Location and Functional Significance of Retinol-binding Sites on the Serine/Threonine Kinase, c-Raf
2005; Elsevier BV; Volume: 280; Issue: 8 Linguagem: Inglês
10.1074/jbc.m412695200
ISSN1083-351X
AutoresBeatrice Hoyos, Sulin Jiang, Ulrich Hämmerling,
Tópico(s)Antioxidant Activity and Oxidative Stress
ResumoRedox activations of serine/threonine kinases represent alternate pathways in which vitamin A plays a crucial co-factor role. Vitamin A binds the zinc finger domain of c-Raf with nanomolar affinity. The retinoid-binding site has been mapped within this structure by scanning mutagenesis. The deduced contact sites were found anchored on Phe-8, counting from the 1st conserved histidine of the zinc finger. These sites agreed with contact amino acids identified by computational docking. The boundaries of a related binding pocket were identified by mutagenesis and partially confirmed by docking trials in the protein kinase C-α C1A zinc finger. They comprised Phe-7, Phe-8, and Trp-22. This trio was absent from the αC1B domain, explaining why the latter did not bind retinol. Reconfiguring at a minimum the two corresponding amino acids of αC1B, Thr-7 and Tyr-22, to conform to αC1A converted this domain to a binder. Deletion of the predicted retinoid-binding site in the full-length molecule created a mutant c-Raf that was deficient in retinol-dependent redox activation but fully responsive to epidermal growth factor. Our findings indicate that ligation of retinol to a specific site embedded in the regulatory domain is an important feature of c-Raf regulation in the redox pathway. Redox activations of serine/threonine kinases represent alternate pathways in which vitamin A plays a crucial co-factor role. Vitamin A binds the zinc finger domain of c-Raf with nanomolar affinity. The retinoid-binding site has been mapped within this structure by scanning mutagenesis. The deduced contact sites were found anchored on Phe-8, counting from the 1st conserved histidine of the zinc finger. These sites agreed with contact amino acids identified by computational docking. The boundaries of a related binding pocket were identified by mutagenesis and partially confirmed by docking trials in the protein kinase C-α C1A zinc finger. They comprised Phe-7, Phe-8, and Trp-22. This trio was absent from the αC1B domain, explaining why the latter did not bind retinol. Reconfiguring at a minimum the two corresponding amino acids of αC1B, Thr-7 and Tyr-22, to conform to αC1A converted this domain to a binder. Deletion of the predicted retinoid-binding site in the full-length molecule created a mutant c-Raf that was deficient in retinol-dependent redox activation but fully responsive to epidermal growth factor. Our findings indicate that ligation of retinol to a specific site embedded in the regulatory domain is an important feature of c-Raf regulation in the redox pathway. The history of vitamin A research contains a medley of observations concerning widespread physiological roles of retinoids other than the well known functions of retinoic acid in transcription and retinaldehyde in vision (reviewed in Ref. 1Sporn M.B. Roberts A.B. Goodman D.S. The Retinoids, Biology, Chemistry and Medicine. Second Ed. Raven Press, Ltd., New York1994: 521-658Google Scholar). Most convincing for the non-nuclear functions of vitamin A are arguments pointing to the evolution of an elaborate retinoid biochemistry and biology in eukaryotic organisms (2Maden M. Acta Biotheor. 1993; 41: 425-445Crossref PubMed Scopus (16) Google Scholar), predating by far the advent of retinoid retinoic acid receptors and retinoid X receptors and the conservation of the vitamin A metabolites, along with the requisite enzymes, from insects to man. Furthermore, essentially all nucleated cells of higher vertebrates store vitamin A in the form of retinyl esters for ready retrieval and conversion to a variety of metabolites. Because these retinoid products, more often than not, exclude retinoic acid, the question arises as to their purpose. The multitude of defects caused by nutritional vitamin A deficiency, not completely reversible by retinoic acid and ranging from multiple developmental abnormalities (3Wilson J.G. Roth C.B. Warkany J. Am. J. Anat. 1953; 92: 189-217Crossref PubMed Scopus (621) Google Scholar, 4Armstrong R.B. Ashenfelter K.O. Eckhoff C. Levin A.A. Shapiro S.S. Sporn M.B. Roberts A.B. Goodman D.S. The Retinoids, Biology, Chemistry and Medicine. Second Ed. Raven Press, Ltd., New York1994: 545-752Google Scholar), to immune defects (5Wolbach S.B. Howe P.R. J. Exp. Med. 1925; 42: 753-763Crossref PubMed Scopus (1083) Google Scholar, 6Green H.N. Mellanby E. Br. J. Exp. Pathol. 1930; 11: 81-89Google Scholar, 7Lassen H.C.A. J. Hyg. 1930; 30: 300-310Crossref PubMed Scopus (12) Google Scholar, 8Ross A.C. Hammerling U. Sporn M.B. Roberts A. Goodman D.W. The Retinoids, Biology, Chemistry and Medicine. Second Ed. Raven Press, Ltd., New York1994: 521-543Google Scholar), and to male sterility (9Coward W. Howell J.M. Thompson J.N. Pitt G.A. Br. J. Nutr. 1969; 23: 619-626Crossref PubMed Scopus (17) Google Scholar, 10van Pelt A.M. deRooij D.G. Endocrinology. 1991; 128: 697-704Crossref PubMed Scopus (174) Google Scholar), was not explainable by a single non-nuclear target. In fact, multiple molecular targets emerged when the serine/threonine kinases were found by us to harbor high affinity retinoid-binding sites (11Hoyos B. Imam A. Chua R. Swenson C. Tong G.C. Levi E. Noy N. Hammerling U. J. Exp. Med. 2000; 192: 835-846Crossref PubMed Scopus (67) Google Scholar). These were encoded within the cysteinerich domains of several PKC 1The abbreviations used are: PKC, protein kinase C; cys, cysteinerich domain; EGF, epidermal growth factor; GST, glutathione-S-transferase; WT, wild type; GTPγS, guanosine 5′-3-O-(thio)triphosphate; MEK, mitogen-activated protein kinase/extracellular signal-regulated kinase kinase; ELISA, enzyme-linked immunosorbent assay. 1The abbreviations used are: PKC, protein kinase C; cys, cysteinerich domain; EGF, epidermal growth factor; GST, glutathione-S-transferase; WT, wild type; GTPγS, guanosine 5′-3-O-(thio)triphosphate; MEK, mitogen-activated protein kinase/extracellular signal-regulated kinase kinase; ELISA, enzyme-linked immunosorbent assay. isoforms and c-Raf and overlapped with known structures intimately involved with kinase regulation. This is where lipid second messengers bind and activate the conventional and novel PKC isoforms (12Kazanietz M. Wang S. Milne G.W.A. Lewin N.E. Liu H.L. Blumberg P.M. J. Biol. Chem. 1995; 270: 21852-21859Abstract Full Text Full Text PDF PubMed Scopus (162) Google Scholar, 13Ono Y. Fuji T. Igarashi K. Takayoshi K. Tanaka C. Kikkawa U. Nishizuka Y. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 4868-4871Crossref PubMed Scopus (391) Google Scholar, 14Burns D.J. Bell R.M. J. Biol. Chem. 1991; 266: 18330-18338Abstract Full Text PDF PubMed Google Scholar) and where, in c-Raf, a crucial half-site is located for recognition of the activating GTP/Ras protein (15Gosh S. Xie W.Q. Quest A.F.G. Mabrouk G.M. Strum J.C. Bell R.M. J. Biol. Chem. 1994; 269: 10000-10007Abstract Full Text PDF PubMed Google Scholar, 16Gosh S. Bell R.M. J. Biol. Chem. 1994; 260: 30785-30788Abstract Full Text PDF Google Scholar, 17Williams J.G. Drugan J.K. Yi G.S. Clark G.J. Der C.J. Campbell S.L. J. Biol. Chem. 2000; 275: 22172-22179Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar). Nevertheless, the purpose of retinoid-binding sites remained elusive as the classical receptor tyrosine kinase pathways leading to PKC and c-Raf activation operated independently of vitamin A. With the discovery of the alternate pathway of serine/threonine kinase activation via reactive oxygen species (18Gopalakrishna R. Anderson W. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 6758-6762Crossref PubMed Scopus (368) Google Scholar, 19Konishi H. Tanaka M. Takemura Y. Matsuzaki H. Ono Y. Kikkawa U. Kikkawa N.Y. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 11233-11237Crossref PubMed Scopus (534) Google Scholar), this situation changed. We could show that vitamin A itself served as an essential co-factor in redox activation of both PKCs and c-Raf (20Hoyos B. Imam A. Korichneva I. Levi E. Chua R. Hammerling U. J. Biol. Chem. 2002; 277: 23949-23957Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar, 21Imam A. Hoyos B. Swenson C. Levi E. Chua R. Viriya E. Hammerling U. FASEB J. 2000; (10.1096/fj.00-0329fje)PubMed Google Scholar). The hypothesis was developed that the binding of vitamin A to the cysteinerich domains was required for the controlled oxidation of defined cysteines (22Imam A. Hoyos B. Swenson C. Levi E. Chua R. Viriya E. Hammerling U. FASEB J. 2001; 15: 28-30Crossref PubMed Scopus (68) Google Scholar). When absent because of nutritional deficiency or when experimentally displaced by non-functional retinoid antagonists, such as anhydroretinol, PKC, and c-Raf, activation by reactive oxygen species was severely compromised. Jakob et al. (23Jakob U. Muse W. Eser M. Bardwell J. Cell. 1999; 96: 341-352Abstract Full Text Full Text PDF PubMed Scopus (425) Google Scholar) proposed that in bacteria, cysteine-rich domains are organized into a zinc finger fold that functions as a molecular hinge. The mammalian counterpart, although more complex, also forms a composite zinc finger (24Mott H.R. Carpenter J. Zhong S. Ghosh S. Bell R. Campbell S. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 8312-8317Crossref PubMed Scopus (157) Google Scholar, 25Zhang G. Kazanietz M.G. Blumberg P.M. Hurley J.H. Cell. 1995; 81: 917-924Abstract Full Text PDF PubMed Scopus (595) Google Scholar). Upon oxidation, the latter has been shown to relinquish the central zinc ions, allowing potentially a conformational change (26Knapp L. Klann E. J. Biol. Chem. 2000; 275: 24136-24145Abstract Full Text Full Text PDF PubMed Scopus (167) Google Scholar, 27Korichneva I. Hoyos B. Chua R. Levi E. Hammerling U. J. Biol. Chem. 2002; 277: 44327-44331Abstract Full Text Full Text PDF PubMed Scopus (146) Google Scholar). Bound retinol accelerated this oxidation process. Surprisingly, phorbol ester and diacylglycerol also caused the release of Zn2+ from the PKCα zinc finger (27Korichneva I. Hoyos B. Chua R. Levi E. Hammerling U. J. Biol. Chem. 2002; 277: 44327-44331Abstract Full Text Full Text PDF PubMed Scopus (146) Google Scholar). Thus, the two chemically dissimilar activators, reactive oxygen species and phorbol ester, nevertheless produced the same outcome: disassembly of the zinc finger, and presumably, a conformational shift. Unfolding has long been postulated as a prelude to the release of the auto-inhibition that the regulatory domain imposes on the catalytic domain (28Heidecker G. Huleihel M. Cleveland J.L. Kolch W. Beck T.W. Lloyd P. Pawson T. Rapp U.R. Mol. Cell. Biol. 1990; 10: 2503-2512Crossref PubMed Scopus (206) Google Scholar, 29Hurley J. Gobler J. Curr. Opin. Struct. Biol. 1997; 7: 557-565Crossref PubMed Scopus (60) Google Scholar). Although the role as co-factor in redox regulation presents a conceptual advance to explain vitamin A action, explorations of biological significance have not kept pace. The reason can be traced to the multitude of target molecules (i.e. the PKC and Raf families) and attendant vitamin A-dependent signal chains. To overcome the difficulties stemming from simultaneous engagement by reactive oxygen species of the diverse vitamin A-dependent signal pathways, we have devised a genetic approach that is predicated on the elimination of retinol-binding sites in select signaling molecules. This was accomplished by mutation of critical contact amino acids of the zinc finger domain. As reported here for the example of c-Raf, the impairment of binding for retinol was paralleled by the selective loss of redox regulation, whereas kinase activation by the classic hormone receptor signals was retained. Additionally, introducing three critical contact residues, copied from the PKCα C1A domain, into the natural non-binding C1B domain conferred retinol binding capacity. Reagents—All-trans retinol, GTPγS, bovine serum albumin (enzyme immunoassay grade), p-nitrophenyl phosphate, glutathione-agarose beads, anti-FLAG® M2-agarose affinity gel, anti-rabbit IgG-alkaline phosphatase conjugate, and epidermal growth factor (EGF) were purchased from Sigma. Rabbit antibody to c-Raf C20-terminal peptide was purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA), and anti-glutathione S-transferase (GST) antibody was purchased from Amersham Biosciences. Cell Culture—COS-7 cells were grown and maintained in Dulbecco's modified Eagle's medium high glucose supplemented with 10% fetal calf serum and l-glutamine without antibiotics. Plasmids and Mutagenesis—The cys domains of c-Raf-(134–190), PKCα C1A-(37–87), and C1B-(102–151) were cloned by PCR into the BamHI and EcoRI sites of GEX-2T vector (Amersham Biosciences). Mutagenesis of FLAG-c-Raf full-length (a gift from Dr. R. J. Davis, University of Massachusetts, Worcester, MA) and the cys domains was performed using the QuikChange® site-directed mutagenesis kit (Stratagene). Transfection and Cell Activation—COS-7 cells were transfected by the calcium phosphate method as described (20Hoyos B. Imam A. Korichneva I. Levi E. Chua R. Hammerling U. J. Biol. Chem. 2002; 277: 23949-23957Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar). To deplete endogenous retinol, cell cultures were first incubated for 30 min with 1 μm anhydroretinol and then cultured with 2 ml of retinoid-free, serum-free, phenol red-free Dulbecco's modified Eagle's medium high glucose for 2½ days (to deplete retinyl esters (30Eppinger T.M. Buck J. Hammerling U. J. Exp. Med. 1993; 178: 1995-2005Crossref PubMed Scopus (44) Google Scholar)). Where indicated, cultures were restored to vitamin A sufficiency by incubation for 20 min with 1 μm retinol with 0.1% bovine serum albumin as carrier. Subsequently, cells were activated by UV-irradiation for 2 min at 400 milliwatt/cm2 using 312-nm wavelength as described (20Hoyos B. Imam A. Korichneva I. Levi E. Chua R. Hammerling U. J. Biol. Chem. 2002; 277: 23949-23957Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar), or alternatively, by the classic receptor tyrosine kinase pathway with human EGF at 100 ng/ml. Cultures were incubated at 37 °C for 10 min after activation and harvested. c-Raf Immunoprecipitation/Kinase Assay—This was carried out as described (20Hoyos B. Imam A. Korichneva I. Levi E. Chua R. Hammerling U. J. Biol. Chem. 2002; 277: 23949-23957Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar). Briefly, COS-7 cells transfected by calcium phosphate were lysed with 100 μl of lysis buffer (50 mm Tris-HCl, pH 7.5, 150 mm NaCl, 2 mm EDTA, 1 mm EGTA, 1% Triton X-100, 25 μg/ml each leupeptin and aprotinin, 1 mmp-methanesulfonyl fluoride, 1 mm vanadate, 30 mm β-glycerophosphate) and precleared. FLAG-c-Raf protein was precipitated using 30 μl of anti-FLAG M2 affinity gel (Sigma). The immunoprecipitates were washed four times with lysis buffer containing 0.5 m NaCl and twice with kinase buffer (35 mm Tris-HCl, pH 7.5, 10 mm MgCl2, 0.5 mm EGTA, and 1 mm vanadate). The kinase reaction was performed in 20 μl of kinase buffer using 200 ng of kinase-disabled His-MEK(K97M) as substrate, 60 μm ATP, and 10 μCi of [γ-32P]ATP (6,000 Ci/mmol). The reaction was carried out for 20 min at 30 °C and terminated by the addition of 10 μl of 5× Laemmli buffer. Quantitation was carried out by densitometry using Quantity-One software (Bio-Rad). Phosphotransferase signals were normalized on the amounts of c-Raf immunoprecipitated. Bacteria Growth and Protein Purification—The c-Raf-cys domain WT and mutants were expressed as GST fusion protein in the BL21/DE3 strain of Escherichia coli (Novagen) (11Hoyos B. Imam A. Chua R. Swenson C. Tong G.C. Levi E. Noy N. Hammerling U. J. Exp. Med. 2000; 192: 835-846Crossref PubMed Scopus (67) Google Scholar). Bacteria were initially grown at 37 °C to an optical density (OD) at 600 nm of 0.5, transferred to room temperature. At an OD660 nm of 0.7–0.8, protein synthesis was induced by 0.5 mm isopropyl-β-d-thiogalactopyranoside, and the cells harvested 2 h later. Bacteria were passed twice through a French press, and the GST fusion proteins were recovered from the lysates by affinity chromatography on the glutathione-agarose matrix. Purity by Coomassie Blue staining of SDS-PAGE was usually >90% by this protocol. Retinoid Binding Assay by Quenching of the Endogenous Protein Fluorescence—Quantitative fluorescence measurement of 250 nm GST fusion protein with retinoid titration at 25 nm increments were performed as described (11Hoyos B. Imam A. Chua R. Swenson C. Tong G.C. Levi E. Noy N. Hammerling U. J. Exp. Med. 2000; 192: 835-846Crossref PubMed Scopus (67) Google Scholar) in phosphate-buffered saline, purged of oxygen by sparging with helium for 15 min, in a JASCO spectrofluorometer (model FP777). The protein solution was excited at 280 nm, and the protein emission was monitored at 330 nm. Binding constants were calculated by non-linear curve fitting according to the theorem by Norris et al. (31Norris A.W. Cheng L. Giguere V. Rosenberger M. Li E. Biochim. Biophys. Acta. 1994; 1209: 10-18Crossref PubMed Scopus (74) Google Scholar). Qualitative retinol binding assays based on vibronic fine structure determinations were performed as described (22Imam A. Hoyos B. Swenson C. Levi E. Chua R. Viriya E. Hammerling U. FASEB J. 2001; 15: 28-30Crossref PubMed Scopus (68) Google Scholar). Ras/c-Raf Binding Assay—Quantitative binding of GST·Raf cys WT and mutants to GTPγS/Ras was measured by the ELISA described by Gosh et al. (15Gosh S. Xie W.Q. Quest A.F.G. Mabrouk G.M. Strum J.C. Bell R.M. J. Biol. Chem. 1994; 269: 10000-10007Abstract Full Text PDF PubMed Google Scholar). Computational Biology Methods—Docking of retinol into c-Raf-1 and PKCα was carried out using the software Autodock3.05 (32Morris G.M. Goodsell D.S. Halliday R.S. Huey R. Hart W.E. Belew R.K. Olson A.J. J. Comput. Chem. 1998; 19: 1639-1662Crossref Scopus (9035) Google Scholar) to find potential binding sites of retinol. Autodock used a Monte Carlo simulated annealing technique for configurational exploration with a rapid energy evaluation using grid-based molecular affinity potentials. It thus combined the advantages of exploring a large search space and a robust energy evaluation. The method has proven to be a powerful approach to the problem of docking a flexible ligand into the binding site of a static protein. Van der Waals interactions were calculated using a Lennard-Jones 12-6 potential, whereas the hydrogen-bonding term was modeled by Lennard-Jones directional 12-10 potential. Electrostatic potential energies were calculated with a distance-dependent dielectric function. Chemical preference terms were also added in its scoring function. The program was tested on a number of protein-substrate complexes, which had been characterized by x-ray crystallography (33Goodsell D.S. Olson A.J. Proteins Struct. Funct. Genet. 1990; 8: 195-202Crossref PubMed Scopus (1021) Google Scholar) as recommended for docking studies. c-Raf structure was taken from Protein Data Bank accession number 1FAR (24Mott H.R. Carpenter J. Zhong S. Ghosh S. Bell R. Campbell S. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 8312-8317Crossref PubMed Scopus (157) Google Scholar), and PKCα C1A structure was modeled using 1PTQ (25Zhang G. Kazanietz M.G. Blumberg P.M. Hurley J.H. Cell. 1995; 81: 917-924Abstract Full Text PDF PubMed Scopus (595) Google Scholar) as template for the program Modeler (34Sali A. Blundell T.L. J. Mol. Biol. 1993; 234: 779-815Crossref PubMed Scopus (10440) Google Scholar) as part of the molecular modeling software package InsightII from Accelrys. NMR structure coordinates for αC1B were kindly provided by Dr. Marcel Luyten (35Hommel U. Zurini M. Luyten M. Nat. Struct. Biol. 1994; 1: 383-388Crossref PubMed Scopus (137) Google Scholar). Modeler is known to produce reliable models when high homologous high resolution crystal structures are available as template, which is the case in our study. Quality of the model was checked with modules available in InsightII package. The GST fusion protein comprising c-Raf amino acids 139–184, spanning the zinc finger domain, encodes one high affinity retinol-binding site (11Hoyos B. Imam A. Chua R. Swenson C. Tong G.C. Levi E. Noy N. Hammerling U. J. Exp. Med. 2000; 192: 835-846Crossref PubMed Scopus (67) Google Scholar) and a half-site for GTP/Ras recognition (16Gosh S. Bell R.M. J. Biol. Chem. 1994; 260: 30785-30788Abstract Full Text PDF Google Scholar, 36Clark G.J. Drugan J.K. Terrell R.S. Bradham C. Der C.J. Bell R.M. Campbell S. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 1577-1581Crossref PubMed Scopus (65) Google Scholar). PKCα C1A and C1B zinc fingers comprise amino acids 37–86, and 102–151, respectively. To maintain a unified numbering system between the c-Raf and PKC zinc fingers, the conserved cysteines and histidines were aligned with each other, requiring a gap for c-Raf since this domain lacks the 4-amino-acid phorbol-binding loop. Numbering began at the 1st histidine. (Fig. 1). To map the retinol-binding site of c-Raf, we converted consecutively all amino acids (except Cys and His of the zinc finger core) to Trp and tested the bacterially expressed mutant peptides for retinol binding by the fluorescence quench assay. This assay registers the amount of bound retinol by the relative decrease of intrinsic protein fluorescence emission caused by fluorescence resonance energy transfer between a tryptophan residue and proximal retinol. Titrations of retinol yielded, after correction for inner filtering, Scatchard plots that allowed us to compute the apparent binding affinities by a theorem developed by Norris et al. (31Norris A.W. Cheng L. Giguere V. Rosenberger M. Li E. Biochim. Biophys. Acta. 1994; 1209: 10-18Crossref PubMed Scopus (74) Google Scholar). Examples are shown for WT and the mutants F8W, T33W, and K37W. The first two mutants were unable to bind retinol, as indicated by the absence of quenching of protein fluorescence (Fig. 2), whereas the control K37W mutant yielded binding comparable with WT. Because point mutations can cause broad structural disruptions, it was desirable to ascertain the integrity of each mutated protein. Otherwise, the loss of retinol binding might merely reflect a general structural collapse. The zinc finger domain encodes a face where GTP/Ras docks (17Williams J.G. Drugan J.K. Yi G.S. Clark G.J. Der C.J. Campbell S.L. J. Biol. Chem. 2000; 275: 22172-22179Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar). The mutated peptides were tested for retention/loss of GTP/Ras binding capacity by an ELISA devised by Gosh et al. (15Gosh S. Xie W.Q. Quest A.F.G. Mabrouk G.M. Strum J.C. Bell R.M. J. Biol. Chem. 1994; 269: 10000-10007Abstract Full Text PDF PubMed Google Scholar). Briefly, GTP/Ras was adsorbed to ELISA plates and incubated with WT and mutant GST fusion proteins. Anti-GST antibody-conjugated phosphatase was used to detect the amount of bound fusion proteins. As shown in Fig. 3 the two retinol-binding loss mutants, F8W and T33W, retained Ras binding capacity comparable with WT, whereas that of the non-permissible framework mutant C34S was at least 4-fold weaker (25Zhang G. Kazanietz M.G. Blumberg P.M. Hurley J.H. Cell. 1995; 81: 917-924Abstract Full Text PDF PubMed Scopus (595) Google Scholar). Table I summarizes the results of a systematic study of all mutants. Of 41 mutations, 12 resulted in substantial loss of retinol binding affinity. However, within this category several, including C34W, S43W, and K45W, were disregarded as they showed weakened Ras binding capacity.Table IScanning mutagenesis of c-Raf zinc-finger to identify contact residues mediating retinol binding Point mutations were systematically introduced in the 45 amino acid stretch from His-139 to Cys-184 comprising the zinc-finger domain (* indicates numbering beginning with first histidine of zinc-finger; a 4-amino-acid gap was allowed for alignment with PKC zinc fingers; ** indicates numbering according to full-length cRaf molecule). Six cysteines and 2 histidines defining the two zinc coordination centers were left unchanged. Mutated peptides were expressed as GST fusion proteins and tested for retinol binding by the quench method (31Norris A.W. Cheng L. Giguere V. Rosenberger M. Li E. Biochim. Biophys. Acta. 1994; 1209: 10-18Crossref PubMed Scopus (74) Google Scholar) (see also Fig. 2 for examples). The dissociation constants and standard deviations of the mean are listed in column 4. Each mutated peptide was also tested for capacity to bind GTP/ras by the ELISA assay of Gosh et al (15Gosh S. Xie W.Q. Quest A.F.G. Mabrouk G.M. Strum J.C. Bell R.M. J. Biol. Chem. 1994; 269: 10000-10007Abstract Full Text PDF PubMed Google Scholar), and results are reported in column 5 as + for binding capacity within 90th percentile of wild type, - for absence of binding, and +/- for intermediate binding (see Fig. 3 for examples).View Large Image Figure ViewerDownload Hi-res image Download (PPT) Open table in a new tab To narrow the search for presumptive contact residues among the remaining 9, a computational docking study was undertaken, using the coordinates of three known PKC and c-Raf zinc finger structures (24Mott H.R. Carpenter J. Zhong S. Ghosh S. Bell R. Campbell S. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 8312-8317Crossref PubMed Scopus (157) Google Scholar, 25Zhang G. Kazanietz M.G. Blumberg P.M. Hurley J.H. Cell. 1995; 81: 917-924Abstract Full Text PDF PubMed Scopus (595) Google Scholar, 35Hommel U. Zurini M. Luyten M. Nat. Struct. Biol. 1994; 1: 383-388Crossref PubMed Scopus (137) Google Scholar). To account for the mutual competition of retinoids for the same site on zinc fingers (21Imam A. Hoyos B. Swenson C. Levi E. Chua R. Viriya E. Hammerling U. FASEB J. 2000; (10.1096/fj.00-0329fje)PubMed Google Scholar, 37Buck J. Grun F. Derguini F. Chen Y. Kimura S. Noy N. Hammerling U. J. Exp. Med. 1993; 178: 675-680Crossref PubMed Scopus (88) Google Scholar, 38O'Connell M.J. Chua R. Hoyos B. Buck J. Chen Y.Q. Derguini F. Hammerling U. J. Exp. Med. 1996; 184: 549-555Crossref PubMed Scopus (55) Google Scholar), retinol was assumed to bind by the β-ionone ring and polyene structure. Therefore the computer was instructed to search for a hydrophobic pocket that would accommodate retinol in headfirst orientation at an energy minimum. A groove was identified in the c-Raf zinc finger with Phe-8 at its head that permitted retinol to dock optimally. Additional contact residues that consistently showed up in the two top ranking docking models were Leu-9, Ala-12, and Arg-30 (Fig. 4, models B and C). These bounded the presumptive pocket that held the head group. In the first model, the residues contacting the tail end were Gly-35, Thr-33, and framework Cys-34 (Fig. 4, model B). Mutations of Thr-33 and Gly-35 both led to impaired retinol binding (Table I). In the alternate model, the orientation and contacts with amino acids for holding the tail end were less well defined. Thus, residues Phe-8 and Thr-33 were identified as retinol contact residues by both docking modeling and site-directed mutagenesis. PKCα zinc fingers are highly homologous in their structures to c-Raf, except for the presence of the loop formed by amino acids 23–26 that defines the phorbol ester-binding site of PKC but is missing in c-Raf. To determine whether the site mapped to a similar topographical region in the C1A domain of PKCα (which is known to bind retinol), comparative docking trials were undertaken. The results identified a phenylalanine at position 8 that furnished the principal contact site for the β-ionone ring. This residue corresponded precisely to Phe-8 in c-Raf. The surrounding amino acids were, however, quite different from those of the c-Raf-binding site, αC1A having Phe-7, instead of Thr-7 found in c-Raf. The PKCα C1B domain does not bind retinol (21Imam A. Hoyos B. Swenson C. Levi E. Chua R. Viriya E. Hammerling U. FASEB J. 2000; (10.1096/fj.00-0329fje)PubMed Google Scholar). Consistent with this, a docking trial yielded no firm assignment of a binding pocket in C1B (Fig. 4, model E). To experimentally confirm predictions from the docking trials, mutations were introduced into the αC1A domain. As shown in Table II, mutating any of the presumptive contact amino acids (Phe-7, Phe-8, or Trp-22) alone was ineffective, unlike in c-Raf, where single point mutations impaired retinol binding significantly. Double mutations of the presumptive hydrophobic amino acids Phe-7 and Phe-8 believed to contact the head group slightly lowered the binding affinity. However, dual substitutions involving either Phe-7 or Phe-8 in combination with Trp-22 drastically diminished retinol binding. Amino acids Thr-7, Tyr-8, and Tyr-22 are characteristic of the non-binding αC1B domain. When copying this motif into the αC1A domain, retinol binding was abolished, whereas phorbol ester binding was preserved (data not shown), indicating that this triple mutation was structurally permissible.Table IIEffect of mutagenesis of predicted contact amino acids of PKCα C1A mediating retinol binding Mutated peptides were expressed as GST fusion proteins and tested for retinol binding by the quench method (31Norris A.W. Cheng L. Giguere V. Rosenberger M. Li E. Biochim. Biophys. Acta. 1994; 1209: 10-18Crossref PubMed Scopus (74) Google Scholar) (legend for Fig. 2). Listed are the dissociation constants and standard deviations of the means. Mutated peptides preserved capacity to bind phorbol ester (data not shown).αC1ARetinol binding constant ± S.E.nmWild type28.3 ± 8F7T73 ± 54F7G69 ± 26F8Y13 ± 4F8G27.2 ± 6.5W22Y30.6 ± 10DoubleF7T/F8Y71 ± 26F7G/F8G55.8 ± 20F7T/W22Y>500F8Y/W22Y>500TripleF7T/F8Y/W22Y>500 Open t
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