Effects on Ligand Interaction and Membrane Translocation of the Positively Charged Arginine Residues Situated along the C1 Domain Binding Cleft in the Atypical Protein Kinase C Isoforms
2006; Elsevier BV; Volume: 281; Issue: 44 Linguagem: Inglês
10.1074/jbc.m606560200
ISSN1083-351X
AutoresYongmei Pu, Megan L. Peach, Susan H. Garfield, Stephen Wincovitch, Víctor E. Márquez, Peter M. Blumberg,
Tópico(s)HER2/EGFR in Cancer Research
ResumoThe C1 domain zinc finger structure is highly conserved among the protein kinase C (PKC) superfamily members. As the interaction site for the second messenger sn-1,2-diacylglycerol (DAG) and for the phorbol esters, the C1 domain has been an important target for developing selective ligands for different PKC isoforms. However, the C1 domains of the atypical PKC members are DAG/phorbol ester-insensitive. Compared with the DAG/phorbol ester-sensitive C1 domains, the rim of the binding cleft of the atypical PKC C1 domains possesses four additional positively charged arginine residues (at positions 7, 10, 11, and 20). In this study, we showed that mutation to arginines of the four corresponding sites in the C1b domain of PKCδ abolished its high potency for phorbol 12,13-dibutyrate in vitro, with only marginal remaining activity for phorbol 12-myristate 13-acetate in vivo. We also demonstrated both in vitro and in vivo that the loss of potency to ligands was cumulative with the introduction of the arginine residues along the rim of the binding cavity rather than the consequence of loss of a single, specific residue. Computer modeling reveals that these arginine residues reduce access of ligands to the binding cleft and change the electrostatic profile of the C1 domain surface, whereas the basic structure of the binding cleft is still maintained. Finally, mutation of the four arginine residues of the atypical PKC C1 domains to the corresponding residues in the δC1b domain conferred response to phorbol ester. We speculate that the arginine residues of the C1 domain of atypical PKCs may provide an opportunity for the design of ligands selective for the atypical PKCs. The C1 domain zinc finger structure is highly conserved among the protein kinase C (PKC) superfamily members. As the interaction site for the second messenger sn-1,2-diacylglycerol (DAG) and for the phorbol esters, the C1 domain has been an important target for developing selective ligands for different PKC isoforms. However, the C1 domains of the atypical PKC members are DAG/phorbol ester-insensitive. Compared with the DAG/phorbol ester-sensitive C1 domains, the rim of the binding cleft of the atypical PKC C1 domains possesses four additional positively charged arginine residues (at positions 7, 10, 11, and 20). In this study, we showed that mutation to arginines of the four corresponding sites in the C1b domain of PKCδ abolished its high potency for phorbol 12,13-dibutyrate in vitro, with only marginal remaining activity for phorbol 12-myristate 13-acetate in vivo. We also demonstrated both in vitro and in vivo that the loss of potency to ligands was cumulative with the introduction of the arginine residues along the rim of the binding cavity rather than the consequence of loss of a single, specific residue. Computer modeling reveals that these arginine residues reduce access of ligands to the binding cleft and change the electrostatic profile of the C1 domain surface, whereas the basic structure of the binding cleft is still maintained. Finally, mutation of the four arginine residues of the atypical PKC C1 domains to the corresponding residues in the δC1b domain conferred response to phorbol ester. We speculate that the arginine residues of the C1 domain of atypical PKCs may provide an opportunity for the design of ligands selective for the atypical PKCs. The protein kinase C (PKC) 3The abbreviations used are: PKC, protein kinase C; cPKC, conventional PKC; nPKC, novel PKC; aPKC, atypical PKC; PS, phosphatidylserine; DAG, sn-1,2-diacylglycerol; PDBu, phorbol 12,13-dibutyrate; PMA, phorbol 12-myristate 13-acetate; PC, phosphatidylcholine; GST, glutathione S-transferase; CHO, Chinese hamster ovary; GFP, green fluorescent protein. isozymes comprise a family of serine/threonine protein kinases that are involved in the transduction of a large number of signals important for the regulation of proliferation, differentiation, apoptosis, and other physiological functions (1Dempsey E.C. Newton A.C. Mochly-Rosen D. Fields A.P. Reyland M.E. Insel P.A. Messing R.O. Am. J. Physiol. 2000; 279: L429-L438Crossref PubMed Google Scholar, 2Newton A.C. Curr. Biol. 1995; 5: 973-976Abstract Full Text Full Text PDF PubMed Scopus (175) Google Scholar). The family is divided into three subfamilies: 1) the conventional PKCs (cPKCs) α, β I, β II, and γ; 2) the novel PKCs (nPKCs) δ, ϵ, η, and θ; and 3) the atypical PKCs (aPKCs) ζ and ι/λ (human PKCι and mouse PKCλ are orthologs). These three subfamilies share a common requirement for phospholipids for their kinase activity but differ in their dependence on other activators (3Newton A.C. Chem. Rev. 2001; 101: 2353-2364Crossref PubMed Scopus (837) Google Scholar). The cPKCs (α, β I, β II, and γ) meet the original definition of PKC as a Ca2+ and phospholipid-dependent protein kinase. Indeed, they require phosphatidylserine (PS), Ca2+, and diacylglycerol (DAG) (or phorbol esters) for their activation. The nPKCs (δ, ϵ, η, and θ) require only DAG/phorbol ester and PS. The activation of aPKCs (ζ and ι/λ) is much different from that of the cPKCs and nPKCs. They require only PS but not Ca2+ and DAG/phorbol ester (4Violin J.D. Newton A.C. IUBMB Life. 2003; 55: 653-660Crossref PubMed Scopus (38) Google Scholar). As an important second messenger, DAG plays the major role in transducing cellular signals to the PKC molecules (5Brose N. Rosenmund C. Rettig J. Curr. Opin. Neurobiol. 2000; 10: 303-311Crossref PubMed Scopus (179) Google Scholar). A highly conserved cysteine-rich motif (the so-called "C1 domain") in the regulatory region of the PKCs acts as the specific receptor for the DAG signal (6Hurley J.H. Newton A.C. Parker P.J. Blumberg P.M. Nishizuka Y. Protein Sci. 1997; 6: 477-480Crossref PubMed Scopus (321) Google Scholar, 7Hall C. Lim L. Leung T. Trends Biochem. Sci. 2005; 30: 169-171Abstract Full Text Full Text PDF PubMed Scopus (20) Google Scholar). All of the three PKC subfamilies contain this C1 domain. The cPKCs and nPKCs have two tandem C1 domains in their N termini, the C1a and C1b domains. They both showed high binding affinities for DAG or phorbol esters in vitro as isolated fragments (8Slater S.J. Kelly M.B. Taddeo F.J. Rubin E. Stubbs C.D. J. Biol. Chem. 1994; 269: 17160-17165Abstract Full Text PDF PubMed Google Scholar, 9Slater S.J. Ho C. Kelly M.B. Larkin J.D. Taddeo F.J. Yeager M.D. Stubbs C.D. J. Biol. Chem. 1996; 271: 4627-4631Abstract Full Text Full Text PDF PubMed Scopus (122) Google Scholar). However, although the aPKCs, PKCζ as well as PKCι/λ, also possess a C1 domain at their N termini, these atypical C1 domains are not sensitive to either DAG or phorbol esters (10Ways D.K. Cook P.P. Webster C. Parker P.J. J. Biol. Chem. 1992; 267: 4799-4805Abstract Full Text PDF PubMed Google Scholar). The activation mechanisms for the cPKCs and nPKCs have been well studied. Briefly, ligand binding to many receptor tyrosine kinases or G-protein-coupled receptors leads to phospholipase C activation and the consequent production of the DAG second messenger at the plasma membrane. The DAG binds to the C1 domains of cPKC and nPKC, inducing the translocation of PKC to the plasma membrane (3Newton A.C. Chem. Rev. 2001; 101: 2353-2364Crossref PubMed Scopus (837) Google Scholar). The C1-DAG interaction and membrane association trigger a conformational change of the PKC molecule, leading to the release of the pseudosubstrate domain from the catalytic site and the final activation of the enzyme. The activation of the aPKCs also involves the release of the pseudosubstrate domain from the catalytic site of the kinase domain (11Suzuki A. Akimoto K. Ohno S. J. Biochem. (Tokyo). 2003; 133: 9-16Crossref PubMed Scopus (113) Google Scholar, 12Hirai T. Chida K. J. Biochem. (Tokyo). 2003; 133: 1-7Crossref PubMed Scopus (264) Google Scholar, 13Toker A. Front. Biosci. 1998; 3: D1134-D1147Crossref PubMed Google Scholar). However, the molecular mechanism of how their activation process is regulated is not clear. Likewise, little is known about whether the C1 domain plays any role in the activation of the aPKCs. Among its multiple roles in various physiological processes, the important role of the PKC family in tumorigenesis has attracted much attention (14Hofmann J. Curr. Cancer Drug Targets. 2004; 4: 125-146Crossref PubMed Scopus (167) Google Scholar, 15Kazanietz M.G. Biochim. Biophys. Acta. 2005; 1754: 296-304Crossref PubMed Scopus (50) Google Scholar). Many recent studies have found that the atypical PKCs are essentially involved in tumorigenesis. For example, PKCι has recently been found overexpressed in human non-small cell lung cancer cells (16Regala R.P. Weems C. Jamieson L. Copland J.A. Thompson E.A. Fields A.P. J. Biol. Chem. 2005; 280: 31109-31115Abstract Full Text Full Text PDF PubMed Scopus (171) Google Scholar, 17Regala R.P. Weems C. Jamieson L. Khoor A. Edell E.S. Lohse C.M. Fields A.P. Cancer Res. 2005; 65: 8905-8911Crossref PubMed Scopus (235) Google Scholar) and ovarian cancer cells (18Eder A.M. Sui X. Rosen D.G. Nolden L.K. Cheng K.W. Lahad J.P. Kango-Singh M. Lu K.H. Warneke C.L. Atkinson E.N. Bedrosian I. Keyomarsi K. Kuo W.L. Gray J.W. Yin J.C. Liu J. Halder G. Mills G.B. Proc. Natl. Acad. Sci. U. S. A. 2005; 102: 12519-12524Crossref PubMed Scopus (211) Google Scholar, 19Zhang L. Huang J. Yang N. Liang S. Barchetti A. Giannakakis A. Cadungog M.G. O'Brien-Jenkins A. Massobrio M. Roby K.F. Katsaros D. Gimotty P. Butzow R. Weber B.L. Coukos G. Cancer Res. 2006; 66: 4627-4635Crossref PubMed Scopus (113) Google Scholar). It has also been proposed to be an attractive target to develop novel therapeutics against colon cancer (20Murray N.R. Jamieson L. Yu W. Zhang J. Gokmen-Polar Y. Sier D. Anastasiadis P. Gatalica Z. Thompson E.A. Fields A.P. J. Cell Biol. 2004; 164: 797-802Crossref PubMed Scopus (126) Google Scholar) and chronic myelogenous leukemia (21Gustafson W.C. Ray S. Jamieson L. Thompson E.A. Brasier A.R. Fields A.P. J. Biol. Chem. 2004; 279: 9400-9408Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar). PKCζ has been reported recently to be associated with chemotaxis of human breast cancer cells (22Sun R. Gao P. Chen L. Ma D. Wang J. Oppenheim J.J. Zhang N. Cancer Res. 2005; 65: 1433-1441Crossref PubMed Scopus (144) Google Scholar). The atypical PKC is thus viewed as a new promising therapeutic target for cancer treatment. An underlying problem with the inhibition of PKC kinase activity as a therapeutic strategy is achievement of sufficient selectivity among serine/threonine-specific protein kinases with homologous catalytic sites. A complementary strategy that we are pursuing has therefore been to design modulators targeted to the C1 domains (23Marquez V.E. Blumberg P.M. Acc. Chem. Res. 2003; 36: 434-443Crossref PubMed Scopus (90) Google Scholar). Using DAG derivatives in which the flexibility of the structure has been constrained to reduce the entropic loss due to binding, we have actually developed a novel DAG lactone derivative, 130C037, which displayed marked selectivity among the recombinant C1a and C1b domains of PKCα and -δ as well as substantial selectivity for RasGRP (another family of the C1 domain-containing proteins) relative to PKCα (24Pu Y. Perry N.A. Yang D. Lewin N.E. Kedei N. Braun D.C. Choi S.H. Blumberg P.M. Garfield S.H. Stone J.C. Duan D. Marquez V.E. J. Biol. Chem. 2005; 280: 27329-27338Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar). By combining the techniques of computational chemistry and molecular biology, we have also successfully modified the structure of some potent DAG lactones and obtained novel compounds (named dioxolanes), which gained an additional point of contact in their binding with the C1b domain of PKCδ. 4Y. S. Choi, J. H. Kang, M. L. Peach, D. M. Sigano, Y. Pu, N. E. Lewin, S. H. Garfield, S. Wincovitch, P. M. Blumberg, and V. E. Marquez, manuscript in preparation. These previous studies provide encouraging evidence that by manipulating the interactions between the C1 domains and the ligands, we might be able to design DAG derivative compounds that can specifically recognize different C1 domains of different PKC isoforms. The atypical C1 domains are DAG/phorbol ester-insensitive. Although the sequence identity between the PKCδ C1b domain and the atypical C1 domain of PKCζ is only 32%, we can feel confident that these domains adopt the same fold, because the zinc-binding residues (His1, Cys14, Cys17, Cys31, Cys34, His39, Cys42, and Cys50) are conserved, as are critical structural residues Phe3, Gln27, and Val38. Many of the ligand binding residues are also conserved: Trp22, Gly23, and Leu24, along with a conservative substitution of isoleucine for leucine at position 21 (25Kazanietz M.G. Wang S. Milne G.W. Lewin N.E. Liu H.L. Blumberg P.M. J. Biol. Chem. 1995; 270: 21852-21859Abstract Full Text Full Text PDF PubMed Scopus (164) Google Scholar). All of the PKC C1 domains carry a net positive charge, but an interesting characteristic of the atypical C1 domains is that they have an overall positive charge of +9, which is more than twice the average charge of the conventional and novel C1 domains. This is mainly due to four extra arginine residues that line the edges of the binding site: Arg7, Arg10, Arg11, and Arg20. No charged residues are found at these positions in the conventional or novel C1 domains. Do these arginine residues interfere with ligand binding? Can we take advantage of these positively charged arginine residues to design compounds that form specific contacts with them? In this study, using the C1b domain of PKCδ as a template, we mutated the residues Asn7, Ser10, Pro11, and Leu20 in the binding cleft into arginines corresponding to those in the atypical C1 domains. We wanted to study the roles of these arginine residues in phorbol ester binding both in vivo and in vitro, to broaden our general understanding of ligand-C1 domain interactions as well as to develop insights into the unique properties of the C1 domains of the atypical PKCs. We expect that with these atypical C1-like mutants, which still maintain some binding activity for the phorbol esters, we will be able to screen DAG analogue compounds that have been specifically designed for interaction with the arginine residues of the atypical PKC C1 domains. Materials—[20-3H]Phorbol 12,13-dibutyrate ([3H]PDBu) (20 Ci/mmol) was purchased from PerkinElmer. PDBu and phorbol 12-myristate 13-acetate (PMA) were from LC Laboratories (Woburn, MA). PS and phosphatidylcholine (PC) were purchased from Avanti Polar Lipids (Alabaster, AL). Reagents for purification of glutathione S-transferase (GST) fusion proteins were obtained from Pierce. Cell culture medium and reagents were obtained from Invitrogen. The LB broth and agents used for bacteria culture were from K. D. Medical, Inc. (Columbia, MD). The DNA primers were obtained from Invitrogen. Site-directed Mutagenesis of the C1b Domain of PKCδ—Site-directed mutagenesis of the Asn7, Ser10, Pro11, and Leu20 residues was performed in both the GST-δC1b and GFP-δC1b fusion proteins using the QuikChange® II site-directed mutagenesis kit (Stratagene, La Jolla, CA), according to the manufacturer's instructions. The δC1b domain containing-pGEX plasmid and pEGFP plasmid, which had been constructed previously in this laboratory (24Pu Y. Perry N.A. Yang D. Lewin N.E. Kedei N. Braun D.C. Choi S.H. Blumberg P.M. Garfield S.H. Stone J.C. Duan D. Marquez V.E. J. Biol. Chem. 2005; 280: 27329-27338Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar), were used as templates for the mutagenesis reactions. For generation of mutants at multiple sites, the mutagenesis was performed stepwise. The mutations were confirmed by DNA sequence analysis (DNA Minicore, Center for Cancer Research, NCI, National Institutes of Health). Construction of the GFP-fused C1 Domains of PKCζ and PKCι—The C1 domains of the atypical PKCζ and -ι were generated by PCR using the Platinum® Pfx DNA polymerase (Invitrogen). The full-length cDNA clones of human PKCζ and -ι (obtained from OriGene (Rockville, MD)) were used as the templates. The blunt-ended PCR products were ligated into a pCR®-Blunt vector using the Zero Blunt® PCR cloning kit (Invitrogen). The pCR®-Blunt vector was digested with EcoRI (New England BioLabs, Inc., Beverly, MA) to produce adhesive ends of the C1 fragments. These fragments were then ligated into the appropriate pEGFP vectors (Clontech) using the EcoRI restriction sites with the sequence of the insert in the intended reading frame. The DNA sequence of the constructs was confirmed by sequence analysis. Site-directed Mutagenesis of the C1 Domains of PKCζ and PKCι—Site-directed mutagenesis was employed to generate the back mutants of the atypical C1 domains of PKCζ and -ι. Briefly, the arginine residues at positions 7, 10, 11, and 20 were mutated back to the corresponding residues of Asn7, Ser10, Pro11, and Leu20, as in the binding cleft of the δC1b domain. The mutagenesis was performed in the GFP-tagged PKCζ and -ι C1 domains using the QuikChange® II site-directed mutagenesis kit. For generation of the quadruple back mutants at multiple sites, the mutagenesis was performed stepwise. The mutations were confirmed by DNA sequence analysis. Expression and Purification of GST Fusion Proteins from Escherichia coli—The recombinant plasmids containing the arginine mutants of the GST-δC1b domains were expressed and purified from BL-21 E. coli as described elsewhere (24Pu Y. Perry N.A. Yang D. Lewin N.E. Kedei N. Braun D.C. Choi S.H. Blumberg P.M. Garfield S.H. Stone J.C. Duan D. Marquez V.E. J. Biol. Chem. 2005; 280: 27329-27338Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar). [3H]PDBu Binding Assays—Scatchard analysis was performed in this study to determine the dissociation constant (Kd values) of the arginine mutants in binding to [3H]PDBu as described elsewhere (26Lewin N.E. Blumberg P.M. Methods Mol. Biol. 2003; 233: 129-156PubMed Google Scholar). Competitive binding assays were also performed in this study to determine Kd values of some mutants with weak potencies for PDBu binding as described elsewhere (27Wang Q.J. Fang T.W. Nacro K. Marquez V.E. Wang S. Blumberg P.M. J. Biol. Chem. 2001; 276: 19580-19587Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar). Expression and Imaging of the Fluorescent Protein-labeled C1 Domains in Live LNCaP and CHO Cells—LNCaP and CHO-K1 cells (obtained from ATCC, Manassas, VA) were cultured at 37 °C in RPMI 1640 medium or F-12 medium containing 10% fetal bovine serum, penicillin (50 units/ml), and streptomycin (0.05 mg/ml) in a 5% CO2 humidified atmosphere. The plasmids of GFP-fused C1 proteins were transfected into the cells using Lipofectamine 2000 (Invitrogen) according to the manufacturer's instructions. The expression of the fluorescent protein was detected 24 h after transfection. Confocal fluorescent images were collected with a Zeiss MRC 1024 confocal scan head (Zeiss) mounted on a Nikon microscope with a ×60 planapochromat lens as described before (24Pu Y. Perry N.A. Yang D. Lewin N.E. Kedei N. Braun D.C. Choi S.H. Blumberg P.M. Garfield S.H. Stone J.C. Duan D. Marquez V.E. J. Biol. Chem. 2005; 280: 27329-27338Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar). Molecular Modeling—Structures for the single mutants N7R, S10R, P11R, and L20R were generated from the crystal structure of the PKCδ C1b domain (28Zhang G. Kazanietz M.G. Blumberg P.M. Hurley J.H. Cell. 1995; 81: 917-924Abstract Full Text PDF PubMed Scopus (603) Google Scholar) by mutating the selected residue to arginine while preserving the backbone and χ1 angles. A conformational search was performed on the four χ angles of each new arginine residue using 5000 steps of systematic torsional sampling in Macromodel 9.1 (29Mohamadi F. Richards N.G.J. Guida W.C. Liskamp R. Lipton M. J. Comp. Chem. 1990; 11: 440-467Crossref Scopus (3933) Google Scholar). The search used the OPLS 2005 force field with implicit water solvent. Each arginine structure in the resulting set was energy-minimized to convergence at a gradient of 0.05, while fixing the rest of the protein structure in place. Duplicate structures, defined as those with less than a 0.5 Å difference in the arginine side chain atoms, were deleted. Structures for the double, triple, and quadruple mutants were built using the lowest energy conformer found for each single arginine side chain. A structure for a phorbol-binding quadruple mutant was built in the same way, with an alternate conformer of Arg20. A homology model for the PKCζ C1 domain was built on the backbone coordinates of the crystal structure of the PKCδ C1b domain (28Zhang G. Kazanietz M.G. Blumberg P.M. Hurley J.H. Cell. 1995; 81: 917-924Abstract Full Text PDF PubMed Scopus (603) Google Scholar). Side chains were constructed using the program SCWRL (30Bower M.J. Cohen F.E. Dunbrack Jr., R.L. J. Mol. Biol. 1997; 267: 1268-1282Crossref PubMed Scopus (488) Google Scholar), which uses a backbone-dependent rotamer library to place residues in their most likely conformation given the backbone φ-ψ angles at that position. Residues homologous to δC1b were left unchanged from their crystallographic positions, and the binding site arginine residues at positions 7, 10, 11, and 20 were given the same lowest energy conformation found in the conformational search above. The model was energy-minimized with harmonic positional restraints on the backbone atoms to eliminate steric clashes in the side chains without inducing deformations in the backbone. The resulting structure was inspected carefully by hand to ensure that residue packing was reasonable and that the orientations of conserved but nonidentical charged residues were similar to the crystal structure of δC1b. Electrostatic potentials were calculated for the wild-type δC1b, ζC1, and all of the arginine mutants in Grasp (31Nicholls A. Sharp K.A. Honig B. Proteins. 1991; 11: 281-296Crossref PubMed Scopus (5318) Google Scholar), using an interior dielectric of 2, an exterior dielectric of 80, a solvent radius of 1.4 Å, an ionic strength of 0.145 m, and an ionic radius of 2.0 Å. PARSE3 atomic charges and radii were used (32Sitkoff D. Sharp K.A. Honig B. J. Phys. Chem. 1994; 98: 1978-1988Crossref Scopus (1874) Google Scholar), modified slightly to include values for zinc (+2 charge, 0.74 Å radius) and to modify the values for the eight zinc-coordinating residues, which were each given a partial charge of -0.5 to set the net charge for each zinc-binding motif to zero. Compared with the DAG/phorbol ester-responsive PKC C1 domains, one of the most striking characteristics of the atypical PKCs (PKCζ as well as PKCι) is that there are several positively charged arginine residues located in the loops that make up the binding site. As shown in Fig. 1, the Arg residues that line the binding cleft in the atypical C1 domains of the human are Arg7, Arg10, Arg11, and Arg20, whose corresponding residues in the PKCδ C1b domain are Asn7, Ser10, Pro11, and Leu20 (see the three-dimensional structure in Fig. 1B). Arg7 in the atypical C1 domain is polar in other C1 domains (Asn, Thr, and Tyr) and in fact the adjacent residue 6 is positively charged in many C1 domains. In the folded structure, Arg7 is also close to Arg26, which is a conserved positively charged residue across all PKC C1 domains. Thus, this region of the structure is generally already positively charged. Arg10 is at the apex of the first loop and points out away from the binding site. Some non-PKC C1 domains (protein kinase D C1b and RasGRP) also have positively charged Arg or Lys at position 10. We expected that this residue was probably the least likely to interfere with natural ligand binding. According to previous studies (25Kazanietz M.G. Wang S. Milne G.W. Lewin N.E. Liu H.L. Blumberg P.M. J. Biol. Chem. 1995; 270: 21852-21859Abstract Full Text Full Text PDF PubMed Scopus (164) Google Scholar), Pro11 and Leu20 are partially relevant for binding. Mutating these two residues into glycine caused 100- and 15-fold reductions, respectively, in the in vitro binding affinity for PDBu. However, it was not known whether the introduction of Arg residues at these two positions, such as is found in the atypical C1 domains (Arg11 and Arg20), would cause any change in the ligand binding affinity. To explore the role of these Arg residues experimentally, we used the potent phorbol ester-sensitive C1b domain of PKCδ as the template, and we mutated the four residues Asn7, Ser10, Pro11, and Leu20 in the binding cleft into Arg, generating all possible combinations of 1-4 mutated residues. The rationale for our approach was that the δC1b domain is biochemically well behaved and extensively characterized by us both structurally and with regard to its ligand interactions. The site-directed mutagenesis was carried out stepwise. The single-site mutations yielded N7R, S10R, P11R, and L20R; the double-site mutations provided N7R/S10R, N7R/P11R, N7R/L20R, S10R/P11R, S10R/L20R, and P11R/L20R; the triple-site mutations yielded N7R/S10R/P11R, N7R/S10R/L20R, N7R/P11R/L20R, and S10R/P11R/L20R; and finally the quadruple-site mutation yielded N7R/S10R/P11R/L20R. These mutants were fused with GST or GFP for use in either in vitro binding assays or in vivo translocation assays. With the GST-tagged mutants, we determined how the binding affinity for [3H]PDBu in vitro was affected by the Arg residues. Then, with the GFP-tagged mutants, we evaluated the effect of the introduction of the Arg residues on the subcellular distribution of the C1 domain in living cultured mammalian cells in the absence of PMA and its response as a function of time upon the addition of PMA. The Single-site Mutations Showed No to Moderate (24-Fold) Reduction in the Binding Affinity for PDBu; the Dependence of Binding on PS Was Differentially and Significantly Increased—We first examined whether the binding affinity for [3H]PDBu was affected by the introduction of an Arg residue along the rim of the binding cleft. The GST-tagged δC1b mutants were expressed and purified from E. coli. A Scatchard assay was employed to measure the binding affinities (represented by Kd values) of these mutants for [3H]PDBu. Under these in vitro assay conditions, binding is evaluated in the presence of 100 μg/ml phosphatidylserine. Our results demonstrated that all the single mutants N7R, S10R, P11R, and L20R maintained considerably potent binding affinities for the phorbol ester PDBu (Table 1).TABLE 1Binding affinities for PDBu of δC1b and the arginine mutantsReceptorKdΔGΔGiΔG+nmkcal/molkcal/molkcal/molWild type0.33 ± 0.05−12.97N7R1.18 ± 0.07−12.210.76S10R0.32 ± 0.10−12.98−0.01P11R1.19 ± 0.19−12.210.76L20R8.0 ± 1.8−11.071.90N7R/S10R1.58 ± 0.04−12.040.18S10R/P11R1.22 ± 0.17−12.010.21S10R/L20R7.67 ± 0.20−11.10−0.02N7R/P11R33.8 ± 1.8−10.221.23N7R/L20R480 ± 44−8.641.67P11R/L20R374 ± 20−8.791.52N7R/S10R/P11R19.6 ± 2.3−10.540.92N7R/S10R/L20R1360 ± 290−8.032.29S10R/P11R/L20R380 ± 45−8.781.54N7R/P11R/L20RNAN7R/S10R/P11R/L20RNA Open table in a new tab As we expected, the mutation at Ser10 (S10R) had no effect on ligand binding. It showed a Kd value (0.32 ± 0.10 nm) similar to that of the wild-type δC1b (0.33 ± 0.05 nm). Strikingly, the P11R mutant retained potent binding affinity for [3H]PDBu with a Kd value of 1.19 ± 0.19 nm. This Kd was thus only 4-fold weaker than that for the wild-type (Kd = 0.33 ± 0.05 nm). Pro11 is highly conserved across all the typical DAG/phorbol ester-responsive C1 domains but not the atypical C1 domains. Previous studies had shown that mutation of the Pro11 residue into glycine (P11G) reduced the binding affinity for PDBu more than 100-fold (25Kazanietz M.G. Wang S. Milne G.W. Lewin N.E. Liu H.L. Blumberg P.M. J. Biol. Chem. 1995; 270: 21852-21859Abstract Full Text Full Text PDF PubMed Scopus (164) Google Scholar). However, we demonstrated here that replacing the proline with a positively charged Arg residue at this site did not significantly interfere with the phorbol ester interaction. This suggests that it may be the increased flexibility of glycine at this site that interferes with binding rather than a change in the overall structure of the loop. In fact, the backbone angles of this proline in the PKCδ C1b crystal structure (28Zhang G. Kazanietz M.G. Blumberg P.M. Hurley J.H. Cell. 1995; 81: 917-924Abstract Full Text PDF PubMed Scopus (603) Google Scholar) are φ =-58.6, ψ = 118.0, ω =-176.7, which is a β-turn conformation that is compatible with any amino acid. Like Ser10, Asn7 is located in a region of the binding site that can be positively charged. However, unlike Ser10, the replacement of Asn7 with an Arg residue (N7R) slightly decreased the binding affinity (4-fold) for PDBu. The most significant change in PDBu binding occurred with the mutation at site 20. The introduction of an Arg residue to this site caused a 24-fold decrease in the binding affinity for PDBu. The Kd was increased from 0.33 ± 0.05 nm (for the wild type) to 8.0 ± 1.8 nm (for the L20R mutant). In general, we conclude that none of the individual Arg residues along the binding cleft of the atypical C1 domain by itself can account for the loss of the potency of the atypical C1 domains for phorbol ester binding. The binding of phorbol ester to the C1 domain represents a ternary interaction between ligand, C1 domain, and phospholipid. We therefore also examined the effect of the introduction of Arg residues along the rim of the binding cleft on the ability of phospholipid to support phorbol ester binding. We found that the dependence of binding on the proportion of PS in the phospholipids was significantly increased when either Asn7, Pro11, or Leu20 was replaced with an Arg, which is positively charged. The total lipid concentration was fixed at 100 μg/ml, and
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