absence of any cofactors suramin activates all PKC isoforms in the order of aPKCzeta >> PKCmu > cPKC, nPKCdelta. As judged by the Vmax/KM ratios (0.5 for PKCmu and 2.2 for PKCzeta) the substrate syntide 2 is phosphorylated by suramin-activated PKCzeta around four times more effectively than by suramin-activated PKCmu. Suramin-activated PKCmu behaves like that activated by phosphatidylserine and the phorbol ester TPA regarding autophosphorylation and differential inhibition by the PKC inhibitors Gö 6976 and Gö 6983. In the presence of activating cofactors, such as phosphatidylserine and TPA or cholesterol sulfate (for PKCzeta), the activity of the aPKCzeta is further stimulated, PKCmu is not significantly affected, and the cPKCs and the nPKCdelta are strongly inhibited by suramin. The differential action of suramin on PKC isoenzymes might play a role in some of its biological effects, as for instance inhibition of proliferation and tumor development. Moreover, due to this property suramin will possibly be a valuable tool for discriminating the activities of PKC isoenzymes in vitro and in vivo.

Regulation of protein kinase Cmu by basic peptides and heparin. Putative role of an acidic domain in the activation of the kinase.

Protein kinase Cmu is a novel member of the protein kinase C (PKC) family that differs from the other isoenzymes in structural and enzymatic properties. No substrate proteins of PKCmu have been identified as yet. Moreover, the regulation of PKCmu activity remains obscure, since a structural region corresponding to the pseudosubstrate domains of other PKC isoenzymes has not been found for PKCmu. Here we show that aldolase is phosphorylated by PKCmu in vitro. Phosphorylation of aldolase and of two substrate peptides by PKCmu is inhibited by various proteins and peptides, including typical PKC substrates such as histone H1, myelin basic protein, and p53. This inhibitory activity seems to depend on clusters of basic amino acids in the protein/peptide structures. Moreover, in contrast to other PKC isoenzymes PKCmu is activated by heparin and dextran sulfate. Maximal activation by heparin is about twice and that by dextran sulfate four times as effective as maximal activation by phosphatidylserine plus 12-O-tetradecanoylphorbol-13-acetate, the conventional activators of c- and nPKC isoforms. We postulate that PKCmu contains an acidic domain, which is involved in the formation and stabilization of an active state and which, in the inactive enzyme, is blocked by an intramolecular interaction with a basic domain. This intramolecular block is thought to be released by heparin and possibly also by 12-O-tetradecanoylphorbol-13-acetate/phosphatidylserine, whereas basic peptides and proteins inhibit PKCmu activity by binding to the acidic domain of the active enzyme.

Determination of the specific substrate sequence motifs of protein kinase C isozymes.

Protein kinase C (PKC) family members play significant roles in a variety of intracellular signal transduction processes, but information about the substrate specificities of each PKC family member is quite limited. In this study, we have determined the optimal peptide substrate sequence for each of nine human PKC isozymes (alpha, betaI, betaII, gamma, delta, epsilon, eta, mu, and zeta) by using an oriented peptide library. All PKC isozymes preferentially phosphorylated peptides with hydrophobic amino acids at position +1 carboxyl-terminal of the phosphorylated Ser and basic residues at position -3. All isozymes, except PKC mu, selected peptides with basic amino acids at positions -6, -4, and -2. PKC alpha, -betaI, -betaII, -gamma, and -eta selected peptides with basic amino acid at positions +2, +3, and +4, but PKC delta, -epsilon, -zeta, and -mu preferred peptides with hydrophobic amino acid at these positions. At position -5, the selectivity was quite different among the various isozymes; PKC alpha, -gamma, and -delta selected peptides with Arg at this position while other PKC isozymes selected hydrophobic amino acids such as Phe, Leu, or Val. Interestingly, PKC mu showed extreme selectivity for peptides with Leu at this position. The predicted optimal sequences from position -3 to +2 for PKC alpha, -betaI, -betaII, -gamma, -delta, and -eta were very similar to the endogenous pseudosubstrate sequences of these PKC isozymes, indicating that these core regions may be important to the binding of corresponding substrate peptides. Synthetic peptides based on the predicted optimal sequences for PKC alpha, -betaI,-delta, -zeta, and -mu were prepared and used for the determination of Km and Vmax for these isozymes. As judged by Vmax/Km values, these peptides were in general better substrates of the corresponding isozymes than those of the other PKC isozymes, supporting the idea that individual PKC isozymes have distinct optimal substrates. The structural basis for the selectivity of PKC isozymes is discussed based on residues predicted to form the catalytic cleft.

Immunological demonstration of protein kinase C mu in murine tissues and various cell lines. Differential recognition of phosphorylated forms and lack of down-regulation upon 12-O-tetradecanoylphorphol-13-acetate treatment of cells.

13 murine tissues and 12 cell lines were tested for the expression of the novel protein kinase C (PKC) isoenzyme mu. Using two different PKC mu antibodies (sc-639 and P26720), PKC mu was detected in all tissues and cells and thus proved to be an ubiquitous PKC isotype. However, in some tissues, PKC mu was recognized only by the antibody P26720 and not by sc-639. Thymus, lung and peripheral blood mononuclear cells expressed the greatest amount of PKC mu. Recognition of PKC mu by the antibody sc-639 was drastically impaired when treating keratinocytes or mouse skin in vivo with the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA), thus mimicking down-regulation of PKC mu. The lack of a decrease in the PKC mu amount and, thus, the lack of down-regulation could be proved using the antibody P26720. This antibody was able to recognize PKC mu in extracts of untreated as well as TPA-treated tissues or cells. Phosphorylation of proteins in a cell-free system (cell or tissue extracts) in the presence and absence of TPA or other PKC activators and various protein kinase inhibitors indicated that phosphorylation of activated PKC mu caused its reduced interaction with the antibody sc-639. Therefore, this antibody might present a well suited tool for the detection of activated PKC mu in vivo. Moreover, our results clearly show that some antibodies, such as sc-639, might be able to selectively detect non-phosphorylated or phosphorylated forms of a protein, and that such properties of an antibody have to be studied carefully before the latter can be used for reliable quantitative determination of this protein. We consider this information important to avoid misinterpretation of data concerning the immunological quantification of proteins such as PKC mu.

Protein kinase C mu is located at the Golgi compartment.

Protein kinase C mu (PKC mu) displays unusual structural features like a pleckstrin homology domain and an amino-terminal hydrophobic region with a putative leader peptide and transmembrane sequence. As a discrete location often is a direct clue to the potential biological function of a kinase, antibodies directed against unique amino- and carboxy-terminal domains of PKC mu were used to localize the protein within intracellular compartments in immunofluorescence and subcellular fractionation studies. Confocal laser scanning microscopy showed colocalization of PKC mu with the resident Golgi marker protein beta 1,4 galactosyltransferase in PKC mu transfectants and in the human hepatocellular carcinoma cell line HepG2, expressing endogenous PKC mu. Long-term treatment of cells with brefeldin A, which disintegrates the Golgi apparatus, disrupted PKC mu-specific staining. Cosegregation of PKC mu with beta 1,4 galactosyltransferase, but not with the endosomal marker rab5, upon density gradient fractionation and Western blot analysis of HepG2 cell extracts, provides independent evidence for a Golgi localization of PKC mu. Moreover, cellular sulfate uptake and Golgi-specific glycosaminoglycan sulfation was enhanced in PKC mu transfectants. Together, these data suggest that PKC mu is a resident protein kinase of the core Golgi compartment and is involved in basal transport processes.

Inhibition of protein kinase C mu by various inhibitors. Differentiation from protein kinase c isoenzymes.

Various inhibitors were tested for their potential to suppress the kinase activity of protein kinase C mu (PKC mu) in vitro and in vivo. Among the staurosporine-derived, rather selective PKC inhibitors the indolocarbazole Gö 6976 previously shown to inhibit preferentially cPKC isotypes proved to be a potent inhibitor of PKC mu with an IC50 of 20 nM, whereas the bisindolylmaleimide Gö 6983 was extremely ineffective in suppressing PKC mu kinase activity with a thousand-fold higher IC50 of 20 microM. Other strong inhibitors of PKC mu were the rather unspecific inhibitors staurosporine and K252a. Contrary to the poor inhibition of PKC mu by Gö 6983, this compound was found to suppress in vitro kinase activity of PKC isoenzymes from all three subgroups very effectively with IC50 values from 7 to 60 nM. Thus, Gö 6983 was able to differentiate between PKC mu and other PKC isoenzymes being useful for selective determination of PKC mu kinase activity in the presence of other PKC isoenzymes.