A phorbol ester receptor/protein kinase, nPKC eta, a new member of the protein kinase C family predominantly expressed in lung and skin

Protein kinase C (PKC)-related cDNA clones isolated from mouse epidermis cDNA library encoded a 78-kDa protein, nPKC eta. nPKC eta contains a characteristic cysteine-rich repeat sequence (C1 region) and a protein kinase domain sequence (C3 region), both of which are conserved among PKC family members. However, nPKC eta lacks a putative Ca2+ binding region (C2 region) that is seen in conventional PKCs (alpha, beta I, beta II, gamma), but not in novel PKCs (nPKC delta, -epsilon, -zeta). nPKC eta shows the highest sequence similarity to nPKC epsilon (59.4% identity). The similarity extends to the NH2-terminal sequence (E region) which corresponds to one of the divergent regions (D1 region). Northern blot analysis showed that the mRNA for nPKC eta is highly expressed in the lung and skin but, in contrast to other members of the PKC family, only slightly expressed in the brain. nPKC eta expressed in COS cells shows phorbol ester binding activity with a similar affinity to nPKC epsilon. Antiserum raised against a COOH-terminal peptide of nPKC eta identified an 82-kDa protein in mouse lung extract as well as in an extract from COS cells transfected with the nPKC eta-cDNA expression plasmid. Autophosphorylation of nPKC eta immunoprecipitated with the specific antiserum was observed, indicating that nPKC eta is a protein kinase. These results clearly demonstrate the existence and the possible importance of nPKC eta as a member of the phorbol ester receptor/protein kinase, PKC, family.     

Autophosphorylation of the C2 domain inhibits translocation of the novel protein kinase C (nPKC) Apl II

Protein kinase Cs (PKCs) are critical signaling molecules controlled by complex regulatory pathways. Herein, we describe an important regulatory role for C2 domain phosphorylation. Novel PKCs (nPKCs) contain an N‐terminal C2 domain that cannot bind to calcium. Previously, we described an autophosphorylation site in the Aplysia novel PKC Apl II that increased the binding of the C2 domain to lipids. In this study, we show that the function of this phosphorylation is to inhibit PKC translocation. Indeed, a phosphomimetic serine‐glutamic acid mutation reduced translocation of PKC Apl II while blocking phosphorylation with a serine‐alanine mutation enhanced translocation and led to the persistence of the kinase at the membrane longer after the end of the stimulation. Consistent with a role for autophosphorylation in regulating kinase translocation, inhibiting PKC activity using bisindolymaleimide 1 increased physiological translocation of PKC Apl II, whereas inhibiting phosphatase activity using calyculin A inhibited physiological translocation of PKC Apl II in neurons. Our results suggest a major role for autophosphorylation‐dependent regulation of translocation.     

The eta isoform of protein kinase C inhibits UV-induced activation of caspase-3 in normal human keratinocytes

Protein kinase C (PKC) fulfills a central role in the decision of cell fate in keratinocytes. Both PKC delta and PKC eta induce growth inhibition and differentiation of normal human keratinocytes (NHK). Here we show that PKC delta and PKC eta play opposite roles in UVB-induced apoptosis in NHK. PKC delta enhanced UVB-induced caspase-3 activity, while overexpression of PKC eta reduced it. In keeping with these observations, the dominant negative mutant of PKC delta significantly inhibited the activation of caspase-3, whereas dominant negative PKC eta increased it in a dose (MOI)-dependent manner. Unlike PKC delta, cleavage and translocation to mitochondria of PKC eta were not observed, resulting in no detection of cytochorome c release. Furthermore, UV-induced activation of p38 MAP kinase, which suppressed the caspase-3 activity in NHK, was blocked by dominant negative PKC eta. These findings suggest that PKC eta negatively regulates UV-induced apoptosis through its localization, resistance to cleavage, and the p38 MAPK pathway.     

Protein kinase C isozymes and their selectivity towards ruboxistaurin

Protein kinase C (PKC) isozymes are an important class of enzymes in cell signaling and as drug targets. They are involved in specific pathways and have selectivity towards certain ligands, despite their high sequence similarities. Ruboxistaurin is a specific inhibitor of PKC-beta. To understand the molecular determinants for the selectivity of ruboxistaurin, we derived the three-dimensional structures of the kinase domains of PKC-alpha, -betaI, and -zeta using homology modeling. Several binding orientations of ruboxistaurin in the binding sites of these PKC catalytic domains were analyzed, and a putative alternative binding site for PKC-zeta was identified in its kinase domain. The calculated free energy of binding correlates well with the IC(50) of the inhibitor against each PKC isozyme. A residue-based energy decomposition analysis attributed the binding free energy to several key residues in the catalytic sites of these enzymes, revealing potential protein-ligand interactions responsible for ligand binding. The contiguous binding site revealed in the catalytic domain of PKC-zeta provides avenues for selective drug design. The details of structural nuances for specific inhibition of PKC isozymes are presented in the context of the three-dimensional structures of this important class of enzymes.     

Classical protein kinases C are regulated by concerted interaction with lipids: the importance of phosphatidylinositol-4,5-bisphosphate

Classical protein kinase C (PKC) enzymes are known to be important factors in cell physiology both in terms of health and disease. They are activated by triggering signals that induce their translocation to membranes. The consensus view is that several secondary messengers are involved in this activation, such as cytosolic Ca²⁺ and diacylglycerol. Cytosolic Ca²⁺ bridges the C2 domain to anionic phospholipids as phosphatidylserine in the membrane, and diacylglycerol binds to the C1 domain. Both diacylglycerol and the increase in Ca²⁺ concentration are assumed to arise from the extracellular signal that triggers the hydrolysis of phosphatidylinositol-4,5-bisphosphate. However, results obtained during the last decade indicate that this phosphoinositide itself is also responsible for modulating classical PKC activity and its localization in the plasma membrane.     

Membrane binding kinetics of protein kinase C betaII mediated by the C2 domain.

Conventional isoforms of protein kinase C (PKC) are activated when their two membrane-targeting modules, the C1 and C2 domains, bind the second messengers diacylglycerol (DG) and Ca2+, respectively. This study investigates the mechanism of Ca2+-induced binding of PKC betaII to anionic membranes mediated by the C2 domain. Stopped-flow fluorescence spectroscopy reveals that Ca2+-induced binding of the isolated C2 domain to anionic vesicles proceeds via at least two steps: (1) rapid binding of two or more Ca2+ ions to the free domain with relatively low affinity and (2) diffusion-controlled association of the Ca2+-occupied domain with vesicles. Ca2+ increases the affinity of the C2 domain for anionic membranes by both decreasing the dissociation rate constant (k(off)) and increasing the association rate constant (k(on)) for membrane binding. For binding to vesicles containing 40 mol % anionic lipid in the presence of 200 microM Ca2+, k(off) and k(on) are 8.9 s(-1) and 1.2 x 10(10) M(-1) x s(-1), respectively. The k(off) value increases to 150 s(-1) when free Ca2+ levels are rapidly reduced, decreasing the average lifetime of the membrane-bound C2 domain (tau = k(off)(-1)) from 110 ms in the presence of Ca2+ to 6.7 ms when Ca2+ is rapidly removed. Experiments addressing the role of electrostatic interactions reveal that they stabilize either the initial C2 domain-membrane encounter complex or the high-affinity membrane-bound complex. Specifically, lowering the phosphatidylserine mole fraction or including MgCl2 in the binding reaction decreases the affinity of the C2 domain for anionic vesicles by both reducing k(on) and increasing k(off) measured in the presence of 200 microM Ca2+. These species do not affect the k(off) value when Ca2+ is rapidly removed. Studies with PKC betaII reveal that Ca2+-induced binding to membranes by the full-length protein proceeds minimally via two kinetically resolvable steps: (1) a rapid bimolecular association of the enzyme with vesicles near the diffusion-controlled limit and, most likely, (2) subsequent conformational changes of the membrane-bound enzyme. As is the case for the C2 domain, k(off) for full-length PKC betaII increases when Ca2+ is rapidly removed, reducing tau from 11 s in the presence of Ca2+ to 48 ms in its absence. Thus, both the C2 domain and the slow conformational change prolong the lifetime of the PKC betaII-membrane ternary complex in the presence of Ca2+, with rapid membrane release triggered by removal of Ca2+. These results provide a molecular basis for cofactor regulation of PKC whereby the C2 domain searches three-dimensional space at the diffusion-controlled limit to target PKC to relatively common anionic phospholipids, whereupon a two-dimensional search is initiated by the C1 domain for the more rare, membrane-partitioned DG.     

Molecular dynamics characterization of the C2 domain of protein kinase Cbeta.

Protein kinase C (PKC) isozymes comprise a family of related enzymes that play a central role in many intracellular eukaryotic signaling events. Isozyme specificity is mediated by association of each PKC isozyme with specific anchoring proteins, termed RACKs. The C2 domain of betaPKC contains at least part of the RACK-binding sites. Because the C2 domain contains also a RACK-like sequence (termed pseudo-RACK), it was proposed that this pseudo-RACK site mediates intramolecular interaction with one of the RACK-binding sites in the C2 domain itself, stabilizing the inactive conformation of betaPKC. BetaPKC depends on calcium for its activation, and the C2 domain contains the calcium-binding sites. The x-ray structure of the C2 domain of betaPKC shows that three Ca(2+) ions can be coordinated by two opposing loops at one end of the domain. Starting from this x-ray structure, we have performed molecular dynamics (MD) calculations on the C2 domain of betaPKC bound to three Ca(2+) ions, to two Ca(2+) ions, and in the Ca(2+)-free state, in order to analyze the effect of calcium on the RACK-binding sites and the pseudo-RACK sites, as well as on the loops that constitute the binding site for the Ca(2+) ions. The results show that calcium stabilizes the beta-sandwich structure of the C2 domain and thus affects two of the three RACK-binding sites within the C2 domain. Also, the interactions between the third RACK-binding site and the pseudo-RACK site are not notably modified by the removal of Ca(2+) ions. On that basis, we predict that the pseudo-RACK site within the C2 domain masks a RACK-binding site in another domain of betaPKC, possibly the V5 domain. Finally, the MD modeling shows that two Ca(2+) ions are able to interact with two molecules of O-phospho-l-serine. These data suggest that Ca(2+) ions may be directly involved in PKC binding to phosphatidylserine, an acidic lipid located exclusively on the cytoplasmic face of membranes, that is required for PKC activation.     

p65 fragments, homologous to the C2 region of protein kinase C, bind to the intracellular receptors for protein kinase C.

Receptors for activated protein kinase C (RACKs) have been isolated from the particulate cell fraction of heart and brain. We previously demonstrated that binding of protein kinase C (PKC) to RACKs requires PKC activators and is via a site on PKC that is distinct from the substrate binding site. Here, we examine the possibility that the C2 region in the regulatory domain of PKC is involved in binding of PKC to RACKs. The synaptic vesicle-specific p65 protein contains two regions homologous to the C2 region of PKC. We found that three p65 fragments, containing either one or two of these PKC C2 homologous regions, bound to highly purified RACKs. Binding of the p65 fragments and PKC to RACKs was mutually exclusive; preincubation of RACKs with the p65 fragments inhibited PKC binding, and preincubation of RACKs with PKC inhibited binding of the p65 fragments. Preincubation of the p65 fragments with a peptide resembling the PKC binding site on RACKs also inhibited p65 binding to RACKs, suggesting that PKC and p65 bind to the same or nearby regions on RACKs. Since the only homologous region between PKC and the p65 fragments is the C2 region, these results suggest that the C2 region on PKC contains at least part of the RACK binding site.     

Reversible oxidative activation and inactivation of protein kinase C by the mitogen/tumor promoter periodate.

The oxidant mitogen/tumor promoter, periodate, was used to selectively modify either the regulatory domain or the catalytic domain of protein kinase C (PKC) to induce oxidative activation or inactivation of PKC, respectively. Periodate, at micromolar concentrations, modified the regulatory domain of PKC as determined by the loss of ability to stimulate kinase activity by Ca2+/phospholipid, and also by the loss of phorbol ester binding. This modification resulted in an increase in Ca2+/phospholipid-independent kinase activity (oxidative activation). However, at higher concentrations (greater than 100 microM) periodate also modified the catalytic domain, resulting in complete inactivation of PKC. The oxidative modification induced by low periodate concentrations (less than 0.5 mM) was completely reversed by a brief treatment with 2 mM dithiothreitol. In this aspect, the modification induced by periodate was different from that of the previously reported irreversible modification of PKC induced by H2O2. However, the inactivation of PKC induced by periodate at concentrations greater than 1 mM was not reversed by dithiothreitol. Among the phospholipids and ligands of the regulatory domain tested, only phosphatidylserine protected the regulatory domain from oxidative modification. In the presence of phosphatidylserine, the catalytic site was selectively modified by periodate, resulting in formation of a form of PKC that exhibited phorbol ester binding but not kinase activity. Both reversible and irreversible oxidative activation and inactivation of PKC also were observed in intact cells treated with periodate. Taken together these results suggest that periodate, by virtue of having a tetrahedral structure, binds to the phosphate-binding regions present within the phosphatidylserine-binding site of the regulatory domain and the ATP-binding site of the catalytic domain, and modifies the vicinal thiols present within these sites. This results in the formation of intramolecular disulfide bridge(s) within the regulatory domain or catalytic domain leading to either reversible activation or inactivation of PKC, respectively. Thus, oxidant mitogen/tumor promoters such as periodate may be able to bypass normal transmembrane signalling systems to directly activate pathways involved in cellular regulation.     

The C-terminal 26-residue peptide of serpin A1 stimulates proliferation of breast and liver cancer cells: role of protein kinase C and CD47

C26, the C-terminal 26 residue peptide of serpin A1, significantly increased cell proliferation in cultures of hepatoma cells, but not in porcine kidney epithelial cells, human skin fibroblasts or keratinocytes. The mitogenic activity of C26 was preferentially inhibited with a protein kinase C (PKC) inhibitor, an antibody against CD47 and CD47 short interfering RNA. The mutant C26-K19R,N22M, imitating a thrombospondin-like cell adhesion motif, increased the mitogenic activity in both Hep G2 cells and MCF-7 breast cancer cells. Phosphorylation of C26 at T24 (a putative PKC phosphorylation site) resulted in a 1.9-2.5 increase in mitogenic activity over C26 in MCF-7 cells.     

Protein kinase C(mu) regulation of the JNK pathway is triggered via phosphoinositide-dependent kinase 1 and protein kinase C(epsilon)

The protein kinase C (PKC)-related enzyme PKC(mu)/PKD (protein kinase D) is activated by activation loop phosphorylation through PKC(eta). Here we demonstrate that PKC(mu) is activated by the direct phosphorylation of PKC(epsilon). PKC(mu) colocalizes with PKC(epsilon) in HEK293 and MCF7 cells as shown by confocal immunofluorescence analyses. PDK1, known as the upstream kinase for several PKC isozymes, associates intracellularly with PKC(epsilon) and PKC(eta). PKC(eta) is phosphorylated by PDK1 in vitro, leading to kinase activation as similarly reported for PKC(epsilon) activation by PDK1. Coexpression of PDK1, PKC(epsilon) and PKC(mu) in HEK293 cells results in PKC(mu) activation. In contrast, the coexpression of PDK1 and PKC(eta) with PKC(mu) does not activate PKC(eta) or consequently PKC(mu). PDK1/PKC(epsilon)-triggered activation of PKC(mu) inhibits JNK, a downstream effector of PKC(mu), whereas upon transient expression of PDK1, PKC(eta), and PKC(mu), JNK is not affected. These data implicate PKC(epsilon) as the biologically important upstream kinase for PKC(mu) in HEK293 cells, regulating downstream effectors. Our results further indicate a PDK1/PKC(eta)/PKC(mu) controlled negative regulation of PKC(eta) kinase activity. In this study, we show that differentially activated kinase cascades involving PDK1 and novel PKC isotypes are responsible for the regulation of PKC(mu) activity and consequently inhibit the JNK pathway.     

Protein kinase C {delta}-specific activity reporter reveals agonist-evoked nuclear activity controlled by Src family of kinases

Conventional and novel protein kinase C (PKC) isozymes transduce the abundance of signals mediated by phospholipid hydrolysis; however redundancy in regulatory mechanisms confounds dissecting the unique signaling properties of each of the eight isozymes constituting these two subgroups. Previously, we created a genetically encoded reporter (C kinase activity reporter (CKAR)) to visualize the rate, amplitude, and duration of agonist-evoked PKC signaling at specific locations within the cell. Here we designed a reporter, δCKAR, that specifically measures the activation signature of one PKC isozyme, PKC δ, in cells, revealing unique spatial and regulatory properties of this isozyme. Specifically, we show two mechanisms of activation: 1) agonist-stimulated activation at the plasma membrane (the site of most robust PKC δ signaling), Golgi, and mitochondria that is independent of Src and can be triggered by phorbol esters and 2) agonist-stimulated activation in the nucleus that requires Src kinase activation and cannot be triggered by phorbol esters. Translocation studies reveal that the G-protein-coupled receptor agonist UTP induces the translocation of PKC δ into the nucleus by a mechanism that depends on the C2 domain and requires Src kinase activity. However, translocation from the cytosol into the nucleus is not required for the Src-dependent regulation of nuclear activity; a construct of PKC δ prelocalized to the nucleus continues to be activated by UTP by a mechanism dependent on Src kinase activity. These data identify the nucleus as a signaling hub for PKC δ that is driven by receptor-mediated signaling pathways (but not phorbol esters) and differs from signaling at plasma membrane and Golgi in that it is controlled by Src family kinases.     

Active site inhibitors protect protein kinase C from dephosphorylation and stabilize its mature form

Conformational changes acutely control protein kinase C (PKC). We have previously shown that the autoinhibitory pseudosubstrate must be removed from the active site in order for 1) PKC to be phosphorylated by its upstream kinase phosphoinositide-dependent kinase 1 (PDK-1), 2) the mature enzyme to bind and phosphorylate substrates, and 3) the mature enzyme to be dephosphorylated by phosphatases. Here we show an additional level of conformational control; binding of active site inhibitors locks PKC in a conformation in which the priming phosphorylation sites are resistant to dephosphorylation. Using homogeneously pure PKC, we show that the active site inhibitor Gö 6983 prevents the dephosphorylation by pure protein phosphatase 1 (PP1) or the hydrophobic motif phosphatase, pleckstrin homology domain leucine-rich repeat protein phosphatase (PHLPP). Consistent with results using pure proteins, treatment of cells with the competitive inhibitors Gö 6983 or bisindolylmaleimide I, but not the uncompetitive inhibitor bisindolylmaleimide IV, prevents the dephosphorylation and down-regulation of PKC induced by phorbol esters. Pulse-chase analyses reveal that active site inhibitors do not affect the net rate of priming phosphorylations of PKC; rather, they inhibit the dephosphorylation triggered by phorbol esters. These data provide a molecular explanation for the recent studies showing that active site inhibitors stabilize the phosphorylation state of protein kinases B/Akt and C.     

Domain shuffling as a tool for investigation of protein function: substitution of the cysteine-rich region of Raf kinase and PKC eta for that of yeast Pkc1p

With the completion of the sequences of entire genomes, the need for functional characterisation of proteins and their domains is becoming acute. Conserved regions within proteins often share overlapping functions but despite this conservation may fulfil quite different tasks in different species. In this work, we investigated the cysteine-rich motif (C1 domain) of yeast protein kinase C (Pkc1p) as a model to establish a test system for domain function. C1 domains activate kinases through binding of either diacylglycerol and/or phosphatidylserine, as in many members of the protein kinase C (PKC) family, or by binding small GTPases, as in Raf kinase. In contrast to other members of the protein kinase C superfamily, Pkc1p of Saccharomyces cerevisiae is activated via binding of the small G-protein Rho1p to its C1 domain. We developed a system for domain shuffling to establish the function of C1 domains from human Raf kinase and rat PKC eta in yeast. Only the C1 domain from Raf kinase enabled the chimeric enzyme to bind Rho1p when substituted for the native yeast domain. Accordingly, a chimeric Pkc1p carrying the C1 from Raf kinase, but not that from PKC eta, was able to partially complement the phenotypes of a yeast pkc1 deletion mutant. We interpret these data as further evidence that interaction with a small GTPase is the main regulatory function of the C1 domain in yeast.     

C2 domains of protein kinase C isoforms alpha,beta, and gamma: activation parameters and calcium stoichiometries of the membrane-bound state

The independently folding C2 domain motif serves as a Ca(2+)-dependent membrane docking trigger in a large number of Ca(2+) signaling pathways. A comparison was initiated between three closely related C2 domains from the conventional protein kinase C subfamily (cPKC, isoforms alpha, beta, and gamma). The results reveal that these C2 domain isoforms exhibit some similarities but are specialized in important ways, including different Ca(2+) stoichiometries. In the absence of membranes, Ca(2+) affinities of the isolated C2 domains are similar (2-fold difference) while Hill coefficients reveal cooperative Ca(2+) binding for the PKC beta C2 domain but not for the PKC alpha or PKC gamma C2 domain (H = 2.3 +/- 0.1 for PKC beta, 0.9 +/- 0.1 for PKC alpha, and 0.9 +/- 0.1 for PKC gamma). When phosphatidylserine-containing membranes are present, Ca(2+) affinities range from the sub-micromolar to the micromolar (7-fold difference) ([Ca(2+)](1/2) = 0.7 +/- 0.1 microM for PKC gamma, 1.4 +/- 0.1 microM for PKC alpha, and 5.0 +/- 0.2 microM for PKC beta), and cooperative Ca(2+) binding is observed for all three C2 domains (Hill coefficients equal 1.8 +/- 0.1 for PKC beta, 1.3 +/- 0.1 for PKC alpha, and 1.4 +/- 0.1 for PKC gamma). The large effects of membranes are consistent with a coupled Ca(2+) and membrane binding equilibrium, and with a direct role of the phospholipid in stabilizing bound Ca(2+). The net negative charge of the phospholipid is more important to membrane affinity than its headgroup structure, although a slight preference for phosphatidylserine is observed over other anionic phospholipids. The Ca(2+) stoichiometries of the membrane-bound C2 domains are detectably different. PKC beta and PKC gamma each bind three Ca(2+) ions in the membrane-associated state; membrane-bound PKC alpha binds two Ca(2+) ions, and a third binds weakly or not at all under physiological conditions. Overall, the results indicate that conventional PKC C2 domains first bind a subset of the final Ca(2+) ions in solution, and then associate weakly with the membrane and bind additional Ca(2+) ions to yield a stronger membrane interaction in the fully assembled tertiary complex. The full complement of Ca(2+) ions is needed for tight binding to the membrane. Thus, even though the three C2 domains are 64% identical, differences in Ca(2+) affinity, stoichiometry, and cooperativity are observed, demonstrating that these closely related C2 domains are specialized for their individual functions and contexts.     

Regulation of insulin receptor substrate 1 pleckstrin homology domain by protein kinase C: role of serine 24 phosphorylation

Phosphorylation of insulin receptor substrate (IRS) proteins on serine residues is an important posttranslational modification that is linked to insulin resistance. Several phosphoserine sites on IRS1 have been identified; the majority are located proximal to the phosphotryosine-binding domain or near key receptor tyrosine kinase substrate- and/or Src-homology 2 domain-binding sites. Here we report on the characterization of a serine phosphorylation site in the N-terminal pleckstrin homology (PH) domain of IRS1. Bioinformatic tools identify serine 24 (Ser24) as a putative substrate site for the protein kinase C (PKC) family of serine kinases. We demonstrate that this site is indeed a bona fide substrate for conventional PKC. In vivo, IRS-1 is also phosphorylated on Ser24 after phorbol 12-myristate 13-acetate treatment of cells, and isoform-selective inhibitor studies suggest the involvement of PKCalpha. By comparing the pharmacological characteristics of phorbol 12-myristate 13-acetate-stimulated Ser24 phosphorylation with phosphorylation at two other sites previously linked to PKC activity (Ser307 and Ser612), we show that PKCalpha is likely to be directly involved in Ser24 phosphorylation, but indirectly involved in Ser307 and Ser612 phosphorylation. Using Ser24Asp IRS-1 mutants to mimic the phosphorylated residue, we demonstrate that the phosphorylation status of Ser24 does play an important role in regulating phosphoinositide binding to, and the intracellular localization of, the IRS1-PH domain, which can ultimately impinge on insulin-stimulated glucose uptake. Hence we provide evidence that IRS1-PH domain function is important for normal insulin signaling and is regulated by serine phosphorylation in a manner that could contribute to insulin resistance.     

The C1 and C2 domains of protein kinase C are independent membrane targeting modules, with specificity for phosphatidylserine conferred by the C1 domain

Protein kinase C is specifically activated by binding two membrane lipids: the second messenger, diacylglycerol, and the amino phospholipid, phosphatidylserine. This binding provides the energy to release an autoinhibitory pseudosubstrate from the active site. Interaction with these lipids recruits the enzyme to the membrane by engaging two membrane-targeting modules: the C1 domain (present as a tandem repeat in most protein kinase Cs) and the C2 domain. Here we dissect the contribution of each domain in recruiting protein kinase C betaII to membranes. Binding analyses of recombinant domains reveal that the C2 domain binds anionic lipids in a Ca(2+)-dependent, but diacylglycerol-independent, manner, with little selectivity for phospholipid headgroup beyond the requirement for negative charge. The C1B domain binds membranes in a diacylglycerol/phorbol ester-dependent, but Ca(2+)-independent manner. Like the C2 domain, the C1B domain preferentially binds anionic lipids. However, in striking contrast to the C2 domain, the C1B domain binds phosphatidylserine with an order of magnitude higher affinity than other anionic lipids. This preference for phosphatidylserine is, like that of the full-length protein, stereoselective for sn-1, 2-phosphatidyl-L-serine. Quantitative analysis of binding constants of individual domains and that of full-length protein reveals that the full-length protein binds membranes with lower affinity than expected based on the binding affinity of isolated domains. In addition to entropic and steric considerations, the difference in binding energy may reflect the energy required to expel the pseudosubstrate from the substrate binding cavity. This study establishes that each module is an independent membrane-targeting module with each, independently of the other, containing determinants for membrane recognition. The presence of each of these modules, separately, in a number of other signaling proteins epitomizes the use of these modules as discreet membrane targets.