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Role of Phorbol Ester Localization in Determining Protein Kinase C Or Rasgrp3 Translocation: Real-Time Analysis Using Fluorescent Ligands and Proteins

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Role of Phorbol Ester Localization in Determining Protein Kinase C Or Rasgrp3 Translocation: Real-Time Analysis Using Fluorescent Ligands and Proteins Molecular Cancer Therapeutics 141 Role of phorbol ester localization in determining protein kinase C or RasGRP3 translocation: Real-time analysis using fluorescent ligands and proteins Derek C. Braun,1,2 Yeyu Cao,4 Shaomeng Wang,4 findings argue that the rate of uptake of phorbol esters Susan H. Garfield,3 Gang Min Hur,2 influences the subsequent pattern of PKCy translocation, and Peter M. Blumberg2 and that the specificity for PKCa translocation is dominated by factors other than the localization of the 1 Department of Biology, Gallaudet University, Washington, ligand. [Mol Cancer Ther 2005;4(1):141–50] District of Columbia; Laboratories of 2Cellular Carcinogenesis and Tumor Promotion and 3Experimental Carcinogenesis, National Cancer Institute, NIH, Bethesda, Maryland; 4Departments of Internal Medicine and Medicinal Chemistry and Comprehensive Introduction Cancer Center, University of Michigan, Ann Arbor, Michigan The lipophilic second messenger sn-1,2-diacylglycerol (DAG) regulates a broad range of cellular functions, Abstract including tumor promotion, apoptosis, differentiation, The diacylglycerol signaling pathway, involving protein and growth. The responses to DAG are mediated by a kinase C (PKC) and RasGRP, is a promising therapeutic superfamily of receptors that include the protein kinase C target for both cancer and other indications. The phorbol (PKC) family of serine/threonine kinases (1, 2) and the esters, ultrapotent diacylglycerol analogues, bind to and RasGRP family of Ras guanyl nucleotide exchange factors activate PKC and RasGRP. Here, using fluorescent (3–6). The DAG-sensitive isozymes of PKC include the h h phorbol esters and complementary fluorescent PKC and calcium-dependent classic PKCs (a, I, II, g) and the y q RasGRP constructs, we determined the localization of the calcium-independent novel PKCs ( , , D, u; 1). The DAG phorbol ester as a function of time after addition and how receptors each contain one or more highly conserved C1 the resultant PKC or RasGRP3 translocation related to domains, zinc finger motifs that act as the binding site for ligand localization. For these studies, we prepared DAG and the phorbol esters, ultrapotent analogues of fluorescently labeled phorbol esters of varying lipo- DAG (7, 8). philicities based on the BODIPY FL (green) or BODIPY Because of its roles in cell proliferation, differentiation, 581/591 (red) fluorophores, and by using fusion con- and apoptosis, PKC has attracted considerable interest as a structs of green fluorescent protein or DsRed with PKC potential target for cancer chemotherapy. Overexpression isoforms or RasGRP3 expressed in Chinese hamster of specific PKC isoforms can cause malignant transforma- ovary cells, we simultaneously compared the kinetics tion of cells, and altered expression of specific PKC and pattern of localization of PKC or RasGRP3 with that isoforms has been described in multiple tumor types (9). of the fluorescent red or green phorbol esters. Binding The effects of PKC are isoform, cell type, and context y assays showed that the fluorescent derivatives were specific. For example, in glioma cells PKC is proapoptotic potent ligands. Uptake followed a one-compartment in response to etoposide but antiapoptotic in response to pharmacokinetic model with a half-time of minutes to Sindbis virus (10). Similarly, PKCa induces hormone- hours, depending on the ligand, and all of the fluorescent independent proliferation in breast cancer (11) but inhibits phorbol esters localized primarily to intracellular mem- proliferation in malignant melanoma (12). In numerous branes, with little plasma membrane localization. The systems, PKCs also stimulate multidrug resistance (13). The fluorescent phorbol esters induced translocation of and context-dependent role of individual PKC isozymes in generally colocalized with PKCy or RasGRP3. However, different cell types affords wide opportunities for design of PKCa and, initially, PKCy, translocated to the plasma drug specificity. membrane, in which little phorbol ester accumulated. The Currently, several chemotherapeutic agents targeting PKC are in various stages of development. There are both examples of agents that activate or inhibit PKC, and some show isoform selectivity. These agents include the PKC activators bryostatin 1, phorbol 12-myristate 13-acetate Received 8/4/04; revised 10/15/04; accepted 11/8/04. (PMA), and ingenol 3-angelate (14–16); the antisense The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked oligonucleotides aprinocarsen and Affinitak (LY900003; advertisement in accordance with 18 U.S.C. Section 1734 solely to ISIS 2521; ref. 17, 18); and agents that potentiate cytotoxic indicate this fact. drugs, such as UCN01, PKC412, and tamoxifen (9, 19). Data Requests for reprints: Peter M. Blumberg, National Cancer Institute, NIH, on the Adriamycin derivative AD 198 show specific PKC Building 37, Room 4048, 37 Convent Drive, MSC 4255, Bethesda, MD 20892-4255. Phone: 301-496-3189; Fax: 301-496-8709. targeting and suggest an improved therapeutic profile over E-mail: [email protected] traditional anthracyclines (20). Finally, drugs targeting PKC Copyright C 2005 American Association for Cancer Research. are being evaluated for pathologies other than cancer: these Mol Cancer Ther 2005;4(1). January 2005 Downloaded from mct.aacrjournals.org on September 28, 2021. © 2005 American Association for Cancer Research. 142 Localization of Phorbol Ester and PKC or RasGRP3 include LY333531 for diabetic retinopathy and renopathy cultured at 37jC in Kaighn’s modification of Ham’s F12 (21) and prostratin and 12-deoxyphorbol 13-phenylacetate medium adjusted to contain 1,500 mg/L of sodium for HIV in conjunction with highly active antiretroviral bicarbonate (ATCC), and supplemented with 10% fetal therapy (22, 23). bovine serum (FBS, Invitrogen) in a humidified atmosphere The hallmark of activation by DAG or phorbol ester is the containing 5% CO2. Cultured cells were grown to 90% translocation of the receptor to a subcellular compartment or confluence before transfection. Transient transfection was membrane, in which the activated receptor may phosphor- done with LipofectAMINE 2000 (Invitrogen) according to ylate substrates or interact with target proteins in a location- the manufacturer’s directions. All experiments were done dependent fashion. Fusion proteins between green fluores- the day after transfection. Escherichia coli DH5a-MCR was cent protein (GFP) and PKC have proven of great value for grown at 37jC in Luria-Bertani medium, either in broth or characterizing this translocation (24). The sites of translo- on agar. Kanamycin was used in Luria-Bertani medium at cation of receptor as well as the rapidity of translocation a concentration of 50 Ag/mL and ampicillin at 100 Ag/mL. following treatment with phorbol ester both differ depend- Recombinant DNA transformation of E. coli DH5a-MCR ing upon the specific phorbol ester (25–27). Using a series was done according to the heat-shock protocol (29). of symmetrical phorbol esters that varied in acyl chain Construction of Plasmids length and lipophilicity, our laboratory previously showed GFP fusions of PKCa, PKCy, and RasGRP3 had been that the pattern of PKCy translocation in response to constructed previously in our laboratory (6, 29). We phorbol ester treatment was dependent on the lipophilicity constructed DsRed fusions by amplifying PKCa, PKCy, of the phorbol ester. The lipophilic phorbol esters induced and RasGRP3 from their parent plasmids, using primers initial translocation of GFP-tagged PKCy to the plasma with compatible flanking restriction sites and then inserting membrane, whereas the more hydrophilic phorbol esters re- the amplicons into either pDsRed-N1 or pDsRed2-N1 sulted in initial translocation to the nuclear membrane (26). (Clontech, Palo Alto, CA). Successful construction was Localization of activated PKCs, as well as their persis- verified by restriction digestion, sequencing, and functional tence within a location, should determine their access to testing to see if the novel plasmids, expressed in CHO-K1 different substrates and thereby may affect their selectivity cells, exhibited translocation of fusion protein after treat- of activation of downstream cellular pathways. A better ment of the cells with 1 Amol/L PMA. understanding of the factors driving PKC and RasGRP3 Molecular Biology Methods translocation may allow us to better understand how Plasmid DNA was purified chromatographically by different phorbol esters show selectivity in their activation using commercial kits from Qiagen (Valencia, CA). of downstream cellular pathways although they may Polymerase chain reactions were done with PfuUltra exhibit similar in vitro binding affinities. This knowledge, high-fidelity DNA polymerase (Stratagene, La Jolla, CA) in turn, should facilitate the design of more selective drug according to the manufacturer’s directions. candidates targeted to the DAG signaling pathway (28). Binding of [3H]PDBu In this study, we did real-time analysis of the pharma- [3H]PDBu binding was measured by using the polyeth- cokinetics and intracellular migration of phorbol esters by ylene glycol precipitation assay developed by our labora- using a series of brightly fluorescent phorbol ester tory (30). Dissociation constants (Ki)ofligandswere derivatives. These fluorescent derivatives have allowed us determined by competition of [3H]PDBu binding to to visualize the entry
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