Simon J. Slater

A novel interaction between perlecan protein core and progranulin: potential effects on tumor growth.

In an in vivo search of novel partners for perlecan, a major heparan sulfate proteoglycan of basement membranes and cell surfaces, we identified progranulin, a secreted growth factor, as a strong interacting protein. Unambiguous interaction, first observed with the yeast two-hybrid system, was corroborated by co-immunoprecipitation studies using cell-free transcription/translation and transient cell transfection assays. The interaction of progranulin with perlecan domain V involved the first two laminin- and epidermal growth factor-like repeats. Within progranulin, the subdomains interacting most with perlecan harbored granulins F and B. Kinetics analysis of the interaction using surface plasmon resonance showed a saturable binding of relative low affinity (KD ∼1 μm). These results were supported by significant expression overlap of these two proteins in a series of ovarian tumor tissue microarrays. Progranulin was present within proliferating blood vessels of ovarian carcinomas and perivascular matrices, with a distribution similar to perlecan. Notably, both progranulin and domain V stimulated the growth of adrenal carcinoma cells. However, when used together in equimolar amounts, the two proteins counteracted each others activity. Thus, progranulin/perlecan interaction could contribute to a fine regulation of tumor angiogenesis and could ultimately affect cancer growth.

Inhibition of protein kinase C by resveratrol

Evidence is emerging that resveratrol (RV), a polyphenolic phytoaxelin present in dietary sources including red wine, may protect against atherosclerosis and cardiovascular disease by enhancing the integrity of the endothelium. In this study, the possibility that such beneficial effects of RV may arise from a modulation of protein kinase C (PKC)-mediated signaling was investigated by determining the effects of RV on the in vitro activities of PKC isozymes. It was found that the Ca(2+)-dependent activities of membrane-associated PKCalpha induced by either phorbol ester or diacylglycerol were potently inhibited by RV, each with an IC(50) of approximately 2 microM. The inhibitory effect of RV was also observed for conventional PKCbetaI, whereas the activities of novel PKC epsilon and atypical PKCzeta were each unaffected. The inhibition of PKCalpha activity was found to be competitive with respect to phorbol ester concentration but noncompetitive with respect to Ca(2+) and phosphatidylserine concentrations, suggesting that the RV may compete for phorbol ester-binding to the C1 domains. Supporting this, it was found that RV bound to a fusion peptide containing the C1A and C1B domains of PKCalpha. Similar to the effects of diacylglycerol and phorbol ester, the interaction of RV with the C1 domains induced the association of PKCalpha with membrane lipid vesicles, although this did not result in activation. Overall, the results suggest that the inhibitory effect of RV on PKC activity, and therefore on the associated signaling networks, may, in part, underlie the mechanism(s) by which this agent exerts its beneficial effects on endothelial and cardiovascular function. Furthermore, the effects of RV on these signaling networks are predicted to differ according to the cellular localization and the regulating PKC isozyme.

Interaction of alcohols and anesthetics with protein kinase Calpha.

The key signal transduction enzyme protein kinase C (PKC) contains a hydrophobic binding site for alcohols and anesthetics (Slater, S. J., Cox, K. J. A., Lombardi, J. V., Ho, C., Kelly, M. B., Rubin, E., and Stubbs, C. D. (1993) Nature 364, 82-84). In this study, we show that interaction of n-alkanols and general anesthetics with PKCα results in dramatically different effects on membrane-associated compared with lipid-independent enzyme activity. Furthermore, the effects on membrane-associated PKCα differ markedly depending on whether activity is induced by diacylglycerol or phorbol ester and also on n-alkanol chain length. PKCα contains two distinct phorbol ester binding regions of low and high affinity for the activator, respectively (Slater, S. J., Ho, C., Kelly, M. B., Larkin, J. D., Taddeo, F. J., Yeager, M. D., and Stubbs, C. D. (1996) J. Biol. Chem. 271, 4627-4631). Short chain n-alkanols competed for low affinity phorbol ester binding to the enzyme, resulting in reduced enzyme activity, whereas high affinity phorbol ester binding was unaffected. Long chain n-alkanols not only competed for low affinity phorbol ester binding but also enhanced high affinity phorbol ester binding. Furthermore, long chain n-alkanols enhanced phorbol ester induced PKCα activity. This effect of long chain n-alkanols was similar to that of diacylglycerol, although the n-alkanols alone were weak activators of the enzyme. The cellular effects of n-alkanols and general anesthetics on PKC-mediated processes will therefore depend in a complex manner on the locality of the enzyme (e.g. cytoskeletal or membrane-associated) and activator type, apart from any isoform-specific differences. Furthermore, effects mediated by interaction with the region on the enzyme possessing low affinity for phorbol esters represent a novel mechanism for the regulation of PKC activity.

The effects of non-lamellar forming lipids on membrane protein-lipid interactions

The role of lipid polymorphism in the regulation of membrane-associated protein function is examined, based on recent studies which showed that changes in the levels of phosphatidylethanolamine (PE), cholesterol and phospholipid unsaturation, modulate the activity of the key signal transduction enzyme, protein kinase C (PKC). It is shown that effects of membrane compositional changes on PKC activity involve a perturbation of protein-lipid interactions with the head group region rather than with the hydrophobic interior of the bilayer. A key determinant in the perturbation of these interactions is suggested to be an elastic curvature energy, termed curvature stress, which results from the unfavorable packing of non-lamellar forming lipids in a planar bilayer. PKC activity is shown to be a biphasic function of curvature stress, with an optimum value of this parameter corresponding to an optimally active PKC conformation. Thus, it is shown that the maximal activity of conformationally distinct PKC isoforms may require a different optimum value of curvature stress. Furthermore, it is hypothesized that curvature stress may have differing effects on the conformation of membrane-associated PKC activity induced by diacylglycerols, phorbol esters or other activators, based on recent studies showing that these agents induce the formation of disparate active conformers of the enzyme.

Polyunsaturation in cell membranes and lipid bilayers and its effects on membrane proteins.

The effect of variation of the degree ofcis-unsaturation on cell membrane protein functioning was investigated using a model lipid bilayer system and protein kinase C (PKC). This protein is a key element of signal transduction. Furthermore it is representative of a class of extrinsic membrane proteins that show lipid dependent interactions with cell membranes. To test for dependence of activity on the phospholipid unsaturation, experiments were devised using a vesicle assay system consisting of phosphatidylcholine (PC) and phosphatidylserine (PS) in which the unsaturation was systematically varied. Highly purified PKCα and ε were obtained using the baculovirus-insect cell expression system. It was shown that increased PC unsaturation elevated the activity of PKCα. By contrast, increasing the unsaturation of PSdecreased the activity of PKCα, and to a lesser extent PKCε. This result immediately rules out any single lipid bilayer physical parameter, such as lipid order, underlying the effect. It is proposed that while PC unsaturation effects are explainable on the basis of a contribution to membrane surface curvature stress, the effects of PS unsaturation may be due to specific protein-lipid interactions. Overall, the results indicate that altered phospholipid unsaturation in cell membranes that occurs in certain disease states such as chronic alcoholism, or by dietary manipulations, are likely to have profound effects on signal transduction pathways involving PKC and similar proteins.

PKCε has an alcohol-binding site in its second cysteine-rich regulatory domain

Alcohols regulate the expression and function of PKC (protein kinase C), and it has been proposed that an alcohol-binding site is present in PKC alpha in its C1 domain, which consists of two cysteine-rich subdomains, C1A and C1B. A PKC epsilon-knockout mouse showed a significant decrease in alcohol consumption compared with the wild-type. The aim of the present study was to investigate whether an alcohol-binding site could be present in PKC epsilon. Here we show that ethanol inhibited PKC epsilon activity in a concentration-dependent manner with an EC50 (equilibrium ligand concentration at half-maximum effect) of 43 mM. Ethanol, butanol and octanol increased the binding affinity of a fluorescent phorbol ester SAPD (sapintoxin-D) to PKC epsilon C1B in a concentration-dependent manner with EC50 values of 78 mM, 8 mM and 340 microM respectively, suggesting the presence of an allosteric alcohol-binding site in this subdomain. To identify this site, PKC epsilon C1B was photolabelled with 3-azibutanol and 3-azioctanol and analysed by MS. Whereas azibutanol preferentially labelled His236, Tyr238 was the preferred site for azioctanol. Inspection of the model structure of PKC epsilon C1B reveals that these residues are 3.46 A (1 A=0.1 nm) apart from each other and form a groove where His236 is surface-exposed and Tyr238 is buried inside. When these residues were replaced by alanine, it significantly decreased alcohol binding in terms of both photolabelling and alcohol-induced SAPD binding in the mutant H236A/Y238A. Whereas Tyr238 was labelled in mutant H236A, His236 was labelled in mutant Y238A. The present results provide direct evidence for the presence of an allosteric alcohol-binding site on protein kinase C epsilon and underscore the role of His236 and Tyr238 residues in alcohol binding.

Fluorescence techniques for probing water penetration into lipid bilayers

Fluorescence spectroscopy can be used as a highly sensitive and localized probe for hydration in lipid bilayers. Water associates with the head-group region, where it participates in an interlipid network of hydrogen bonds. Deeper in the bilayer, water is contained within acyl-chain packing defects. Fluorescence methodology is available to probe both the interstitial and head-group hydration in lipid bilayers, and results are in good agreement with other techniques. Using fluorescence spectroscopic approaches, cholesterol is shown to dehydrate the acyl-chain region, while hydrating the head-group region. Membrane proteins appear to increase acyl-chain hydration at the protein-lipid interface. Overall fluorescence spectroscopic techniques may be most effective in studying the water content of lipid bilayers and especially of biological membranes.

The use of time-resolved fluorescence imaging in the study of protein kinase C localisation in cells

BackgroundTwo-photon-excitation fluorescence lifetime imaging (2P-FLIM) was used to investigate the association of protein kinase C alpha (PKCα) with caveolin in CHO cells. PKCα is found widely in the cytoplasm and nucleus in most cells. Upon activation, as a result of increased intracellular Ca2+ and production of DAG, through G-protein coupled-phospholipase C signalling, PKC translocates to a variety of regions in the cell where it phosphorylates and interacts with many signalling pathways. Due to its wide distribution, discerning a particular interaction from others within the cell is extremely difficultResultsFluorescence energy transfer (FRET), between GFP-PKCα and DsRed-caveolin, was used to investigate the interaction between caveolin and PKC, an aspect of signalling that is poorly understood. Using 2P-FLIM measurements, the lifetime of GFP was found to decrease (quench) in certain regions of the cell from ~2.2 ns to ~1.5 ns when the GFP and DsRed were sufficiently close for FRET to occur. This only occurred when intracellular Ca2+ increased or in the presence of phorbol ester, and was an indication of PKC and caveolin co-localisation under these conditions. In the case of phorbol ester stimulated PKC translocation, as commonly used to model PKC activation, three PKC areas could be delineated. These included PKCα that was not associated with caveolin in the nucleus and cytoplasm, PKCα associated with caveolin in the cytoplasm/perinuclear regions and probably in endosomes, and PKC in the peripheral regions of the cell, possibly indirectly interacting with caveolin.ConclusionBased on the extent of lifetime quenching observed, the results are consistent with a direct interaction between PKCα and caveolin in the endosomes, and possibly an indirect interaction in the peripheral regions of the cell. The results show that 2P-FLIM-FRET imaging offers an approach that can provide information not only confirming the occurrence of specific protein-protein interactions but where they occur within the cell.

Inhibition of membrane lipid-independent protein kinase Calpha activity by phorbol esters, diacylglycerols, and bryostatin-1.

The activity of membrane-associated protein kinase C (PKC) has previously been shown to be regulated by two discrete high and low affinity binding regions for diacylglycerols and phorbol esters (Slater, S. J., Ho, C., Kelly, M. B., Larkin, J. D., Taddeo, F. J., Yeager, M. D., and Stubbs, C. D. (1996) J. Biol. Chem. 271, 4627–4631). PKC is also known to interact with both cytoskeletal and nuclear proteins; however, less is known concerning the mode of activation of this non-membrane form of PKC. By using the fluorescent phorbol ester, sapintoxin D (SAPD), PKCα, alone, was found to possess both low and high affinity phorbol ester-binding sites, showing that interaction with these sites does not require association with the membrane. Importantly, a fusion protein containing the isolated C1A/C1B (C1) domain of PKCα also bound SAPD with low and high affinity, indicating that the sites may be confined to this domain rather than residing elsewhere on the enzyme molecule. Both high and low affinity interactions with native PKCα were enhanced by protamine sulfate, which activates the enzyme without requiring Ca2+ or membrane lipids. However, this “non-membrane” PKC activity was inhibited by the phorbol ester 4β-12-O-tetradecanoylphorbol-13-acetate (TPA) and also by the fluorescent analog, SAPD, opposite to its effect on membrane-associated PKCα. Bryostatin-1 and the soluble diacylglycerol, 1-oleoyl-2-acetylglycerol, both potent activators of membrane-associated PKC, also competed for both low and high affinity SAPD binding and inhibited protamine sulfate-induced activity. Furthermore, the inactive phorbol ester analog 4α-TPA (4α-12-O-tetradecanoylphorbol-13-acetate) also inhibited non-membrane-associated PKC. In keeping with these observations, although TPA could displace high affinity SAPD binding from both forms of the enzyme, 4α-TPA was only effective at displacing high affinity SAPD binding from non-membrane-associated PKC. 4α-TPA also displaced SAPD from the isolated C1 domain. These results show that although high and low affinity phorbol ester-binding sites are found on non-membrane-associated PKC, the phorbol ester binding properties change significantly upon association with membranes.

The use of fluorescent phorbol esters in studies of protein kinase C-membrane interactions.

The family of protein kinase C (PKC) isozymes belongs to a growing class of proteins that become active by associating with membranes containing anionic phospholipids, such as phosphatidylserine. Depending on the particular PKC isoform, this process is mediated by Ca(2+)-binding to a C2 domain and interaction of activators such as 1,2-diacyl-sn-glycerol or phorbol esters with tandem C1 domains. This cooperation between the C1 and C2 domains in inducing the association of PKC with lipid membranes provides the energy for a conformational change that consists of the release of a pseudosubstrate sequence from the active site, culminating in activation. Thus, the properties of the interactions of the C1 and C2 domains with membranes, both as isolated domains, and as modules in the full length PKC isoforms, have been the subject of intense scrutiny. Here, we review the findings of studies in which fluorescent phorbol esters have been utilized to probe the properties of the C1 domains of PKC with respect to the interaction with activators, the subsequent interaction with membranes, and the role of the activating conformational change that leads to activation.