FRINK Linked Data Fragments server

Matches in SemOpenAlex for { <https://semopenalex.org/work/W2008575184> ?p ?o ?g. }

• W2008575184 endingPage "12146" @default.
• W2008575184 startingPage "12136" @default.
• W2008575184 abstract "Our previous study showed differential subcellular localization of protein kinase C (PKC) δ by phorbol esters and related ligands, using a green fluorescent protein-tagged construct in living cells. Here we compared the abilities of a series of symmetrically substituted phorbol 12,13-diesters to translocate PKC δ. In vitro, the derivatives bound to PKC with similar potencies but differed in rate of equilibration. In vivo, the phorbol diesters with short, intermediate, and long chain fatty acids induced distinct patterns of translocation. Phorbol 12,13–dioctanoate and phorbol 12,13-nonanoate, the intermediate derivatives and most potent tumor promoters, showed patterns of translocation typical of phorbol 12-myristate 13-acetate, with plasma membrane and subsequent nuclear membrane translocation. The more hydrophilic compounds (phorbol 12,13–dibutyrate and phorbol 12,13–dihexanoate) induced a patchy distribution in the cytoplasm, more prominent nuclear membrane translocation, and little plasma membrane localization at all concentrations examined (100 nm to 10 μm). The highly lipophilic derivatives, phorbol 12,13–didecanoate and phorbol 12,13-diundecanoate, at 1 μm caused either plasma membrane translocation only or no translocation at incubation times up to 60 min. Our results indicate that lipophilicity of phorbol esters is a critical factor contributing to differential PKC δ localization and thereby potentially to their different biological activities. Our previous study showed differential subcellular localization of protein kinase C (PKC) δ by phorbol esters and related ligands, using a green fluorescent protein-tagged construct in living cells. Here we compared the abilities of a series of symmetrically substituted phorbol 12,13-diesters to translocate PKC δ. In vitro, the derivatives bound to PKC with similar potencies but differed in rate of equilibration. In vivo, the phorbol diesters with short, intermediate, and long chain fatty acids induced distinct patterns of translocation. Phorbol 12,13–dioctanoate and phorbol 12,13-nonanoate, the intermediate derivatives and most potent tumor promoters, showed patterns of translocation typical of phorbol 12-myristate 13-acetate, with plasma membrane and subsequent nuclear membrane translocation. The more hydrophilic compounds (phorbol 12,13–dibutyrate and phorbol 12,13–dihexanoate) induced a patchy distribution in the cytoplasm, more prominent nuclear membrane translocation, and little plasma membrane localization at all concentrations examined (100 nm to 10 μm). The highly lipophilic derivatives, phorbol 12,13–didecanoate and phorbol 12,13-diundecanoate, at 1 μm caused either plasma membrane translocation only or no translocation at incubation times up to 60 min. Our results indicate that lipophilicity of phorbol esters is a critical factor contributing to differential PKC δ localization and thereby potentially to their different biological activities. protein kinase C 1,2-diacyl-sn-glycerol phorbol 12,13-dibutyrate green fluorescent protein phorbol 12-myristate 13-acetate Chinese hamster ovary dibutyrate dihexanoate dioctanoate dinonanoate didecanoate diundecanoate. Protein kinase C (PKC),1the primary target of the phorbol ester tumor promoters, consists of a family of 12 members that are classified into four subfamilies. The classical PKCs (α, βI, βII, γ) are Ca2+- and 1,2-diacyl-sn-glycerol (DAG)-dependent, whereas the novel PKCs (δ, ε, η, θ) are Ca2+-independent but DAG-responsive. The atypical PKCs (ζ, λ/ι) lack the responses to both Ca2+ and DAG. The fourth subfamily (μ, ν) is most divergent in structure and function from all other PKC members but maintains the C1 domains in the regulatory region that bind DAG (1.Nishizuka Y. Nature. 1984; 308: 693-698Crossref PubMed Scopus (5757) Google Scholar, 2.Nishizuka Y. Nature. 1988; 334: 661-665Crossref PubMed Scopus (3534) Google Scholar, 3.Parker P.J. Dekker L.V. Protein Kinase C. R. G. Landes, Austin, TX1997Google Scholar, 4.Newton A.C. J. Biol. Chem. 1995; 270: 28495-28498Abstract Full Text Full Text PDF PubMed Scopus (1468) Google Scholar). The structural module in PKC that binds DAG and phorbol esters is a 50-amino acid-long lipid-interacting domain that coordinates two Zn2+ ions, termed a C1 domain (5.Hurley J.H. Newton A.C. Parker P.J. Blumberg P.M. Nishizuka Y. Protein Sci. 1997; 6: 477-480Crossref PubMed Scopus (319) Google Scholar). It is expressed in tandem in the regulatory regions of the classical, novel, and PKC μ subfamilies of PKCs (4.Newton A.C. J. Biol. Chem. 1995; 270: 28495-28498Abstract Full Text Full Text PDF PubMed Scopus (1468) Google Scholar). Phorbol esters and their derivatives bind the classical and novel PKCs with high affinities in vitro; their inhibitory equilibrium dissociation constants are in the lower nanomolar range (6.Blumberg P.M. Cancer Res. 1988; 48: 1-8PubMed Google Scholar). Phorbol derivatives with different lipophilicities exhibit different biological activities and potencies. It is known that high tumor promoting activity by phorbol derivatives requires an optimal length of fatty acid side chains. Phorbol 12,13-diC8 is most potent among the symmetrically substituted phorbol 12,13-diesters (25.Schmidt R. Hecker E. Hecker E. Fusenig N.E. Kunz W. Marks F. Thielmann H.W. Cocarcinogenesis and Biological Effects of Tumor Promoters. Raven Press, New York1982: 57-63Google Scholar), and the 12-myristoyl derivative shows the greatest potency among phorbol 12-acyl ester 13-acetate derivatives (7.Hecker E. Cancer Res. 1968; 28: 2338-2348PubMed Google Scholar). Among 12-deoxyphorbol 13-monoesters, the 13-tetradecanoate derivative displays potent tumor promoting activity (8.Zayed S. Sorg B. Hecker E. Planta Medica. 1984; 4: 65-69Crossref Scopus (47) Google Scholar), whereas the 13-phenylacetate and 13-acetate derivatives actually inhibit tumor promotion (9.Szallasi Z. Krsmanovic L. Blumberg P.M. Cancer Res. 1993; 53: 2507-2512PubMed Google Scholar). In in vitro studies, we have reported previously that highly lipophilic phorbol esters inhibit [3H]PDBu binding to cytosolic PKCs from mouse brain homogenate with different potencies when added to the aqueous or lipid phases (10.Sharkey N.A. Blumberg P.M. Cancer Res. 1985; 45: 19-24PubMed Google Scholar); similar high potencies for the different derivatives were seen when applied in the lipid phase. Using a PKC δ fusion to green fluorescent protein (GFP) to visualize PKC δ dynamics in live cells, we reported recently that two 12-deoxyphorbol 13-monoesters, 12-deoxyphorbol 13-tetradecanoate and 12-deoxyphorbol 13-phenylacetate, with similar high affinities for PKC but different lipophilicities, induced distinct patterns of translocation (11.Wang Q.J. Bhattacharyya D. Garfield S. Nacro K. Marquez V.E. Blumberg P.M. J. Biol. Chem. 1999; 274: 37233-37239Abstract Full Text Full Text PDF PubMed Scopus (170) Google Scholar). The promoting derivative 12-deoxyphorbol 13-tetradecanoate closely resembled PMA in its pattern of translocation, whereas the 12-deoxyphorbol 13-phenylacetate, which is antihyperplastic and antipromoting, caused punctate intracellular accumulation of δ-PKC-GFP as well as nuclear membrane localization (11.Wang Q.J. Bhattacharyya D. Garfield S. Nacro K. Marquez V.E. Blumberg P.M. J. Biol. Chem. 1999; 274: 37233-37239Abstract Full Text Full Text PDF PubMed Scopus (170) Google Scholar). Distinct patterns of localization of PKC, by controlling its access to substrates, provide one mechanism driving distinct patterns of biological response to ligands. In the present study, we have focused on one type of phorbol esters, the symmetrically substituted phorbol 12,13-diesters, to systematically explore the influence of hydrophobicity on the dynamics of PKC δ localization in live cells. The effects of acyl ester chain length in these compounds on their inherent binding affinities and on their translocation of PKC δ fusion protein were studied. Our results indicated that the phorbol 12,13-diesters of different lipophilicities translocated PKC δ with distinct patterns and kinetics. PDBu and phorbol 12,13-diC10 were obtained from Alexis Biochemicals (Pittsburgh, PA). Phorbol 12,13-diC6 was from LC Services Corp. (Woburn, MA). Phorbol 12,13-diC8, phorbol 12,13-diC9, and phorbol 12,13-diC11 were synthesized as described by Bresch et al. (12.Bresch H.M. Kreibich G. Kubinyi H. Schairer H.-U. Thielmann H.W. Hecker E. Z. Naturforsch. 1974; 23b: 538-546Google Scholar). Briefly, the phorbol 12,13,20-triesters were prepared from phorbol (LC Services) and the corresponding acid chlorides in the presence of pyridine. The phorbol 12,13,20-triesters were then converted to the phorbol 12,13-diesters by transesterification in methanol in the presence of perchloric acid. The compounds were purified by silica gel chromatography using hexane-ethyl acetate. The octanol/water partition coefficients (log P) were calculated according to the fragment-based program KOWIN 1.63 (Syracuse Research Corp.). CHO-K1 cells (CCL 61) were obtained from the American Type Culture Collection (Manassas, VA). The cells were cultured at 37 °C in Dulbecco's modified Eagle's medium supplemented with 4500 mg/liter glucose, 4 mml-glutamine, 50 units/ml penicillin, 50 μg/ml streptomycin (Advanced Biotechnologies Inc., Columbia, MD), and 10% fetal bovine serum (Life Technologies, Inc.) in a humidified atmosphere containing 5% CO2. This plasmid was prepared as described previously (11.Wang Q.J. Bhattacharyya D. Garfield S. Nacro K. Marquez V.E. Blumberg P.M. J. Biol. Chem. 1999; 274: 37233-37239Abstract Full Text Full Text PDF PubMed Scopus (170) Google Scholar). Briefly, a plasmid pEGFP-N1 encoding the GFP was purchased fromCLONTECH Laboratories, Inc. (Palo Alto, CA). AMluI restriction site was generated by inserting aMluI linker into the plasmid digested with SmaI. A mouse PKC δ cDNA fragment with XhoI andMluI sites was subcloned into the expression vector pEGFP-N1 with the GFP attached to the 3′ end of PKC δ. The junction of PKC δ and GFP in the constructed plasmid was verified by sequencing, which was performed by the DNA minicore, Division of Basic Sciences, NCI, National Institutes of Health. CHO-K1 cells were grown on 40-mm round coverslips (Bioptechs, Inc., Butler, PA) to 50–75% confluence. Transient transfection was conducted using LipofectAMINE Plus (Life Technologies) according to the manufacturer's standard protocol. The fluorescence became detectable 24 h after transfection, and all experiments were performed 3 days after transfection. Prior to observation, transiently transfected CHO-K1 cells were washed twice with standard medium (Dulbecco's modified Eagle's medium without phenol red supplemented with 1% fetal bovine serum) prewarmed to 37 °C. All PKC activators were diluted to the specified concentrations in the same medium, and the final concentration of solvent was always less than 0.01%. For live cell imaging, a Bioptechs Focht Chamber System (FCS2) was inverted and attached to the microscope stage with a custom stage adapter. The cells cultured on a 40-mm round coverslip were introduced into the chamber system, which was connected to a temperature controller set at 37 °C, and medium was perfused through the chamber with a model P720 microperfusion pump (Instech, Plymouth Meeting, PA). As indicated, the perfusate to the chamber was changed to that containing the specified ligand for PKC, and sequential images of the same cell were then collected at 1-min intervals using LaserSharp software through a Bio-Rad MRC 1024 confocal scan head mounted on a Nikon Optiphot microscope with a 60× planapochromat lens. A krypton-argon gas laser provided excitation at 488 nm with a 522/32 emission filter for green fluorescence. [3H]PDBu binding to PKC δ was measured using the polyethylene glycol precipitation assay developed in our laboratory (10.Sharkey N.A. Blumberg P.M. Cancer Res. 1985; 45: 19-24PubMed Google Scholar) with minor modifications. Dissociation constants (K i) of ligands were determined by competition of [3H]PDBu binding to PKC δ. Recombinant PKC δ was expressed in Sf9 insect cells and purified as described previously (13.Kazanietz M.G. Areces L.B. Bahador A. Mischak H. Goodnight J. Mushinski J.F. Blumberg P.M. Mol. Pharmacol. 1993; 44: 298-307PubMed Google Scholar). The assay mixture (250 ml) contained 50 mm Tris-Cl (pH 7.4), 100 μg/ml 100% phosphatidylserine, 4 mg/ml bovine immunoglobulin G, [3H]PDBu, and variable concentrations of competing ligand. Incubation was carried out at 37 °C for 5 min or 30 min. Samples were chilled to 0 °C for 10 min, 200 ml of 35% polyethylene glycol in 50 mm Tris-Cl (pH 7.4) was added, and the samples were incubated at 0 °C for an additional 15 min. The tubes were centrifuged in a Beckman 12 microcentrifuge at 4 °C (12,000 rpm, 15 min). A 100-μl aliquot of the supernatant was removed for the determination of the free concentration of [3H]PDBu, and the pellet was carefully dried. The tip of the centrifuge tube containing the pellet was cut off and transferred to a scintillation vial for the determination of the total bound [3H]PDBu. Aquasol was added both to aliquots of the supernatants and to the pellets, and radioactivity was determined by scintillation counting. Nonspecific binding was measured using an excess of nonradioactive PDBu (30 μm). Specific binding was calculated as the difference between total and nonspecific binding. In a typical competition assay, 6–8 concentrations of the competing ligand were used, ID50 values were determined from the competition curve, and the K i for the competing ligand was calculated from its ID50 using the relationshipK i = ID50/(1 +L/K d), where L is the concentration of free [3H]PDBu and K dis the dissociation constant. For compounds directly applied to the aqueous phase, binding was conducted for 5 or 30 min at 37 °C as indicated. Compounds incorporated into the lipid phase were mixed with the phosphatidylserine in organic solvent, the solvent was removed under a stream of nitrogen, and the compound-phosphatidylserine mixture was resuspended in 50 mm Tris-Cl (pH 7.4) as described (14.Kazanietz M.G. Krausz K.W. Blumberg P.M. J. Biol. Chem. 1992; 267: 20878-20886Abstract Full Text PDF PubMed Google Scholar). The binding was then assayed using a 30-min incubation time at 37 °C. Values represent the mean of n experiments, as indicated, with triplicate determinations of each point in each competition curve in each experiment. The confocal images were processed and analyzed using Scion Image (Scion Corp., Frederick, MD). Fixed small, representative areas in the cytoplasm and nucleus as well as the plasma and nuclear membranes were selected, and the mean fluorescent intensities were determined. The extent of membrane translocation was calculated using the ratio of (I m −I cyto)/I cyto, whereI m represents the mean fluorescent intensity on the plasma or nuclear membrane in a given area, and Icytois the mean fluorescent intensity in a comparable area of the cytoplasm or the nucleoplasm, respectively. The fusion protein δ-PKC-GFP was characterized in our previous study (11.Wang Q.J. Bhattacharyya D. Garfield S. Nacro K. Marquez V.E. Blumberg P.M. J. Biol. Chem. 1999; 274: 37233-37239Abstract Full Text Full Text PDF PubMed Scopus (170) Google Scholar). Briefly, δ-PKC-GFP could be transiently expressed in CHO-K1 cells. It showed the expected molecular size on Western blots and was stable in the presence of ligand for the duration of the experiment. Similar observations have been made by others (15.Ohmori S. Shirai Y. Sakai N. Fujii M. Konishi H. Kikkawa U. Saito N. Mol. Cell. Biol. 1998; 18: 5263-5271Crossref PubMed Google Scholar). We first examined the apparent potencies of the series of phorbol 12,13-diesters with fatty acid side chains varying in length from 4 to 11 carbon atoms for inhibiting [3H]PDBu binding to PKC δ. The lipophilicities of these compounds depend on the length of the fatty acid side chains. Their calculated octanol-water partition coefficients range from a log P value of 3.43 for PDBu (phorbol 12,13-diC4) to 10.31 for phorbol 12,13-diC11 (TableI). To ensure that the compounds were fully incorporated into the lipid phase, we prepared mixed liposomes of phosphatidylserine and ligand and incubated PKC δ for 30 min in the presence of these mixed liposomes to measure binding activity. The phorbol diesters from diC6 to diC11 inhibited [3H]PDBu binding with similar high affinities (Table I). The most hydrophilic compound in the series, phorbol 12,13-diC4, exhibited a modestly higherK i value than that of the others, presumably reflecting in part its release from the liposomes into the aqueous phase under our assay conditions.Table IThe apparent affinities (Ki) of phorbol 12,13-diesters for inhibition of [ 3 H]PDBu binding to PKC δLigandsLogPK i, lipid phase (30-min incubation)K i, aqueous phase30-min incubation5-min incubationnmnmPDBu3.432.40 ± 0.20 (3)1.50 ± 0.13 (3)1.38 ± 0.03 (3)Phorbol 12,13-DiC65.390.26 ± 0.03 (3)0.33 ± 0.05 (3)0.32 ± 0.03 (3)Phorbol 12,13-DiC87.360.21 ± 0.08 (3)0.28 ± 0.04 (4)1.28 ± 0.08 (3)Phorbol 12,13-DiC98.340.19 ± 0.05 (3)0.29 ± 0.02 (3)2.08 ± 0.38 (3)Phorbol 12,13-DiC109.320.41 ± 0.12 (5)0.73 ± 0.21 (5)3.81 ± 0.62 (3)Phorbol 12,13-DiC1110.310.54 ± 0.13 (5)4.62 ± 1.04 (7)15.93 ± 2.68 (3)Ki values of compounds added to the aqueous or lipid phase were determined with 5- or 30-min incubations at 37 °C as specified. Values represent the mean ± S.E. The number of experiments is indicated in parentheses. P, octanol/water partition coefficient. Open table in a new tab Ki values of compounds added to the aqueous or lipid phase were determined with 5- or 30-min incubations at 37 °C as specified. Values represent the mean ± S.E. The number of experiments is indicated in parentheses. P, octanol/water partition coefficient. In biological experiments, ligands are added to the culture medium and must equilibrate with the cell membranes. PDBu and phorbol 12,13-diC6 are the most hydrophilic compounds and should equilibrate rapidly from the aqueous solution. However, compounds with long chain fatty acids, such as phorbol 12,13-diC10 and phorbol 12,13-diC11, dissolve poorly in aqueous solutions as reported (16.Jacobson K. Wenner C.E. Kemp G. Papahadjopoulos D. Cancer Res. 1975; 35: 2991-2995PubMed Google Scholar) and are expected to equilibrate much more slowly from the aqueous solution. Therefore, we determined the apparent binding affinities of the phorbol diesters applied directly into the aqueous phase and assayed with 30- or 5-min times of incubation at 37 °C. As illustrated in Table I, a 30-min incubation time permitted complete equilibration of the diesters with chain lengths up to diC9 (log P = 8.34). The diC10 derivative showed 2-fold weaker apparent affinity than observed when the ligand was added directly to the lipid, and for the diC11 derivative this difference increased to 8.5-fold. If only a 5-min incubation time was used, then complete equilibration was only obtained for diesters with chain lengths up to diC6 (log P = 5.39). For diC11, the apparent affinity was 30-fold weaker than that obtained for the ligand added directly to the lipid. Reflecting that the measurements were not being made under equilibrium conditions, the apparent K i values for phorbol 12,13-diC10 and phorbol 12,13-diC11 showed greater variability when added to the aqueous phase. Based on the in vitro binding analysis, we used similar single doses of phorbol 12,13-diesters to induce the translocation of δ-PKC-GFP (Fig. Figure 1, Figure 2, Figure 3, Figure 4, Figure 5, Figure 6, Figure 7). Distinctive patterns of translocation were observed for phorbol 12, 13-diesters with short, intermediate, and long chain fatty acids.Figure 1PDBu at 1 μm induced translocation of δ-PKC-GFP expressed in CHO-K1 cells.Fluorescent images of CHO cells expressing δ-PKC-GFP after 0-, 5-, 10-, or 20-min treatment of 1 μm PDBu. Images obtained from three independent experiments, specified as 1,2, and 3, are shown. The bottom panels illustrate the line density profile across one cell in a given image. The representative intensity profiles are shown at each time point.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Typical sequential images of δ-PKC-GFP translocation induced by treatment with phorbol diesters are illustrated in Figs. 1,2, 4, 5, 7, and 8. The three series of images presented for each ligand were from three independent experiments to show the range of variability. The same instrument parameters for confocal microscopy were used in all experiments; images were from cells with optimal levels of GFP fusion protein under a laser intensity that gave rise to negligible photobleaching.Figure 4Phorbol 12,13-diC8 (PDiC8) induced translocation of δ-PKC-GFP expressed in CHO-K1 cells at 1 μm. CHO cells expressing δ-PKC-GFP were treated with 1 μmphorbol 12,13-diC8 for 20 min, and images collected at 0-, 5-, 10-, and 20-min time points are shown. Images are from three independent experiments, specified as 1, 2, and 3. The bottom panels illustrate the line density profile across one cell in a given image. The representative intensity profiles are shown at each time point.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Figure 5Phorbol 12,13-diC9 (PDiC9) induced translocation of δ-PKC-GFP expressed in CHO-K1 cells at 1 μm.Fluorescent images of CHO cells expressing δ-PKC-GFP after 0-, 5-, 10-, 20-, and 30-min treatment of 1 μm phorbol 12,13-diC9 are shown. Images were obtained from three independent experiments, specified as 1, 2, and 3. Thebottom panels illustrate the line intensity profile across one cell in a given image. The representative intensity profiles are shown at each time point.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Figure 8Phorbol 12,13-diC11 (PDiC11) induced translocation of δ-PKC-GFP expressed in CHO-K1 cells at 1 μm. CHO cells expressing δ-PKC-GFP were treated with 1 μmphorbol 12,13-diC11 for 60 min, and images at 0, 5, 10, 20, and 60 min of treatment are shown. Data represent images obtained from three independent experiments, specified as 1, 2, and3. The bottom panels illustrate the line density profile across one cell in a given image. The representative intensity profiles are shown at each time point.View Large Image Figure ViewerDownload Hi-res image Download (PPT) As illustrated in Figs. 1 and 2, the more hydrophilic compounds, PDBu and phorbol 12,13-diC6, induced translocation of PKC δ primarily to the nuclear membrane together with patchy cytoplasmic distribution. Both sites of translocation were apparent very rapidly after ligand addition (1–2 min), and the translocation appeared complete within 5–10 min. The nuclear translocation by PDBu at 1 μm was less extensive compared with that by phorbol 12,13-diC6. Higher concentrations (10 μm) of PDBu gave similar results. After a 20-min treatment with PDBu, small portions of δ-GFP-PKC located to the plasma membrane in some cells examined. Phorbol 12,13-diC6 induced some plasma membrane translocation in the majority of cells coincident with the nuclear membrane translocation; this plasma membrane translocation peaked at 10–20 min and gradually disappeared (data not shown). The degree and timing of nuclear translocation induced by 1 μm PDBu and phorbol 12,13-diC6 were quantitated as shown in Fig.3 A. For both ligands, translocation peaked at 10 min, although the degree of translocation caused by phorbol 12,13-diC6 was 4-fold higher compared with that by PDBu. The pattern of this nuclear translocation appeared more transient compared with that of phorbol 12,13-diC8 and phorbol 12,13-diC9 (Fig.3 B). The translocation of δ-PKC-GFP induced by phorbol 12,13-diesters with fatty acid side chains of intermediate length, viz. phorbol 12,13-diC8 and phorbol 12,13-diC9, bore great resemblance to that of PMA, as described previously (11.Wang Q.J. Bhattacharyya D. Garfield S. Nacro K. Marquez V.E. Blumberg P.M. J. Biol. Chem. 1999; 274: 37233-37239Abstract Full Text Full Text PDF PubMed Scopus (170) Google Scholar). As shown in Figs.Figure 4, Figure 5, Figure 6, both phorbol 12,13-diC8 and phorbol 12,13-diC9 (1 μm) generated rapid plasma membrane translocation that peaked at 5 and 10 min, respectively, followed by slower but more extensive nuclear membrane translocation. Between 10 and 20 min, nuclear membrane translocation exceeded the level of translocation to the plasma membrane and continued to rise, while the level of plasma membrane translocation decreased. The kinetics of translocation by phorbol 12,13-diC8 are most comparable with that of PMA (log P= 7.36), in agreement with their similar lipophilicities (Fig. 6). Phorbol 12,13-diesters with long chain fatty acids, phorbol 12,13-diC10 and phorbol 12,13-diC11, showed weak activity in translocating δ-PKC-GFP (Fig. 7 and8). At 1 μm, phorbol 12,13-diC10 caused only plasma membrane translocation visible after 20 min of treatment, while no apparent nuclear membrane translocation was observed even after 45 min of treatment. A 10-fold higher concentration gave a response resembling that of 1 μm phorbol 12,13-diC9. Phorbol 12,13-diC11 failed to translocate δ-PKC-GFP at 1 μm even after 60 min of treatment. Higher concentrations of phorbol 12,13-diC11 (10 and 100 μm) gave weak responses (Fig. 9). Concentrations higher than 100 μm generated signs of cytotoxicity (data not shown). A potential factor contributing to the isozyme- and ligand-specific activities of PKC is their distinct patterns of subcellular localization. The differential localization of various PKC isoforms in live cells in real time has been reported using GFP fused to PKC (15.Ohmori S. Shirai Y. Sakai N. Fujii M. Konishi H. Kikkawa U. Saito N. Mol. Cell. Biol. 1998; 18: 5263-5271Crossref PubMed Google Scholar, 17.Sakai N.K. Sasaki N. Ikegaki Y. Ono Y. Saito N. J. Cell Biol. 1997; 139: 1465-1476Crossref PubMed Scopus (198) Google Scholar, 18.Oancea E. Teruel M.N. Quest F.G.A. Meyer T. J. Cell Biol. 1998; 140: 485-498Crossref PubMed Scopus (291) Google Scholar, 19.Shirai Y. Kashiwagi K. Yagi K. Sakai N. Saito N. J. Cell Biol. 1998; 143: 511-521Crossref PubMed Scopus (123) Google Scholar). Distinctive patterns of translocation of GFP-tagged PKC δ have been reported in response to treatment with ATP, PMA, and H2O2 in CHO-K1 cells (15.Ohmori S. Shirai Y. Sakai N. Fujii M. Konishi H. Kikkawa U. Saito N. Mol. Cell. Biol. 1998; 18: 5263-5271Crossref PubMed Google Scholar). Saito and collaborators have shown that fatty acids as co-activators affect the translocation of PKC-γ and -ε differently (19.Shirai Y. Kashiwagi K. Yagi K. Sakai N. Saito N. J. Cell Biol. 1998; 143: 511-521Crossref PubMed Scopus (123) Google Scholar). Anchoring proteins, RICKs, RACKs, and PICKs, that interact with the inactive and activated PKCs at different subcellular locations have been identified and characterized (20.Mochly-Rosen D. Henrich C.J. Cheever L. Khaner H. Simpson P.C. Mol. Biol. Cell. 1990; 1: 693-706Crossref Scopus (224) Google Scholar, 21.Mochly-Rosen D. Gordon A.S. FASEB J. 1998; 12: 35-42Crossref PubMed Scopus (509) Google Scholar, 22.Hyatt S.L. Liao L. Chapline C. Jaken S. Biochemistry. 1994; 33: 1223-1228Crossref PubMed Scopus (80) Google Scholar, 23.Dong L. Stevens J.L. Jaken S. Cell Growth Differ. 1993; 4: 793-798PubMed Google Scholar) and provide a mechanism for cell type-specific control of localization. In our previous studies, we had described a series of phorbol esters and related compounds with different patterns of biological response that target a GFP fusion protein of PKC δ to different subcellular locations inside the cell. The interaction of phorbol esters with the C1 domain of PKC involves insertion into the hydrophilic cleft within the C1 domain by the hydrophilic head group of the phorbol esters, hydrophobic interaction with the rim of the cleft, and the further hydrophobic interaction between the fatty acid side chains of phorbol esters and the lipid bilayer or proteins associated with it (5.Hurley J.H. Newton A.C. Parker P.J. Blumberg P.M. Nishizuka Y. Protein Sci. 1997; 6: 477-480Crossref PubMed Scopus (319) Google Scholar, 24.Zhang G. Kazanietz M.G. Blumberg P.M. Hurley J.H. Cell. 1995; 81: 917-924Abstract Full Text PDF PubMed Scopus (598) Google Scholar). In the present study, the effect of the latter hydrophobic interaction on the translocation of PKC was addressed. By maintaining the phorbol ester pharmacophore while varying the lipophilicity with different lengths of fatty acid side chains in the phorbol 12,13-diesters, we are able to show that lipophilicity of ligands plays a critical role in the targeting of PKC in live cells. The GFP fusion protein of PKC δ transiently expressed in CHO-K1 cells was shown to be intact and stable and behave as native PKC δ (11.Wang Q.J. Bhattacharyya D. Garfield S. Nacro K. Marquez V.E. Blumberg P.M. J. Biol. Chem. 1999; 274: 37233-37239Abstract Full Text Full Text PDF PubMed Scopus (170) Google Scholar,15.Ohmori S. Shirai Y. Sakai N. Fujii M. Konishi H. Kikkawa U. Saito N. Mol. Cell. Biol. 1998; 18: 5263-5271Crossref PubMed Google Scholar). To better characterize the in vitro pharmacology of the phorbol 12,13-diesters, we tested the binding properties of phorbol 12,13-diesters directly incorporated into the lipid phase or added to the aqueous phase and evaluated at different times of incubation. Phorbol 12,13-diC6 to phorbol 12,13-diC11 showed the same high inherent binding affinities of 0.2–0.3 nm when incorporated into the lipid phase, in contrast to a 50-fold difference when added in the aqueous phase and evaluated after only a 5-min incubation time. Our results indicated that the length of the fatty acid side chains had little effect on the affinity of the phorbol diesters under these assay conditions, although the change of lipophilicity significantly affected its kinetics of interaction with PKC. The more lipophilic the compound was, the longer it needed to equilibrate. The K i for PDBu in our study gave modestly lower affinity than expected (K d = 0.75 nm). The data are consistent with our previous analysis of even more lipophilic phorbol diesters (10.Sharkey N.A. Blumberg P.M. Cancer Res. 1985; 45: 19-24PubMed Google Scholar). In that study, phorbol 12,13-diC10 was shown to equilibrate within 30 min, whereas phorbol 12,13-dioleate, for example, showed a 3000-fold weaker potency when added to the aqueous phase rather than the lipid phase. For the pattern of translocation of PKC δ in response to phorbol diesters differing only in lipophilicity, our core finding is that hydrophilic derivatives caused PKC δ to translocate differently in the cell than did the more hydrophobic derivatives. The hydrophilic derivatives caused reduced plasma membrane translocation and accumulation of PKC δ with a patchy cytoplasmic distribution, as well as the nuclear membrane distribution common to both these derivatives and the more hydrophobic ones. These studies thus begin to reveal differential structural requirements for ligand-driven translocation of PKC δ to different membrane compartments. Our results are consistent with our recently reported observation for a pair of 12-deoxyphorbol 13-monoesters, which likewise differed in lipophilicity, although they were not fully homologous (11.Wang Q.J. Bhattacharyya D. Garfield S. Nacro K. Marquez V.E. Blumberg P.M. J. Biol. Chem. 1999; 274: 37233-37239Abstract Full Text Full Text PDF PubMed Scopus (170) Google Scholar). Whereas 12-deoxyphorbol 13-tetradecanoate (log P = 7.89) translocated PKC δ with a pattern similar to PMA (log P = 7.36), the more hydrophilic derivative 12-deoxyphorbol 13-phenylacetate (logP = 3.45), like phorbol 12,13-diC4 (logP = 3.43), caused patchy cytoplasmic distribution together with nuclear membrane localization. A prediction from the localization studies is that the differential pattern of localization should translate into differential access to substrates and different biology. Limited evidence from the literature supports different biology for more hydrophilic derivatives. The most dramatic example is that 12-deoxyphorbol 13-phenylacetate is an inhibitor of tumor promotion, whereas 12-deoxyphorbol 13-tetradecanoate is a complete tumor promoter (9.Szallasi Z. Krsmanovic L. Blumberg P.M. Cancer Res. 1993; 53: 2507-2512PubMed Google Scholar). Interestingly, Schmidt and Hecker had likewise reported that phorbol 12,13-diacetate, -dipropionate, or -diC4 inhibited promotion by PMA when coapplied (25.Schmidt R. Hecker E. Hecker E. Fusenig N.E. Kunz W. Marks F. Thielmann H.W. Cocarcinogenesis and Biological Effects of Tumor Promoters. Raven Press, New York1982: 57-63Google Scholar). As another example, phorbol 12,13-diC4 was only 5.6-fold less potent than the diC8 derivative for mouse ear inflammation but was 53-fold less potent for tumor promotion (26.Thielmann H.W. Hecker E. Schmidt G.C. Fortschritte der Krebsforschung. Schattauer, Stuttgart1969: 171-179Google Scholar). On the other hand, the structural activity for stimulation of deoxyglucose uptake in chick embryo fibroblasts and binding affinity showed good agreement over a broad range of lipophilicities (27.Driedger P.E. Blumberg P.M. Proc. Natl. Acad. Sci. U. S. A. 1980; 77: 567-571Crossref PubMed Scopus (374) Google Scholar). Obviously, interpretation of complex responses is clouded by the potential role of pharmacokinetics. In addition, different PKC isoforms show different patterns of localization and different ligand dependence for their localization. PKC β1, for example, translocates to the plasma membrane with all derivatives examined. 2Q. J. Wang and P. M. Blumberg, unpublished observations. Responses coupled to PKC β would thus be expected to show different ligand dependence from those coupled to PKC δ. In any case, our current findings argue that this area deserves close examination. A second, not unexpected finding of our studies is that the kinetics of PKC δ translocation in response to ligands is dependent on their lipophilicity. Murphy et al. (28.Murphy T.V. Prountzos C. Kotsonis P. Iannazzo L. Majewski H. Eur. J. Pharmacol. 1999; 381: 77-84Crossref PubMed Scopus (16) Google Scholar) recently showed that the binding of PDBu was similar in intact synaptosomes and in synaptosomal membranes, whereas the rate of binding of PMA in the intact synaptosomes was slower than in the membranes, suggesting that lipophilicity slowed the rate of penetration through the membrane. They further correlated the kinetics with the biology of noradrenaline release. Our results directly demonstrate that the more lipophilic derivatives required longer to induce nuclear translocation. Since the hydrophilic derivatives did not induce plasma membrane translocation consistently, the kinetics of translocation to this site could not be compared. For responses that desensitize rapidly, different rates of onset of a response could be reflected in different absolute extents of response before desensitization. For the highly lipophilic derivatives, we did not explore very long times of incubation because we were concerned that interpretation would be clouded by possible hydrolysis of the compounds to the more hydrophilic 13-monoesters. Comparison of binding activity to PKC and translocation by the symmetrically substituted phorbol 12,13-diesters with their tumor promoting activity (26.Thielmann H.W. Hecker E. Schmidt G.C. Fortschritte der Krebsforschung. Schattauer, Stuttgart1969: 171-179Google Scholar) shows substantial correspondence (Fig.10). The loss of tumor promoting activity at longer chain lengths occurs as the compounds become unable to rapidly equilibrate in aqueous solution. The loss of tumor promoting ability at short chain lengths is reflected in the decreased intrinsic affinity for ligand added directly to the lipid phase. The kinetics of plasma membrane translocation approximately parallel the relative potencies for tumor promotion. Because of its central role in signal transduction, PKC has been an attractive target for drug development, both for cancer chemotherapy and for other applications. Bryostatin 1, a natural product that interacts with the C1 domain, is currently in phase II clinical trials for melanoma, multiple myeloma, renal cell carcinoma, and B-cell chronic lymphocytic leukemia (29.Lilly M. Brown C. Pettit G. Kraft A. Leukemia. 1991; 5: 283-287PubMed Google Scholar). LY333531, a PKC β-selective kinase inhibitor, is currently in clinical trials for vascular proliferation in the retina and kidney associated with diabetes (30.Ishii H. Jirousek M.R. Koya D. Takagi C. Xia P. Clermont A. Bursell S.E. Kern T.S. Ballas L.M. Heath W.F. Stramm L.E. Feener E.P. King G.L. Science. 1996; 272: 728-731Crossref PubMed Scopus (1081) Google Scholar, 31.Ishii H. Koya D. King G.L. J. Mol. Med. 1998; 76: 21-31Crossref PubMed Scopus (256) Google Scholar). Design of drugs with maximal selectivity for a specific PKC pathway remains a major objective. For ligands targeted to the regulatory domain, the cellular context in which PKC is found has emerged as a dramatic determinant of specificity. Thus, differences of 2 orders of magnitude in isotype selectivity were found between in vitro binding assays and translocation in cultured cells (32.Kazanietz M.g. Areces L.B. Bahador A. Mischak H. Goodnight J. Mushinski J.F. Blumberg P.M. Mol. Pharmacol. 1993; 44: 298-307PubMed Google Scholar, 33.Szallasi Z. Kosa K. Smith C.B. Dlugosz A.A. Williams E.K. Yuspa S.H. Blumberg P.M. Mol. Pharmacol. 1995; 47: 258-265PubMed Google Scholar, 34.Szallasi Z. Smith C.B. Blumberg P.M. J. Biol. Chem. 1994; 269 (17162): 27159Abstract Full Text PDF PubMed Google Scholar). Similarly, marked differences in selectivity for PKC translocation were found between different cell types (35.Szallasi Z. Smith C.B. Pettit G.R. Blumberg P.M. J. Biol. Chem. 1994; 269: 2118-2124Abstract Full Text PDF PubMed Google Scholar, 36.Szallasi Z. Denning M.F. Smith C.B. Dlugosz A.A. Yuspa S.H. Pettit G.R. Blumberg P.M. Mol. Pharmacol. 1994; 46: 840-850PubMed Google Scholar). Those studies assessed translocation in terms of a shift for the cytosol to the particulate fraction. Our current findings demonstrate further opportunity for selectivity, by controlling the specific site to which PKC is translocated." @default.
• W2008575184 created "2016-06-24" @default.
• W2008575184 creator A5007763690 @default.
• W2008575184 creator A5009584633 @default.
• W2008575184 creator A5010714518 @default.
• W2008575184 creator A5040455211 @default.
• W2008575184 creator A5048501263 @default.
• W2008575184 creator A5074142145 @default.
• W2008575184 creator A5084821891 @default.
• W2008575184 date "2000-04-01" @default.
• W2008575184 modified "2023-10-03" @default.
• W2008575184 title "The Lipophilicity of Phorbol Esters as a Critical Factor in Determining the Pattern of Translocation of Protein Kinase C δ Fused to Green Fluorescent Protein" @default.
• W2008575184 cites W1503469366 @default.
• W2008575184 cites W1522765298 @default.
• W2008575184 cites W1606913060 @default.
• W2008575184 cites W1926511387 @default.
• W2008575184 cites W1966419887 @default.
• W2008575184 cites W1972223792 @default.
• W2008575184 cites W1993282564 @default.
• W2008575184 cites W2001692359 @default.
• W2008575184 cites W2010994547 @default.
• W2008575184 cites W2013539562 @default.
• W2008575184 cites W2019705381 @default.
• W2008575184 cites W2020192398 @default.
• W2008575184 cites W2027235796 @default.
• W2008575184 cites W2066489411 @default.
• W2008575184 cites W2079097093 @default.
• W2008575184 cites W2089475281 @default.
• W2008575184 cites W2137882621 @default.
• W2008575184 cites W2147956365 @default.
• W2008575184 cites W4232940172 @default.
• W2008575184 cites W4243517899 @default.
• W2008575184 doi "https://doi.org/10.1074/jbc.275.16.12136" @default.
• W2008575184 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/10766849" @default.
• W2008575184 hasPublicationYear "2000" @default.
• W2008575184 type Work @default.
• W2008575184 sameAs 2008575184 @default.
• W2008575184 citedByCount "71" @default.
• W2008575184 countsByYear W20085751842012 @default.
• W2008575184 countsByYear W20085751842013 @default.
• W2008575184 countsByYear W20085751842014 @default.
• W2008575184 countsByYear W20085751842015 @default.
• W2008575184 countsByYear W20085751842016 @default.
• W2008575184 countsByYear W20085751842017 @default.
• W2008575184 countsByYear W20085751842018 @default.
• W2008575184 countsByYear W20085751842019 @default.
• W2008575184 countsByYear W20085751842020 @default.
• W2008575184 countsByYear W20085751842021 @default.
• W2008575184 crossrefType "journal-article" @default.
• W2008575184 hasAuthorship W2008575184A5007763690 @default.
• W2008575184 hasAuthorship W2008575184A5009584633 @default.
• W2008575184 hasAuthorship W2008575184A5010714518 @default.
• W2008575184 hasAuthorship W2008575184A5040455211 @default.
• W2008575184 hasAuthorship W2008575184A5048501263 @default.
• W2008575184 hasAuthorship W2008575184A5074142145 @default.
• W2008575184 hasAuthorship W2008575184A5084821891 @default.
• W2008575184 hasBestOaLocation W20085751841 @default.
• W2008575184 hasConcept C104317684 @default.
• W2008575184 hasConcept C121332964 @default.
• W2008575184 hasConcept C12554922 @default.
• W2008575184 hasConcept C138626823 @default.
• W2008575184 hasConcept C142613039 @default.
• W2008575184 hasConcept C184235292 @default.
• W2008575184 hasConcept C185592680 @default.
• W2008575184 hasConcept C186518008 @default.
• W2008575184 hasConcept C195794163 @default.
• W2008575184 hasConcept C201399114 @default.
• W2008575184 hasConcept C3017695315 @default.
• W2008575184 hasConcept C55493867 @default.
• W2008575184 hasConcept C62520636 @default.
• W2008575184 hasConcept C86803240 @default.
• W2008575184 hasConcept C91881484 @default.
• W2008575184 hasConcept C95444343 @default.
• W2008575184 hasConcept C97029542 @default.
• W2008575184 hasConceptScore W2008575184C104317684 @default.
• W2008575184 hasConceptScore W2008575184C121332964 @default.
• W2008575184 hasConceptScore W2008575184C12554922 @default.
• W2008575184 hasConceptScore W2008575184C138626823 @default.
• W2008575184 hasConceptScore W2008575184C142613039 @default.
• W2008575184 hasConceptScore W2008575184C184235292 @default.
• W2008575184 hasConceptScore W2008575184C185592680 @default.
• W2008575184 hasConceptScore W2008575184C186518008 @default.
• W2008575184 hasConceptScore W2008575184C195794163 @default.
• W2008575184 hasConceptScore W2008575184C201399114 @default.
• W2008575184 hasConceptScore W2008575184C3017695315 @default.
• W2008575184 hasConceptScore W2008575184C55493867 @default.
• W2008575184 hasConceptScore W2008575184C62520636 @default.
• W2008575184 hasConceptScore W2008575184C86803240 @default.
• W2008575184 hasConceptScore W2008575184C91881484 @default.
• W2008575184 hasConceptScore W2008575184C95444343 @default.
• W2008575184 hasConceptScore W2008575184C97029542 @default.
• W2008575184 hasIssue "16" @default.
• W2008575184 hasLocation W20085751841 @default.
• W2008575184 hasOpenAccess W2008575184 @default.
• W2008575184 hasPrimaryLocation W20085751841 @default.
• W2008575184 hasRelatedWork W1518872013 @default.
• W2008575184 hasRelatedWork W1977525265 @default.
• W2008575184 hasRelatedWork W1979774143 @default.

• next