The C2 domain of protein kinase calpha is directly involved in the diacylglycerol-dependent binding of the C1 domain to the membrane

Protein kinase Calpha (PKCalpha), which is known to be critical for the control of many cellular processes, was submitted to site-directed mutagenesis in order to test the functionality of several amino acidic residues. Thus, D187, D246 and D248, all of which are located at the Ca(2+) binding site of the C2 domain, were substituted by N. Subcellular fractionation experiments demonstrated that these mutations are important for both Ca(2+)-dependent and diacylglycerol-dependent membrane binding. The mutants are not able to phosphorylate typical PKC substrates, such as histone and myelin basic protein. Furthermore, using increasing concentrations of dioleylglycerol, one of the mutants (D246/248N) was able to recover total activity although the amounts of dioleylglycerol it required were larger than those required by wild type protein. On the other hand, the other mutants (D187N and D187/246/248) only recovered 50% of their activity. These data suggest that there is a relationship between the C1 domain, where dioleylglycerol binds, and the C2 domain, and that this relationship is very important for enzyme activation. These findings led us to propose a mechanism for PKCalpha activation, where C1 and C2 domains cannot be considered independent membrane binding modules.     

Mechanism of diacylglycerol-induced membrane targeting and activation of protein kinase Cdelta

The regulatory domains of novel protein kinases C (PKC) contain two C1 domains (C1A and C1B), which have been identified as the interaction site for sn-1,2-diacylglycerol (DAG) and phorbol ester, and a C2 domain that may be involved in interaction with lipids and/or proteins. Although recent reports have indicated that C1A and C1B domains of conventional PKCs play different roles in their DAG-mediated membrane binding and activation, the individual roles of C1A and C1B domains in the DAG-mediated activation of novel PKCs have not been fully understood. In this study, we determined the roles of C1A and C1B domains of PKCdelta by means of in vitro lipid binding analyses and cellular protein translocation measurements. Isothermal titration calorimetry and surface plasmon resonance measurements showed that isolated C1A and C1B domains of PKCdelta have opposite affinities for DAG and phorbol ester; i.e. the C1A domain with high affinity for DAG and the C1B domain with high affinity for phorbol ester. Furthermore, in vitro activity and membrane binding analyses of PKCdelta mutants showed that the C1A domain is critical for the DAG-induced membrane binding and activation of PKCdelta. The studies also indicated that an anionic residue, Glu(177), in the C1A domain plays a key role in controlling the DAG accessibility of the conformationally restricted C1A domain in a phosphatidylserine-dependent manner. Cell studies with enhanced green fluorescent protein-tagged PKCdelta and mutants showed that because of its phosphatidylserine specificity PKCdelta preferentially translocated to the plasma membrane under the conditions in which DAG is randomly distributed among intracellular membranes of HEK293 cells. Collectively, these results provide new insight into the differential roles of C1 domains in the DAG-induced membrane activation of PKCdelta and the origin of its specific subcellular localization in response to DAG.     

The Pleckstrin homology domain of CK2 interacting protein-1 is required for interactions and recruitment of protein kinase CK2 to the plasma membrane

CKIP-1 is a recently identified interaction partner of protein kinase CK2 with a number of protein-protein interaction motifs, including an N-terminal pleckstrin homology domain. To test the hypothesis that CKIP-1 has a role in targeting CK2 to specific locations, we examined the effects of CKIP-1 on the localization of CK2. These studies demonstrated that CKIP-1 can recruit CK2 to the plasma membrane. Furthermore, the pleckstrin homology domain of CKIP-1 was found to be required for interactions with CK2 and for the recruitment of CK2 to the plasma membrane. In this regard, point mutations in this domain abolish membrane localization and compromise interactions with CK2. In addition, replacement of the pleckstrin homology domain with a myristoylation signal was insufficient to elicit any interaction with CK2. An investigation of the lipid binding of CKIP-1 reveals that it has broad specificity. A comparison with other pleckstrin homology domains revealed that the pleckstrin homology domain of CKIP-1 is distinct from other defined classes of pleckstrin homology domains. Finally, examination of CK2alpha for a region that mediates interactions with CKIP-1 revealed a putative HIKE domain, a complex motif found exclusively in proteins that bind pleckstrin homology domains. However, mutations within this motif were not able to abolish CKIP-1-CK2 interactions suggesting that this motif by itself may not be sufficient to mediate interactions. Overall, these results provide novel insights into how CK2, a predominantly nuclear enzyme, is targeted to the plasma membrane, and perhaps more importantly how it may be regulated.     

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.     

Identification of a general anesthetic binding site in the diacylglycerol-binding domain of protein kinase Cdelta

Protein kinase C (PKC) is an important signal transduction protein that has been proposed to interact with general anesthetics at its cysteine-rich diacylglycerol/phorbol ester-binding domain C1, a tandem repeat of C1A and C1B subdomains. To test this hypothesis, we expressed, purified, and characterized the high affinity phorbol-binding subdomain, C1B, of mouse protein kinase Cdelta, and studied its interaction with general anesthetic alcohols. When the fluorescent phorbol ester, sapintoxin-D, bound to PKCdelta C1B in buffer at a molar ratio of 1:2, its fluorescence emission maximum, lambda(max), shifted from 437 to 425 nm. The general anesthetic alcohols, butanol and octanol, further shifted lambda(max) of the PKCdelta C1B-bound sapintoxin-D in a concentration-dependent, saturable manner to approximately 415 nm, suggesting that alcohols interact at a discrete allosteric binding site. To identify this site, PKCdelta C1B was photolabeled with three photo-activable diazirine alcohol analogs, 3-azioctanol, 7-azioctanol, and 3-azibutanol. Mass spectrometry showed photoincorporation of all three alcohols in PKCdelta C1B at a stoichiometry of 1:1 in the labeled fraction. The photolabeled PKCdelta C1B was subjected to tryptic digest, the fragments were separated by online chromatography and sequenced by mass spectrometry. Each azialcohol photoincorporated at Tyr-236. Inspection of the known structure of PKCdelta C1B shows that this residue is situated adjacent to the phorbol ester binding pocket, and within approximately 10 A of the bound phorbol ester. The present results provide direct evidence for an allosteric anesthetic site on protein kinase C.     

Identification of an alcohol binding site in the first cysteine-rich domain of protein kinase Cdelta

Protein kinase C (PKC) is an important signal transduction protein whose cysteine-rich regulatory domain C1 has been proposed to interact with general anesthetics in both of its diacylglycerol/phorbol ester-binding subdomains, the tandem repeats C1A and C1B. Previously, we identified an allosteric binding site on one of the two cysteine-rich domains, PKCdelta C1B. To test the hypothesis that there is an additional anesthetic site on the other cysteine-rich subdomain, C1A, we subcloned, expressed in Escherichia coli, purified, and characterized mouse PKCdelta C1A. Octanol and butanol both quenched the intrinsic fluorescence of PKCdelta C1A in a saturable manner, suggesting the presence of a binding site. To locate this site, PKCdelta C1A was photolabeled with three diazirine-containing alkanols, 3-azioctanol, 7-azioctanol, and 3-azibutanol. Mass spectrometry revealed that at low concentrations all three photoincorporated into PKCdelta C1A with a stoichiometry of 1:1 in the labeled fraction, but higher stoichiometries occurred at higher concentrations, particularly with azibutanol. Photocomplexes of PKCdelta C1A with azioctanols were separated from the unlabeled protein by HPLC, reduced, alkylated, digested with trypsin, and sequenced by mass spectrometry. All the azioctanols photolabeled PKCdelta C1A at residue Tyr-29, corresponding to Tyr-187 of the full-length PKCdelta, and at a neighboring residue, Lys-40, suggesting there is an alcohol site in this vicinity. In addition, Glu-2 was photolabeled more efficiently by 3-azibutanol than by the azioctanols, suggesting the existence of a second, smaller site.     

Alkyl cinnamates as regulator for the C1 domain of protein kinase C isoforms

The protein kinase C (PKC) family of serine/threonine kinases is an attractive drug target for the treatment of cancer and other diseases. Natural product curcumin is known to interact with PKC isoforms through the C1 domain and modulate PKC activity. The reported results demonstrate that the symmetric curcumin molecule might act as two separate units during its recognition of C1 domains. To understand the importance of the two halves of curcumin in PKC binding and to develop effective PKC regulators, we synthesized a series of alkyl cinnamates (1-8), characterized absorption and fluorescence properties and measured binding affinities with the C1b subdomains of PKC isoforms. The binding parameters of the monomeric compounds and liposomes containing compounds confirmed their interaction with the C1b subdomains of PKCδ and PKCθ. The molecular docking analysis with PKCδ and PKCθ C1b subdomains revealed that the alkyl cinnamates form hydrogen bond with the backbone of the protein at the same binding site as that of diacylglycerol and phorbol esters. The results show that the alkyl cinnamates bind to the activator binding site of PKCs and both methoxy and hydroxyl groups play important roles in the binding process.     

Interplay of C1 and C2 domains of protein kinase C-alpha in its membrane binding and activation.

The regulatory domain of conventional protein kinase C (PKC) contains two membrane-targeting modules, the C2 domain that is responsible for Ca2+-dependent membrane binding of protein, and the C1 domain composed of two cysteine-rich zinc fingers (C1a and C1b) that bind diacylglycerols and phorbol esters. To understand the individual roles and the interplay of the C1 and C2 domains in the membrane binding and activation of PKC, we functionally expressed isolated C1 and C2 domains of PKC-alpha and measured their vesicle binding and monolayer penetration. Results indicate that the C2 domain of PKC-alpha is responsible for the initial Ca2+- and phosphatidylserine-dependent electrostatic membrane binding of PKC-alpha, whereas the C1 domain is involved in subsequent membrane penetration and diacylglycerol binding, which eventually lead to enzyme activation. To determine the roles of individual zinc fingers in the C1 domain, we also mutated hydrophobic residues in the C1a (Trp58 and Phe60) and C1b (Tyr123 and Leu125) domains of the native PKC-alpha molecule and measured the effects of mutations on vesicle binding, enzyme activity and monolayer penetration. Results show that the hydrophobic residues in the C1a domain are essential for the membrane penetration and activation of PKC-alpha, whereas those in the C1b domain are not directly involved in these processes. Based on these results in conjunction with our previous structure-function studies of the C2 domain (Medkova, M., and Cho, W. (1998) J. Biol. Chem. 273, 17544-17552), we propose a mechanism for the in vitro membrane binding and activation of conventional PKC that accounts for the temporal and spatial sequences of PKC activation.     

Development of diacyltetrol lipids as activators for the C1 domain of protein kinase C

The protein kinase C (PKC) family of serine/threonine kinases is an attractive drug target for the treatment of cancer and other diseases. Diacylglycerol (DAG), phorbol esters and others act as ligands for the C1 domain of PKC isoforms. Inspection of the crystal structure of the PKCδ C1b subdomain in complex with phorbol-13-O-acetate shows that one carbonyl group and two hydroxyl groups play pivotal roles in recognition of the C1 domain. To understand the importance of two hydroxyl groups of phorbol esters in PKC binding and to develop effective PKC activators, we synthesized DAG like diacyltetrols (DATs) and studied binding affinities with C1b subdomains of PKCδ and PKCθ. DATs, with the stereochemistry of natural DAGs at the sn-2 position, were synthesized from (+)-diethyl L-tartrate in four to seven steps as single isomers. The calculated EC(50) values for the short and long chain DATs varied in the range of 3-6 μM. Furthermore, the fluorescence anisotropy values of the proteins were increased in the presence of DATs in a similar manner to that of DAGs. Molecular docking of DATs (1b-4b) with PKCδ C1b showed that the DATs form hydrogen bonds with the polar residues and backbone of the protein, at the same binding site, as that of DAG and phorbol esters. Our findings reveal that DATs represent an attractive group of C1 domain ligands that can be used as research tools or further structurally modified for potential drug development.     

Specific translocation of protein kinase Calpha to the plasma membrane requires both Ca2+ and PIP2 recognition by its C2 domain

The C2 domain of protein kinase Calpha (PKCalpha) controls the translocation of this kinase from the cytoplasm to the plasma membrane during cytoplasmic Ca2+ signals. The present study uses intracellular coimaging of fluorescent fusion proteins and an in vitro FRET membrane-binding assay to further investigate the nature of this translocation. We find that Ca2+-activated PKCalpha and its isolated C2 domain localize exclusively to the plasma membrane in vivo and that a plasma membrane lipid, phosphatidylinositol-4,5-bisphosphate (PIP2), dramatically enhances the Ca2+-triggered binding of the C2 domain to membranes in vitro. Similarly, a hybrid construct substituting the PKCalpha Ca2+-binding loops (CBLs) and PIP2 binding site (beta-strands 3-4) into a different C2 domain exhibits native Ca2+-triggered targeting to plasma membrane and recognizes PIP2. Conversely, a hybrid containing the CBLs but lacking the PIP2 site translocates primarily to trans-Golgi network (TGN) and fails to recognize PIP2. Similarly, PKCalpha C2 domains possessing mutations in the PIP2 site target primarily to TGN and fail to recognize PIP2. Overall, these findings demonstrate that the CBLs are essential for Ca2+-triggered membrane binding but are not sufficient for specific plasma membrane targeting. Instead, targeting specificity is provided by basic residues on beta-strands 3-4, which bind to plasma membrane PIP2.     

Importance of C1B domain for lipid messenger-induced targeting of protein kinase C

The molecular mechanisms by which arachidonic acid (AA) and ceramide elicit translocation of protein kinase C (PKC) were investigated. Ceramide translocated epsilonPKC from the cytoplasm to the Golgi complex, but with a mechanism distinct from that utilized by AA. Using fluorescence recovery after photobleaching, we showed that, upon treatment with AA, epsilonPKC was tightly associated with the Golgi complex; ceramide elicited an accumulation of epsilonPKC which was exchangeable with the cytoplasm. Stimulation with ceramide after AA converted the AA-induced Golgi complex staining to one elicited by ceramide alone; AA had no effect on the ceramide-stimulated localization. Using point mutants and deletions of epsilonPKC, we determined that the epsilonC1B domain was responsible for the ceramide- and AA-induced translocation. Switch chimeras, containing the C1B from epsilonPKC in the context of deltaPKC (delta(epsilonC1B)) and vice versa (epsilon(deltaC1B)), were generated and tested for their translocation in response to ceramide and AA. delta(epsilonC1B) translocated upon treatment with both ceramide and AA; epsilon(deltaC1B) responded only to ceramide. Thus, through the C1B domain, AA and ceramide induce different patterns of epsilonPKC translocation and the C1B domain defines the subtype specific sensitivity of PKCs to lipid second messengers.     

Structure of the C2 domain from novel protein kinase Cepsilon. A membrane binding model for Ca(2+)-independent C2 domains

Protein kinase Cepsilon (PKCepsilon) is a member of the novel PKCs which are activated by acidic phospholipids, diacylglycerol and phorbol esters, but lack the calcium dependence of classical PKC isotypes. The crystal structures of the C2 domain of PKCepsilon, crystallized both in the absence and in the presence of the two acidic phospholipids, 1,2-dicaproyl-sn-phosphatidyl-l-serine (DCPS) and 1,2-dicaproyl-sn-phosphatidic acid (DCPA), have now been determined at 2.1, 1.7 and 2.8 A resolution, respectively. The central feature of the PKCepsilon-C2 domain structure is an eight-stranded, antiparallel, beta-sandwich with a type II topology similar to that of the C2 domains from phospholipase C and from novel PKCdelta. Despite the similar topology, important differences are found between the structures of C2 domains from PKCs delta and epsilon, suggesting they be considered as different PKC subclasses. Site-directed mutagenesis experiments and structural changes in the PKCepsilon-C2 domain from crystals with DCPS or DCPA indicate, though phospholipids were not visible in these structures, that loops joining strands beta1-beta2 and beta5-beta6 participate in the binding to anionic membranes. The different behavior in membrane-binding and activation between PKCepsilon and classical PKCs appears to originate in localized structural changes, which include a major reorganization of the region corresponding to the calcium binding pocket in classical PKCs. A mechanism is proposed for the interaction of the PKCepsilon-C2 domain with model membranes that retains basic features of the docking of C2 domains from classical, calcium-dependent, PKCs.     

Identification of an amino acid residue in the protein kinase C C1b domain crucial for its localization to the Golgi network

Protein kinase C (PKC) isoforms have been reported to be targeted to the Golgi complex via their C1 domains. We have shown recently that the regulatory domain of PKC induces apoptosis in neuroblastoma cells and that this effect is correlated to Golgi localization via the C1b domain. This study was designed to identify specific residues in the C1 domains that mediate Golgi localization. We demonstrate that the isolated C1b domains from PKCalpha, -delta, -epsilon, -eta, and - are targeted to the Golgi complex, whereas the corresponding C1a domains localize throughout the cell. Sequence alignment showed that amino acid residues corresponding to Glu-246 and Met-267 in PKC are conserved among C1b but absent from C1a domains. Mutation of Met-267, but not of Glu-246, to glycine abolished the Golgi localization of the isolated C1b domain and the regulatory domain of PKC. The mutated PKC regulatory domain constructs lacking Golgi localization were unable to induce apoptosis, suggesting a direct correlation between Golgi localization and apoptotic activity of PKC regulatory domain. Mutation of analogous residues in the C1b domain of PKCepsilon abrogated its Golgi localization, demonstrating that this effect is not restricted to one PKC isoform. The abolished Golgi localization did not affect neurite induction by PKCepsilon. However, the PKCepsilon mutant did not relocate to the Golgi network in response to ceramide and ceramide did not suppress the neurite-inducing capacity of the protein. Thus, the specific mutations in the C1b domain influence both the localization and function of full-length PKCepsilon.     

Translocation of protein kinase Cepsilon and protein kinase Cdelta to membrane is required for ultraviolet B-induced activation of mitogen-activated protein kinases and apoptosis.

UV-induced signal transduction may be involved in tumor promotion and induction of apoptosis. The role of protein kinase C (PKC) in UVB-induced signal transduction is not well understood. This study showed that UVB markedly induced translocation of membrane-associated PKCepsilon and PKCdelta, but not PKCalpha, from cytosol to membrane. Dominant negative mutant (DNM) PKCepsilon or PKCdelta inhibited UVB-induced translocation of PKCepsilon and PKCdelta, respectively. UVB-induced activation of extracellular signal-regulated protein kinases (Erks) and c-Jun NH2-terminal kinases (JNKs) was strongly inhibited by DNM PKCepsilon and PKCdelta, whereas the DNM of PKCalpha was less effective on the UVB-induced phosphorylation of Erks and JNKs. Among the PKC inhibitors used only rottlerin, a selective inhibitor of PKCdelta, markedly inhibited the UVB-induced activation of Erks and JNKs, but not p38 kinases. Safingol, a selective inhibitor for PKCalpha, did not show any inhibitory effect on UVB-induced mitogen-activated protein kinase activation. GF109203X is a stronger inhibitor of classical PKC than novel PKC. Lower concentrations of GF109203X (<10 microM) had no effect on UVB-induced activation of Erks or JNKs. However, at higher concentrations (over 20 microM), GF109203X inhibited UVB-induced activation of JNKs, Erks, and even p38 kinases. Meanwhile, rottlerin and GF109203X markedly inhibited UVB-induced apoptosis of JB6 cells, whereas safingol had little inhibitory effect. DNM-Erk2 cells and PD98059, a selective inhibitor for mitogen-activated protein kinase/extracellular signal-regulated kinase 1 that directly activates Erks, inhibited UVB-induced apoptosis. DNM-JNK1 cells also blocked UVB-induced apoptosis, whereas SB202190, a specific inhibitor for p38 kinases, did not produce the inhibitory effect. These data demonstrate that PKCdelta and PKCepsilon, but not PKCalpha, mediate UVB-induced signal transduction and apoptosis in JB6 cells through activation of Erks and JNKs.     

Dynamics of glucose-induced membrane recruitment of protein kinase C beta II in living pancreatic islet beta-cells

The mechanisms by which glucose may affect protein kinase C (PKC) activity in the pancreatic islet beta-cell are presently unclear. By developing adenovirally expressed chimeras encoding fusion proteins between green fluorescent protein and conventional (betaII), novel (delta), or atypical (zeta) PKCs, we show that glucose selectively alters the subcellular localization of these enzymes dynamically in primary islet and MIN6 beta-cells. Examined by laser scanning confocal or total internal reflection fluorescence microscopy, elevated glucose concentrations induced oscillatory translocations of PKCbetaII to spatially confined regions of the plasma membrane. Suggesting that increases in free cytosolic Ca(2+) concentrations ([Ca(2+)](c)) were primarily responsible, prevention of [Ca(2+)](c) increases with EGTA or diazoxide completely eliminated membrane recruitment, whereas elevation of cytosolic [Ca(2+)](c) with KCl or tolbutamide was highly effective in redistributing PKCbetaII both to the plasma membrane and to the surface of dense core secretory vesicles. By contrast, the distribution of PKCdelta.EGFP, which binds diacylglycerol but not Ca(2+), was unaffected by glucose. Measurement of [Ca(2+)](c) immediately beneath the plasma membrane with a ratiometric "pericam," fused to synaptic vesicle-associated protein-25, revealed that depolarization induced significantly larger increases in [Ca(2+)](c) in this domain. These data demonstrate that nutrient stimulation of beta-cells causes spatially and temporally complex changes in the subcellular localization of PKCbetaII, possibly resulting from the generation of Ca(2+) microdomains. Localized changes in PKCbetaII activity may thus have a role in the spatial control of insulin exocytosis.     

C2 domain of protein kinase C alpha: elucidation of the membrane docking surface by site-directed fluorescence and spin labeling

The C2 domain is a conserved signaling motif that triggers membrane docking in a Ca(2+)-dependent manner, but the membrane docking surfaces of many C2 domains have not yet been identified. Two extreme models can be proposed for the docking of the protein kinase C alpha (PKC alpha) C2 domain to membranes. In the parallel model, the membrane-docking surface includes the Ca(2+) binding loops and an anion binding site on beta-strands 3-4, such that the beta-strands are oriented parallel to the membrane. In the perpendicular model, the docking surface is localized to the Ca(2+) binding loops and the beta-strands are oriented perpendicular to the membrane surface. The present study utilizes site-directed fluorescence and spin-labeling to map out the membrane docking surface of the PKC alpha C2 domain. Single cysteine residues were engineered into 18 locations scattered over all regions of the protein surface, and were used as attachment sites for spectroscopic probes. The environmentally sensitive fluorescein probe identified positions where Ca(2+) activation or membrane docking trigger measurable fluorescence changes. Ca(2+) binding was found to initiate a global conformational change, while membrane docking triggered the largest fluorescein environmental changes at labeling positions on the three Ca(2+) binding loops (CBL), thereby localizing these loops to the membrane docking surface. Complementary EPR power saturation measurements were carried out using a nitroxide spin probe to determine a membrane depth parameter, Phi, for each spin-labeled mutant. Positive membrane depth parameters indicative of membrane insertion were found for three positions, all located on the Ca(2+) binding loops: N189 on CBL 1, and both R249 and R252 on CBL 3. In addition, EPR power saturation revealed that five positions near the anion binding site are partially protected from collisions with an aqueous paramagnetic probe, indicating that the anion binding site lies at or near the surface of the headgroup layer. Together, the fluorescence and EPR results indicate that the Ca(2+) first and third Ca(2+) binding loops insert directly into the lipid headgroup region of the membrane, and that the anion binding site on beta-strands 3-4 lies near the headgroups. The data support a model in which the beta-strands are tilted toward the parallel orientation relative to the membrane surface.     

Possible involvement of microfilaments in protein kinase C translocation

We investigated the role of microfilaments in stimulus-induced translocation of protein kinase C (PKC) in polymorphonuclear leukocytes (PMNs) from C57BL/6 mice. Cytochalasin B and dihydrocytochalasin B almost completely inhibited PKC translocation induced by either TPA or Ca2+ ionophore after pretreatment of cells for 30 min. In addition, ML-9, a potent inhibitor of Ca2+/calmodulin-dependent myosin light chain kinase which regulate microfilament contraction, and a calmodulin antagonist W-7, also inhibited PKC translocation. These findings suggest the possibility that microfilaments are involved in the translocation of PKC.