cAMP-independent effects of cholera toxin on B cell activation. I. A possible role for cell surface ganglioside GM1 in B cell activation

Cholera toxin has been used as a tool to study the effects of cAMP on the activation of B cells but may have effects independent of its ability to elevate cAMP. We found five lines of evidence which suggested that cholera toxin suppressed mitogen-stimulated B cell activation through a cAMP-independent pathway. 1) Cholera toxin (1 microgram/ml) was consistently more suppressive than forskolin (100 microM) despite the induction of higher intracellular cAMP levels by forskolin. 2) Cholera toxin was more suppressive at 1 microgram/ml than at 0.1 microgram/ml despite equivalent elevations of cAMP. 3) Washing B cells following their incubation with cholera toxin reversed much of the inhibition without altering intracellular cAMP levels. 4) The A subunit of cholera toxin, which at high concentrations (10 micrograms/ml) induced levels of cAMP comparable to those induced by cholera toxin (1 and 0.1 microgram/ml), did not inhibit B cell activation. 5) cAMP derivatives at high concentrations were much less effective than was cholera toxin in suppressing B cell activation. Although the elevation of cAMP may cause a mild inhibition of B cell proliferation, we found that even a marked elevation of cAMP did not suppress B cell proliferation, unless the elevation was persistent. We did, however, observe that the degree of toxin inhibition more closely paralleled binding of the toxin to B cells than toxin stimulation of cAMP. This result raised the possibility that binding of cholera toxin to its ganglioside GM1 receptor mediated an inhibitory signal which suppressed B cell proliferation.     

Study of the cytolethal distending toxin (CDT)-activated cell cycle checkpoint. Involvement of the CHK2 kinase

The bacterial cytolethal distending toxin (CDT) triggers a G2/M cell cycle arrest in eukaryotic cells by inhibiting the CDC25C phosphatase-dependent CDK1 dephosphorylation and activation. We report that upon CDT treatment CDC25C is fully sequestered in the cytoplasmic compartment, an effect that is reminiscent of DNA damage-dependent checkpoint activation. We show that the checkpoint kinase CHK2, an upstream regulator of CDC25C, is phosphorylated and activated after CDT treatment. In contrast to what is observed with other DNA damaging agents, we demonstrate that the activation of CHK2 can only take place during S-phase. Use of wortmannin and caffeine suggests that this effect is not dependent on ATM but rather on another as yet unidentified PI3 kinase family member. These results confirm that the CDT is therefore responsible for specific genomic injuries that block cell proliferation by activating a cell cycle checkpoint.     

Genetic constructions,hyperexpressing recombinant fragments of cytolethal distending toxin of Aggregatibacter actinomycetemcomitans

Aggregatibacter actinomycetemcomitans is a gram negative bacterium and an infectious agent of human diseases with severe oral and extra oral manifestations. One of the major virulence factors in this microorganism is cytolethal distending toxin (CDT). This toxin consists of three subunits—A, B, and C and is able to disrupt cell cycle by intrinsic DNAse activity of its B-subunit. Due to the fact that this protein can represent an important component of diagnostic, prophylactic and therapeutic preparations, production of CDT subunits in preparative quantities is of considerable practical importance. In the current study we demonstrated that deletion of NH₂-terminal regions from the molecules of CDT-A, -B, or -C resulted in 20–400-fold increase in production of the corresponding subunits. These truncated molecules were used as immunogens to raise monospecific sera, which were shown in western blot to react specifically with the homologous subunits of cytolethal distending toxin.     

In vivo virulence properties of bacterial cytolethal-distending toxin

Multiple pathogenic Gram-negative bacteria produce cytolethal-distending toxins (CDTs). CDT is typically composed of three subunits: the catalytic subunit CdtB has DNase I-like activity, whereas CdtA and CdtC are binding proteins for delivering CdtB into target cells. Translocation of CdtB to the nucleus induces genotoxic effects on host DNA, triggering DNA repair cascades that lead to cell cycle arrest and eventual cell death. Several lines of evidence indicate that this toxin contributes to the pathogenicity of CDT-producing bacteria in vivo . Helicobacter hepaticus and Campylobacter jejuni CDTs are essential for persistent infection of the gastrointestinal tract and increase the severity of mucosal inflammation or liver disease in susceptible mouse strains. Haemophilus ducreyi CDT may contribute to the pathogenesis of chancroid in rabbits. Recently, H. hepaticus CDT has been shown to play a crucial role in promoting the progression of infectious hepatitis to pre-malignant, dysplastic lesions via activation of a pro-inflammatory NF-κB pathway and increased proliferation of hepatocytes, providing the first evidence that CDT has carcinogenic potential in vivo . Thus, both in vitro and in vivo data indicate that CDT is a bacterial virulence factor.     

Escherichia coli cyclomodulin Cif induces G2 arrest of the host cell cycle without activation of the DNA-damage checkpoint-signalling pathway

The cycle inhibiting factor (Cif) belongs to a family of bacterial toxins and effector proteins, the cyclomodulins, that deregulate the host cell cycle. Upon injection into HeLa cells by the enteropathogenic Escherichia coli (EPEC) type III secretion system, Cif induces a cytopathic effect characterized by the recruitment of focal adhesion plates and the formation of stress fibres, an irreversible cell cycle arrest at the G(2)/M transition, and sustained inhibitory phosphorylation of mitosis inducer, CDK1. Here, we report that the reference typical EPEC strain B171 produces a functional Cif and that lipid-mediated delivery of purified Cif into HeLa cells induces cell cycle arrest and actin stress fibres, implying that Cif is necessary and sufficient for these effects. EPEC infection of intestinal epithelial cells (Caco-2, IEC-6) also induces cell cycle arrest and CDK1 inhibition. The effect of Cif is strikingly similar to that of cytolethal distending toxin (CDT), which inhibits the G(2)/M transition by activating the DNA-damage checkpoint pathway. However, in contrast to CDT, Cif does not cause phosphorylation of histone H2AX, which is associated with DNA double-stranded breaks. Following EPEC infection, the checkpoint effectors ATM/ATR, Chk1 and Chk2 are not activated, the levels of the CDK-activating phosphatases Cdc25B and Cdc25C are not affected, and Cdc25C is not sequestered in host cell cytoplasm. Hence, Cif activates a DNA damage-independent signalling pathway that leads to inhibition of the G(2)/M transition.     

Clostridium difficile binary toxin (CDT) and diarrhea

Clostridium difficile is a major enteropathogen of humans. It produces two main virulence factors, toxins A and B. A third, less well known toxin, C. difficile toxin (CDT), is a binary toxin composed of distinct enzymatic (CdtA) and cell binding/translocation (CdtB) proteins. We used a novel enzyme linked immunoassay (EIA) to detect CdtB protein in feces and culture fluids. Additionally, PCR was used to assay C. difficile isolates from fecal samples for the CDT locus (CdtLoc). Although the results from 80 isolates suggest no relationship between toxin concentrations in situ and in vitro, there is a good correlation between PCR detection of the cdtB gene and EIA detection of CdtB protein in vitro. Possible implications of the detection of CDT in patients are discussed.     

Variation in binding of Bacillus sphaericus toxin and wheat germ agglutinin to larval midgut cells of six species of mosquitoes

Bacillus sphaericus toxin labeled with fluorescein isothiocyanate was readily ingested by Culex pipiens, Aedes aegypti, Anopheles stephensi, Anopheles gambiae, Anopheles quadrimaculatus, and Anopheles albimanus larvae. Fluorescent toxin bound to the luminal cell surface in discrete regions of the posterior midgut and gastric caecum in C. pipiens. In Anopheles spp., toxin bound in a variable pattern to these structures and central and anterior midgut as well. The toxin did not bind to midgut cells of A. aegypti. The toxin was internalized in bright fluorescent vesicles in C. pipiens, but was not internalized in Anopheles spp. and appeared to be weakly bound in these larvae, leaking rapidly from the gut surface. The lectin, wheat germ agglutinin, which interferes with binding of the B. sphaericus toxin, bound to the posterior midgut and gastric caecum of all species, but was not internalized. These results suggest that the sugar moiety of the receptor is not solely responsible for specificity of this toxin, and that binding to Culex spp. midgut cells may be highly specific and of high affinity, whereas binding to Anopheles spp. cells may be nonspecific and/or of low affinity.     

Clostridium difficile Toxin CDT Induces Formation of Microtubule-Based Protrusions and Increases Adherence of Bacteria

Clostridium difficile causes antibiotic-associated diarrhea and pseudomembranous colitis by production of the Rho GTPase-glucosylating toxins A and B. Recently emerging hypervirulent Clostridium difficile strains additionally produce the binary ADP-ribosyltransferase toxin CDT (Clostridium difficile transferase), which ADP-ribosylates actin and inhibits actin polymerization. Thus far, the role of CDT as a virulence factor is not understood. Here we report by using time-lapse- and immunofluorescence microscopy that CDT and other binary actin-ADP-ribosylating toxins, including Clostridium botulinum C2 toxin and Clostridium perfringens iota toxin, induce redistribution of microtubules and formation of long (up to >150 µm) microtubule-based protrusions at the surface of intestinal epithelial cells. The toxins increase the length of decoration of microtubule plus-ends by EB1/3, CLIP-170 and CLIP-115 proteins and cause redistribution of the capture proteins CLASP2 and ACF7 from microtubules at the cell cortex into the cell interior. The CDT-induced microtubule protrusions form a dense meshwork at the cell surface, which wrap and embed bacterial cells, thereby largely increasing the adherence of Clostridia. The study describes a novel type of microtubule structure caused by less efficient microtubule capture and offers a new perspective for the pathogenetic role of CDT and other binary actin-ADP-ribosylating toxins in host–pathogen interactions.     

Characterization of Putative Cholesterol Recognition/Interaction Amino Acid Consensus-Like Motif of Campylobacter jejuni Cytolethal Distending Toxin C

Cytolethal distending toxin (CDT) produced by Campylobacter jejuni comprises a heterotrimeric complex formed by CdtA, CdtB, and CdtC. Among these toxin subunits, CdtA and CdtC function as essential proteins that mediate toxin binding to cytoplasmic membranes followed by delivery of CdtB into the nucleus. The binding of CdtA/CdtC to the cell surface is mediated by cholesterol, a major component in lipid rafts. Although the putative cholesterol recognition/interaction amino acid consensus (CRAC) domain of CDT has been reported from several bacterial pathogens, the protein regions contributing to CDT binding to cholesterol in C. jejuni remain unclear. Here, we selected a potential CRAC-like region present in the CdtC from C. jejuni for analysis. Molecular modeling showed that the predicted functional domain had the shape of a hydrophobic groove, facilitating cholesterol localization to this domain. Mutation of a tyrosine residue in the CRAC-like region decreased direct binding of CdtC to cholesterol rather than toxin intermolecular interactions and led to impaired CDT intoxication. These results provide a molecular link between C. jejuni CdtC and membrane-lipid rafts through the CRAC-like region, which contributes to toxin recognition and interaction with cholesterol.     

Binary toxin producing Clostridium difficile strains

Clostridium difficile produces three toxins, TcdA, TcdB and CDT. TcdA and TcdB are single-stranded molecules acting as glucosyltransferases specific for small GTPases. CDT is an actin specific ADP-ribosylating binary toxin characteristically composed of two independent components, enzymatic CDTa (48 kDa) and binding CDTb (99 kDa). The cdtA and cdtB genes were sequenced in two CDT-positive strains of C. difficile (CD 196 and 8864) and at least two CDT-negative strains with truncated form of binary toxin genes are known (VPI 10463 and C. difficile genome strain 630). The prevalence of binary toxin producing strains is estimated to be from 1.6% to 5.5%, although a much higher proportion has been reported in some studies. The role of the binary toxin as an additional virulence factor is discussed.     

Role of the ATM-Checkpoint Kinase 2 Pathway in CDT-Mediated Apoptosis of Gingival Epithelial Cells

The cytolethal distending toxin (CDT) of the oral pathogen Aggregatibacter actinomycetemcomitans induces cell cycle arrest and apoptosis in various cell types. Western analysis, pharmacological inhibition and siRNA silencing were performed in human immortalized gingival keratinocytes (HIGK) to dissect the functional role of the ataxia telangiectasia mutated (ATM) pathway in the signal transduction steps triggered by the CDT. Infection of HIGK was associated with a time-dependent induction of cytoplasmic histone-associated DNA fragmentation. However, in the absence of CDT, infected HIGK underwent reversible DNA strand breaks but not apoptosis, while caspase 3 activity, p21 levels, and HIGK viability were unaffected. Caspase 9 activity was attenuated in the CDT mutant-infected HIGK compared to wild-type infected cells. Pharmacological inhibition and siRNA-silencing of the ATM downstream effector, the protein kinase checkpoint kinase 2 (Chk2), significantly impacted CDT-mediated apoptosis. Together, these findings provide insight on the specificity of the ATM-Chk2 pathway in response to the CDT of A. actinomycetemcomitans in oral epithelial cells, which ultimately leads to apoptosis. We further propose the existence of an unidentified factor that is distinct from the CDT, and involved with a reversible DNA fragmentation that does not trigger terminal apoptosis in oral epithelial cells. This model potentially explains conflicting reports on the biological activity of the A. actinomycetemcomitans CDT.     

Evidence for an essential role of long chain acyl-CoA synthetase in animal cell proliferation. Inhibition of long chain acyl-CoA synthetase by triacsins caused inhibition of Raji cell proliferation

Triacsins A, B, C, and D are new inhibitors of long chain acyl-CoA synthetase (EC 6.2.1.3) and possess different inhibitory potencies against the enzyme (Tomoda, H., Igarashi, K., and Omura, S. (1987) Biochim. Biophys. Acta 921, 595-598). Acyl-CoA synthetase activity in the membrane fraction of Raji cells was also inhibited by triacsins. The same hierarchy of inhibitory potency as that against the enzyme from other sources, triacsin C greater than triacsin A much greater than triacsin D greater than or equal to triacsin B, was observed. When Raji cells were cultivated in the presence of triacsins, cell proliferation was inhibited in a dose-dependent fashion. The drug concentrations required for 50% inhibition of cell growth at day 2 were calculated to be 1.8 microM for triacsin A, much greater than 20 microM for triacsin B, 1.0 microM for triacsin C, and much greater than 15 microM for triacsin D, demonstrating a hierarchy for inhibitory potency of triacsins similar to that against the acyl-CoA synthetase activity. To understand the role of long chain acyl-CoA synthetase in animal cells, the effect of triacsins on the lipid metabolism of Raji cells was studied. When intact Raji cells were incubated with [14C]oleate in the presence of individual triacsins, the incorporation of [14C]oleate into each of the lipid fractions such as phosphatidylcholine, phosphatidylethanolamine, and triacylglycerol was inhibited to an analogous extent. A common hierarchy, triacsin C greater than triacsin A much greater than triacsin D greater than triacsin B, was shown for the inhibition in each synthesis of the three lipids, which was identical with that for acyl-CoA synthetase. These findings indicate that the inhibition of acyl-CoA synthetase is well correlated with the inhibition of lipid synthesis. Taken together, the data strongly suggest that the inhibition of acyl-CoA synthetase by triacsins leads to the inhibition of lipid synthesis and eventually to the inhibition of proliferation of Raji cells.     

Binary toxin production in Clostridium difficile is regulated by CdtR, a LytTR family response regulator

Clostridium difficile binary toxin (CDT) is an actin-specific ADP-ribosyltransferase that is produced by various C. difficile isolates, including the "hypervirulent" NAP1/027 epidemic strains. In contrast to the two major toxins from C. difficile, toxin A and toxin B, little is known about the role of CDT in virulence or how C. difficile regulates its production. In this study we have shown that in addition to the cdtA and cdtB toxin structural genes, a functional cdt locus contains a third gene, here designated cdtR, which is predicted to encode a response regulator. By introducing functional binary toxin genes into cdtR(+) and cdtR-negative strains of C. difficile, it was established that the CdtR protein was required for optimal expression of binary toxin. Significantly increased expression of functional binary toxin was observed in the presence of a functional cdtR gene; an internal deletion within cdtR resulted in a reduction in binary toxin production to basal levels. Strains that did not carry intact cdtAB genes or cdtAB pseudogenes also did not have cdtR, with the entire cdt locus, or CdtLoc, being replaced by a conserved 68-bp sequence. These studies have shown for the first time that binary toxin production is subject to strict regulatory control by the response regulator CdtR, which is a member of the LytTR family of response regulators and is related to the AgrA protein from Staphylococcus aureus.     

The B cell surface molecule B1 is functionally linked with B cell activation and differentiation

The B1 molecule is a 32,000 m.w. phosphorylated cell surface protein expressed exclusively by B cells from the mid pre-B until the plasma cell stage of differentiation. Two monoclonal antibodies (gamma 2a and mu) reactive with this molecule were used to assess the role of B1 in B cell activation, proliferation, and differentiation. The anti-B1 antibodies at concentrations ranging from 0.1 to 100 micrograms/ml significantly inhibited B cell proliferation induced by anti-mu antibodies, Staphylococcus aureus Cowan strain 1, activated T cells, and Epstein Barr virus. Although capable of inhibiting proliferation, anti-B1 antibody in soluble form or coupled to beads did not activate B cells or induce proliferation. Antibodies of comparable isotypes or against other B cell-restricted antigens, including B2, B4, B5, and HB-5, did not inhibit activation. Pretreatment of B cells with anti-B1 antibody did not inhibit activation, indicating that B cells had to be cultured with anti-B1 antibody for anti-B1-mediated inhibition to occur. Maximum inhibition was obtained when anti-B1 antibody was added at the initiation of culture. In agreement with this, growth factor-dependent proliferation of preactivated B cells was not inhibited by anti-B1 antibodies. Comparable inhibition of B cell activation was noted with antibodies reactive with class II antigens of the major histocompatibility complex with the exception that anti-B1 antibody inhibited immunoglobulin secretion in pokeweed mitogen assays, whereas anti-DR antibody did not. These results suggest that the B1 molecule may serve a central role in the regulation of B cell activation and differentiation.     

Profile of toxigenicity of Clostridium difficile strains isolated from paediatric patients with clinical diagnosis of antibiotic associated diarrhea (AAD)

This study was performed to determine profile of toxigenicity of 18 Clostridium difficile strains isolated from paeditric patients suffering from antibiotic associated diarrhea (AAD). Toxigenicity of C. difficile strains was tested for detection toxin A and toxin B by phenotypic methods and for detection of the tcdA and tcdB genes using of PCR. Changes in the repeating regions of the tcdA genes were detected with the NK9/NKV011 primer pairs. For detection of binary toxin (CDT) cdtA and cdtB genes, cdtApos/cdtArev i cdtBpos/cdtBrev two pair primers in PCR was used. Among C. difficile strains was detected three profiles of toxigenicity: C. difficile strains possesing of tcdA and tcdB genes but not possesing cdtA and cdtB genes of binary toxin (A+B+CDT-), strains possesing tcdA and tcdB and cdtA and cdtB genes (A+B+CDT+), strains with deletion of toxin A gene (A-B+CDT-). This is the first report on the occurence of binary positive C. difficile strains isolated from paediatric patients.     

Clostridium difficile toxin A induces multinucleation in the human leukemic T cell line JURKAT.

Clostridium difficile toxin A is a cytotoxic enterotoxin known to be active on all mammalian cell lines tested up to now. It induces a disruption of the cytoskeleton, particularly the microfilament system, leading to inhibition of cell proliferation. Here, we describe some effects of toxin A on the leukemic T cell line JURKAT. Cells exposed to the toxin did not divide, as cell numbers remained constant for 3 days in the presence of 0.5 to 1.0 micrograms/ml of the toxin. However, these cells were found to become multinucleated, a phenomenon which was time- and dose-dependent. After treatment for 72 h with 0.5 micrograms/ml toxin A, 95% of the cells were multinucleated and had a considerably increased cell diameter. These effects in JURKAT cells were partially reversible upon removal of the toxin within 12 h after the beginning of toxin exposure, but irreversible after 24 h of toxin treatment. These results suggest a continuing nuclear division in the absence of cytoplasmic division, i.e., an effect of toxin A on contractile ring formation. The JURKAT cell is the first cell type reported to respond to toxin A with multinucleation.     

G protein modulation of N-type calcium channels is facilitated by physical interactions between syntaxin 1A and Gbetagamma

The direct modulation of N-type calcium channels by G protein betagamma subunits is considered a key factor in the regulation of neurotransmission. Some of the molecular determinants that govern the binding interaction of N-type channels and Gbetagamma have recently been identified (see, i.e., Zamponi, G. W., Bourinet, E., Nelson, D., Nargeot, J., and Snutch, T. P. (1997) Nature 385, 442-446); however, little is known about cellular mechanisms that modulate this interaction. Here we report that a protein of the presynaptic vesicle release complex, syntaxin 1A, mediates a crucial role in the tonic inhibition of N-type channels by Gbetagamma. When syntaxin 1A was coexpressed with (N-type) alpha(1B) + alpha(2)-delta + beta(1b) channels in tsA-201 cells, the channels underwent a 18 mV negative shift in half-inactivation potential, as well as a pronounced tonic G protein inhibition as assessed by its reversal by strong membrane depolarizations. This tonic inhibition was dramatically attenuated following incubation with botulinum toxin C, indicating that syntaxin 1A expression was indeed responsible for the enhanced G protein modulation. However, when G protein betagamma subunits were concomitantly coexpressed, the toxin became ineffective in removing G protein inhibition, suggesting that syntaxin 1A optimizes, rather than being required for G protein modulation of N-type channels. We also demonstrate that Gbetagamma physically binds to syntaxin 1A, and that syntaxin 1A can simultaneously interact with Gbetagamma and the synprint motif of the N-type channel II-III linker. Taken together, our experiments suggest a mechanism by which syntaxin 1A mediates a colocalization of G protein betagamma subunits and N-type calcium channels, thus resulting in more effective G protein coupling to, and regulation of, the channel. Thus, the interactions between syntaxin, G proteins, and N-type calcium channels are part of the structural specialization of the presynaptic terminal.     

Isoform-specific regulation of immune cell reactivity by the catalytic subunit of protein kinase A (PKA)

There are two major genes encoding the catalytic subunits of protein kinase A, Cα and Cβ. The functional significance of these isoforms is enigmatic. Lymphoid cells of the immune system express both Cα and Cβ. In this study we tested the role of Cα and Cβ in regulating immune cell reactivity to antigens using mice carrying a targeted disruption of the Cα and Cβ gene respectively. Cα and Cβ ablation both resulted in a 50% reduction in PKA-specific kinase activity and the level of PKA type I but not PKA type II. Moreover, despite that C subunit ablation did not affect immune cell development and homeostasis, Cα but not Cβ ablation augmented expression of the activation marker CD69 on lymphocytes. CD69 induction coincided with immune cell hyperresponsiveness and was associated with reduced sensitivity to cAMP-mediated inhibition of anti-CD3 induced T cell proliferation. Our results imply that Cα is required for normal immune cell reactivity and demonstrates isoform-specific effects and non-redundant functions of C subunit isoforms expressed in the same cell.     

Critical role for protein phosphatase 2A heterotrimers in mammalian cell survival

The predominant forms of protein phosphatase 2A (PP2A), one of the major Ser/Thr phosphatases, are dimers of catalytic (C) and scaffolding (A) subunits and trimers with an additional variable regulatory subunit. In mammals, catalytic and scaffolding subunits are encoded by two genes each (alpha/beta), whereas three gene families (B, B', and B') with a total of 12 genes contribute PP2A regulatory subunits. We generated stable PC12 cell lines in which the major scaffolding Aalpha subunit can be knocked down by inducible RNA interference (RNAi) to study its role in cell viability. Aalpha RNAi decreased total PP2A activity as well as protein levels of C, B, and B' but not B' subunits. Inhibitor experiments indicate that monomeric C and B subunits are degraded by the proteosome. Knock-down of Aalpha triggered cell death by redundant apoptotic and non-apoptotic mechanisms because the inhibition of RNAi-associated caspase activation failed to stall cell death. PP2A holoenzymes positively regulate survival kinase signaling, because RNAi reduced basal and epidermal growth factor-stimulated Akt phosphorylation. RNAi-resistant Aalpha cDNAs rescued RNAi-induced loss of the C subunit, and Aalpha point mutants prevented regulatory subunit degradation as predicted from each mutant's binding specificity. In transient, stable, and stable-inducible rescue experiments, both wild-type Abeta and Aalpha mutants capable of binding to at least one family of regulatory subunits were able to delay Aalpha RNAi-induced death of PC12 cells. However, only the expression of wild-type Aalpha restored viability completely. Thus, heterotrimeric PP2A holoenzymes containing the Aalpha subunit and members of all three regulatory subunit families are necessary for mammalian cell viability.     

Bimodal effect of phorbol ester on B cell activation. Implication for the role of protein kinase C.

The role of protein kinase C PKC in B cell activation is controversial. These studies were undertaken to determine whether protein kinase C has a stimulatory or inhibitory role in B cell activation. We found that treatment of B cells for a short period of time (30 min) with the PKC activator phorbol 12,13-dibutyrate (PDBU) primed the cells for enhanced proliferative responses to anti-immunoglobulin (anti-Ig) antibody whereas treatment for a longer period of time (3 h or more) resulted in suppression of proliferation. The enhanced proliferative response to treatment of B cells with PDBU for short periods of time was associated with inhibition of anti-Ig-stimulated increases in phosphatidyl 4,5-bisphosphate (PIP2) hydrolysis and inhibition of increases in [Ca2+]i, indicating that activation of PKC per se might be sufficient for enhancing B cell activation. The time-dependent effect of phorbol esters on the inhibition of B cell proliferation was found to be closely correlated with the kinetics of disappearance of PKC as measured by Western blot and by enzymatic activity but not with inhibition of [Ca2+]i and PIP2. These data demonstrate a bimodal time-dependent effect of PDBU on B cell activation and suggest that (a) the inhibitory effect of phorbol ester on anti-Ig-induced proliferation may be due to the disappearance of PKC rather than to the inhibition of PIP2 and Ca2+; and (b) the early activation of PKC is a stimulatory rather than an inhibitory signal in the induction of B lymphocyte proliferation by anti-Ig.