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• W2023006281 abstract "Myristoylated alanine-rich C kinase substrate (MARCKS) is an unfolded protein that contains well characterized actin-binding sites within the phosphorylation site domain (PSD), yet paradoxically, we now find that intact MARCKS does not bind to actin. Intact MARCKS also does not bind as well to calmodulin as does the PSD alone. Myristoylation at the N terminus alters how calmodulin binds to MARCKS, implying that, despite its unfolded state, the distant N terminus influences binding events at the PSD. We show that the free PSD binds with site specificity to MARCKS, suggesting that long-range intramolecular interactions within MARCKS are also possible. Because of the unusual primary sequence of MARCKS with an overall isoelectric point of 4.2 yet a very basic PSD (overall charge of +13), we speculated that ionic interactions between oppositely charged domains of MARCKS were responsible for long-range interactions within MARCKS that sterically influence binding events at the PSD and that explain the observed differences between properties of the PSD and MARCKS. Consistent with this hypothesis, chemical modifications of MARCKS that neutralize negatively charged residues outside of the PSD allow the PSD to bind to actin and increase the affinity of MARCKS for calmodulin. Similarly, both myristoylation of MARCKS and cleavage of MARCKS by calpain are shown to increase the availability of the PSD so as to activate its actin-binding activity. Because abundant evidence supports the conclusion that MARCKS is an important protein in regulating actin dynamics, our data imply that post-translational modifications of MARCKS are necessary and sufficient to regulate actin-binding activity. Myristoylated alanine-rich C kinase substrate (MARCKS) is an unfolded protein that contains well characterized actin-binding sites within the phosphorylation site domain (PSD), yet paradoxically, we now find that intact MARCKS does not bind to actin. Intact MARCKS also does not bind as well to calmodulin as does the PSD alone. Myristoylation at the N terminus alters how calmodulin binds to MARCKS, implying that, despite its unfolded state, the distant N terminus influences binding events at the PSD. We show that the free PSD binds with site specificity to MARCKS, suggesting that long-range intramolecular interactions within MARCKS are also possible. Because of the unusual primary sequence of MARCKS with an overall isoelectric point of 4.2 yet a very basic PSD (overall charge of +13), we speculated that ionic interactions between oppositely charged domains of MARCKS were responsible for long-range interactions within MARCKS that sterically influence binding events at the PSD and that explain the observed differences between properties of the PSD and MARCKS. Consistent with this hypothesis, chemical modifications of MARCKS that neutralize negatively charged residues outside of the PSD allow the PSD to bind to actin and increase the affinity of MARCKS for calmodulin. Similarly, both myristoylation of MARCKS and cleavage of MARCKS by calpain are shown to increase the availability of the PSD so as to activate its actin-binding activity. Because abundant evidence supports the conclusion that MARCKS is an important protein in regulating actin dynamics, our data imply that post-translational modifications of MARCKS are necessary and sufficient to regulate actin-binding activity. Myristoylated alanine-rich C kinase substrate (MARCKS) 1The abbreviations used are: MARCKS, myristoylated alanine-rich C kinase substrate; PSD, phosphorylation site domain; Rh-PSD, rhodamine-labeled phosphorylation site domain; CaM, calmodulin; MIANS, 2-(4-maleimidoanilino)naphthalene-6-sulfonic acid; EDC, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide; MES, 4-morpholineethanesulfonic acid.1The abbreviations used are: MARCKS, myristoylated alanine-rich C kinase substrate; PSD, phosphorylation site domain; Rh-PSD, rhodamine-labeled phosphorylation site domain; CaM, calmodulin; MIANS, 2-(4-maleimidoanilino)naphthalene-6-sulfonic acid; EDC, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide; MES, 4-morpholineethanesulfonic acid. is a well characterized, charge-polarized, natively unfolded molecule (1Wang J. Arbuzova A. Hangyas-Mihalyne G. McLaughlin S. J. Biol. Chem. 2001; 276: 5012-5019Abstract Full Text Full Text PDF PubMed Scopus (151) Google Scholar, 2Arbuzova A. Schmitz A.A. Vergères G. Biochem. J. 2002; 362: 1-12Crossref PubMed Scopus (279) Google Scholar, 3Sundaram M. Cook H.W. Byers D.M. Biochem. Cell Biol. 2004; 82: 191-200Crossref PubMed Scopus (79) Google Scholar) with a centrally located active site known as the phosphorylation site domain (PSD). Consistent with the paradigm for natively unfolded proteins, MARCKS is thought to interact with several ligands so as to integrate information from various signal transduction pathways to produce an output signal that regulates cell motile and contractile function. Numerous studies of the MARCKS protein have utilized a peptide with a sequence that corresponds to the PSD peptide as a substitute for studying interactions between the intact protein and its multiple ligands (3Sundaram M. Cook H.W. Byers D.M. Biochem. Cell Biol. 2004; 82: 191-200Crossref PubMed Scopus (79) Google Scholar, 4Rauch M.E. Ferguson C.G. Prestwich G.D. Cafiso D.S. J. Biol. Chem. 2002; 277: 14068-14076Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar). Although this approach intuitively appears to be logical, given the unfolded state of the native protein, the substitution of the PSD peptide for the intact protein has never been rigorously justified. In fact, there are several reported experiments that imply that the PSD peptide behaves differently from intact MARCKS. The non-phosphorylated PSD peptide is known to have extended structure, to nucleate polymerization, and to cross-link F-actin filaments (5Hartwig J.H. Thelen M. Rosen A. Janmey P.A. Nairn A.C. Aderem A. Nature. 1992; 356: 618-622Crossref PubMed Scopus (617) Google Scholar, 6Bubb M.R. Lenox R.H. Edison A.S. J. Biol. Chem. 1999; 274: 36472-36478Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar, 7Wohnsland F. Schmitz A.A. Steinmetz M.O. Aebi U. Vergères G. J. Biol. Chem. 2000; 275: 20873-20879Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar), presumably because of two binding sites with a site-specific Kd of ∼0.5 μm for F-actin (8Yarmola E.G. Edison A.R. Lenox R.H. Bubb M.R. J. Biol. Chem. 2001; 276: 22351-22358Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar). Although the PSD of MARCKS and its homolog MARCKS-related protein have both been shown to bind to actin with similar affinity, intact recombinant MARCKS-related protein, with or without myristoylation, exhibits a lower affinity for actin (much greater than 1 μm) and does not cross-link F-actin or induce G-actin polymerization (9Wohnsland F. Steinmetz M.O. Aebi U. Vergères G. J. Struct. Biol. 2000; 131: 217-224Crossref PubMed Scopus (14) Google Scholar). Although full-length MARCKS has been shown to bind and bundle F-actin (4Rauch M.E. Ferguson C.G. Prestwich G.D. Cafiso D.S. J. Biol. Chem. 2002; 277: 14068-14076Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar, 10Müller U. Brain Res. 1997; 757: 24-30Crossref PubMed Scopus (7) Google Scholar), the published data are only semi-quantitative. Comparison of the available data also suggests that intact MARCKS binds to vesicles containing acidic phospholipids with 104-fold lower affinity than does the PSD peptide alone (11Kim J. Shishido T. Jiang X. Aderem A. McLaughlin S. J. Biol. Chem. 1994; 269: 28214-28219Abstract Full Text PDF PubMed Google Scholar). Calcium-dependent interactions between the PSD peptide and the Ca2+-binding protein calmodulin have been extensively characterized, and a crystallographic structure is available that reveals that the phenylalanine residues of the PSD are buried in a hydrophobic tunnel of calmodulin and that the highly charged termini of the peptide interact with patches of opposite charge on the surface of calmodulin (12Yamauchi E. Nakatsu T. Matsubara M. Kato H. Taniguchi H. Nat. Struct. Biol. 2003; 10: 226-231Crossref PubMed Scopus (99) Google Scholar). Once again, the PSD peptide is said to interact with higher affinity than the intact protein (Kd = 3.8 versus 12.7 nm, respectively, in 0.1 m KCl) with this ligand (13Matsubara M. Titani K. Taniguchi H. Hayashi N. J. Biol. Chem. 2003; 278: 48898-48902Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar). Recently, it has been speculated that myristoylation of MARCKS adds a second, low affinity, calmodulin-binding site to MARCKS without evidence of cooperativity (13Matsubara M. Titani K. Taniguchi H. Hayashi N. J. Biol. Chem. 2003; 278: 48898-48902Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar). Importantly, the addition of a second non-cooperative binding site cannot explain the long-known result that myristoylated MARCKS binds to calmodulin with higher affinity than does non-myristoylated MARCKS (14Manenti S. Sorokine O. Van Dorsselaer A. Taniguchi H. J. Biol. Chem. 1993; 268: 6878-6881Abstract Full Text PDF PubMed Google Scholar) unless both binding sites can interact with a single calmodulin molecule simultaneously or myristoylation itself changes the accessibility of the PSD to bind to calmodulin. Independent binding would change the stoichiometry of the interaction to two calmodulin molecules/MARCKS, but could not significantly increase the apparent affinity unless the calmodulin ligand was multivalent (oligomerized or attached to a bead). A recent crystallographic structure of a myristoylated peptide bound to calmodulin (15Matsubara M. Nakatsu T. Kato H. Taniguchi H. EMBO J. 2004; 23: 712-718Crossref PubMed Scopus (78) Google Scholar), with the myristoyl group in the same hydrophobic tunnel and interacting with many of the same residues as the phenylalanines of the PSD peptide, suggests that both the N-terminal myristoyl- and PSD-binding sites could not simultaneously interact with calmodulin without significant steric effects. However, the hydrophobic tunnel through calmodulin has been shown to be quite flexible, and there are no experimental data to rule out the possibility that calmodulin could adjust to accommodate both putative binding regions. Of note, myristoylation of MARCKS is likely a dynamically regulated post-translational event (16Manenti S. Sorokine O. Van Dorsselaer A. Taniguchi H. Biochem. Soc. Trans. 1995; 23: 561-564Crossref PubMed Scopus (9) Google Scholar). Non-myristoylated MARCKS has been isolated from bovine brain (14Manenti S. Sorokine O. Van Dorsselaer A. Taniguchi H. J. Biol. Chem. 1993; 268: 6878-6881Abstract Full Text PDF PubMed Google Scholar), and a demyristoylase activity has been characterized (13Matsubara M. Titani K. Taniguchi H. Hayashi N. J. Biol. Chem. 2003; 278: 48898-48902Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar), thus making the acronym somewhat of a misnomer. For the purposes of this study, except in instances in which there may be some confusion, we refer to MARCKS as the non-myristoylated protein, for which, because of its natively unfolded structure, there is reason to believe that the native protein is equivalent to the recombinant protein. Many in vivo studies have implicated MARCKS in an actin-regulating function (18Song J.C. Hrnjez B.J. Farokhzad O.C. Matthews J.B. Am. J. Physiol. 1999; 277: C1239-C1249Crossref PubMed Google Scholar, 19Ohmori S. Sakai N. Shirai Y. Yamamoto H. Miyamoto E. Shimizu N. Saito N. J. Biol. Chem. 2000; 275: 26449-26457Abstract Full Text Full Text PDF PubMed Scopus (94) Google Scholar, 20Spizz G. Blackshear P.J. J. Biol. Chem. 2001; 276: 32264-32273Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar, 21Rose S.D. Lejen T. Zhang L. Trifaro J.M. J. Biol. Chem. 2001; 276: 36757-36763Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar, 22Iioka H. Ueno N. Kinoshita N. J. Cell Biol. 2004; 164: 169-174Crossref PubMed Scopus (56) Google Scholar), but the evidence is indirect and could in all cases be explained by invoking schemes in which MARCKS alters events that in turn regulate cytoskeletal dynamics. However, in several specific examples, MARCKS colocalizes with F-actin, and dissociation from actin is temporally associated with alterations in actin dynamics (22Iioka H. Ueno N. Kinoshita N. J. Cell Biol. 2004; 164: 169-174Crossref PubMed Scopus (56) Google Scholar, 23Lu D. Yang H. Lenox R.H. Raizada M.K. J. Cell Biol. 1998; 142: 217-227Crossref PubMed Scopus (49) Google Scholar), or MARCKS localization is altered after treatment that disrupts actin filaments (18Song J.C. Hrnjez B.J. Farokhzad O.C. Matthews J.B. Am. J. Physiol. 1999; 277: C1239-C1249Crossref PubMed Google Scholar, 22Iioka H. Ueno N. Kinoshita N. J. Cell Biol. 2004; 164: 169-174Crossref PubMed Scopus (56) Google Scholar). Such data are most readily interpreted as direct effects of MARCKS on actin. One target of MARCKS, phosphatidylinositol 4,5-bisphosphate, has been implicated in controlling the actin cytoskeleton by binding to many other actin-regulating proteins such as neural Wiskott-Aldrich syndrome protein (24Sechi A.S. Wehland J. J. Cell Sci. 2000; 113: 3685-3695Crossref PubMed Google Scholar), suggesting one possible indirect mechanism of actin control by MARCKS. MARCKS may sequester phosphatidylinositol 4,5-bisphosphate in the plasma membrane by reversible PSD binding (25Wang J. Gambhir A. Hangyas-Mihalyne G. Murray D. Golebiewska U. McLaughlin S. J. Biol. Chem. 2002; 277: 34401-34412Abstract Full Text Full Text PDF PubMed Scopus (181) Google Scholar). However, it should be noted that published estimates of the intracellular MARCKS concentration (12 μm) (26Blackshear P.J. J. Biol. Chem. 1993; 268: 1501-1504Abstract Full Text PDF PubMed Google Scholar) are based on the yield of a bovine forebrain preparation, but the original data are probably more consistent with an intracellular concentration of 1.2 μm (27Albert K.A. Nairn A.C. Greengard P. Proc. Natl. Acad. Sci. U. S. A. 1987; 84: 7046-7050Crossref PubMed Scopus (99) Google Scholar). Thus, although there may not be sufficient MARCKS to globally regulate phosphatidylinositol 4,5-bisphosphate metabolism, local pools of phosphatidylinositol 4,5-bisphosphate could potentially be regulated by MARCKS. If the PSD of MARCKS is incompletely accessible to its many ligands in the intact protein, then two questions emerge that are addressed in this study. 1) How can a natively unfolded protein maintain the PSD in a buried unavailable position? 2) Do mechanisms exist to alter the availability of the PSD? Here, we test a novel structural hypothesis related to the unusual charge distribution in the primary sequence of MARCKS and attribute physical significance to post-translational modifications that are now shown to regulate the actin-binding functions of MARCKS. Materials—Rabbit skeletal muscle actin was prepared from frozen muscle (Pel-Freeze Biologicals, Rogers, AR) in 5.0 mm Tris-HCl, 0.2 mm ATP, 0.2 mm dithiothreitol, 0.1 mm CaCl2, and 0.01% sodium azide (pH 7.8) (28Kang F. Laine R.O. Bubb M.R. Southwick F.S. Purich D.L. Biochemistry. 1997; 36: 8384-8392Crossref PubMed Scopus (104) Google Scholar), and pyrenyl-actin (actin labeled at Cys374 with N-(1-pyrene)iodoacetamide)) was prepared with 0.5–0.95 mol of label/mol of protein using the method of Kouyama and Mihashi (29Kouyama T. Mihashi K. Eur. J. Biochem. 1981; 114: 33-38Crossref PubMed Scopus (719) Google Scholar). Peptides were synthesized by solid-phase Fmoc (N-(9-fluorenyl)methoxycarbonyl) chemistry at the University of Florida (8Yarmola E.G. Edison A.R. Lenox R.H. Bubb M.R. J. Biol. Chem. 2001; 276: 22351-22358Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar). The PSD peptide has the sequence KKKKKRFSFKKSFKLSGFSFKKSKK. The modified rhodamine-labeled PSD peptide (Rh-PSD) (8Yarmola E.G. Edison A.R. Lenox R.H. Bubb M.R. J. Biol. Chem. 2001; 276: 22351-22358Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar) was N-terminally modified through an amide link with 5(6)-carboxytetramethylrhodamine succinimidyl ester and C-terminally labeled with Oregon Green 488 succinimidyl ester (5-isomer). Calpain I purified from porcine erythrocytes was obtained from Calbiochem. Calmodulin (CaM) isolated from bovine brain was purchased from Sigma. CaM-Sepharose beads were purchased from Amersham Biosciences. Wheat germ CaM (Mr 16,800) fluorescently labeled with 2-(4-maleimidoanilino)naphthalene-6-sulfonic acid (MIANS) at Cys27 (MIANS-CaM) was a gift from Dr. J. M. Chalovich (East Carolina University). Preparation of MARCKS and Myristoylated MARCKS—Murine full-length MARCKS DNA (GenBank™/EBI accession number M60474) was inserted into the pMW172 vector (30Way M. Pope B. Gooch J. Hawkins M. Weeds A.G. EMBO J. 1990; 9: 4103-4109Crossref PubMed Scopus (194) Google Scholar) and transformed into Escherichia coli BL21(DE3) competent cells. Cells (300 ml) were grown overnight in LB medium and used to inoculate 2 liters of culture. After 3 h, protein expression was further induced by the addition of isopropyl β-d-thiogalactopyranoside (100 μm) for 1 h. Cultures were spun down and frozen at –80 °C. Frozen cultures were resuspended in buffer consisting of 10 mm Tris-HCl, 100 μm EGTA, 5 mm β-mercaptoethanol, 2 mm EDTA, 100 μm phenylmethylsulfonyl fluoride, and 0.6 mm diisopropyl fluorophosphate (pH 7.9). Resuspended cells were sonicated, heated to 85 °C for 10 min, and centrifuged at 38,000 rpm for 1 h. The supernatant was loaded onto a DEAE column, and fractions were collected with a 100–400 mm KCl gradient in 10 mm Tris-HCl (pH 7.9). Fractions containing MARCKS (as shown by SDS-PAGE and Western blotting using goat anti-MARCKS polyclonal antibody raised against a C-terminal synthetic peptide (Serotec Inc., Raleigh, NC)) were combined and concentrated on a hydroxylapatite column and further purified by gel filtration on Sephacryl 300 HR. Concentration was determined by UV absorption at 258 nm (ϵM = 1100 at 258 nm) or by amino acid analysis. MARCKS typically eluted as a monomer at 40–70 μm and was stored at –80 °C in the column buffer, which contained 5 mm Tris-HCl, 5.0 mm β-mercaptoethanol, and 50 mm KCl (pH 7.9) (MARCKS buffer). For myristoylated MARCKS, E. coli strain BL21 was transformed with both the pBB131NMT plasmid (a gift from Dr. J. I. Gordon, Washington University), which contains the gene for yeast N-myristoyltransferase (31Duronio R.J. Knoll L.J. Gordon J.I. J. Cell Biol. 1992; 117: 515-529Crossref PubMed Scopus (59) Google Scholar), and the pMW172-MARCKS plasmid described above and selected in the presence of 50 μg/ml kanamycin and ampicillin. A frozen stock of transformed colonies was used to inoculate 200 ml of LB medium containing 50 μg/ml kanamycin and ampicillin. The overnight culture (2 liters) was grown to log phase; 400 mm isopropyl β-d-thiogalactopyranoside was added to induce protein expression; and the culture was grown for an additional 3 h. Myristoylated MARCKS was purified according to the protocol for MARCKS described above. Myristoylated MARCKS runs at ∼83 kDa on SDS-polyacrylamide gel (a slightly higher apparent molecular mass than that of MARCKS). It was characterized by mass spectroscopy with a peak at 29,874 Da compared with 29,664 Da for non-myristoylated MARCKS, and these correspond to the predicted masses based on the sequence. To test for the propensity of MARCKS to aggregate, gel-filtered monomeric MARCKS was concentrated to 300 μm in an Microcon filtration device (Millipore Corp., Billerica, MA) and then diluted to 100, 18, or 3 μm in MARCKS buffer. Samples were centrifuged at 150,000 × g for 15 min through a 20% sucrose cushion either 1 or 12 h after dilution. Pellets and supernatants were analyzed by SDS-PAGE with loading volumes inversely proportional to the protein concentration. Assays of Actin-binding Function—Binding of MARCKS, covalently modified MARCKS, myristoylated MARCKS, or calpain-digested MARCKS to F-actin was detected by a high speed centrifugation assay. Mg2+-F-actin was prepared by converting Ca2+-G-actin to Mg2+-G-actin by the addition of 0.125 mm EGTA and 0.05 mm MgCl2 for 10 min at room temperature and then polymerizing by the addition of MgCl2 to a 2.0 mm final concentration. MARCKS (0–10 μm) was added to Mg2+-F-actin (0–40 μm) in a total volume of 100 μl and equilibrated for varying times (20 min to 24 h). F-actin was pelleted at 140,000 × g in a tabletop ultracentrifuge for 1 h. Supernatants (60 μl) were removed, and pellets were washed gently three times to remove trapped unbound protein. Supernatants and pellets were then analyzed by SDS-PAGE or by fluorescence spectrometry to determine bound (pellet) or free (supernatant) MARCKS. For SDS-PAGE of covalently cross-linked MARCKS, 12% polyacrylamide gels were stained with SYPRO Ruby protein stain (Molecular Probes, Inc., Eugene, OR) after the samples were concentrated in a filtration device. Actin filament cross-linking or bundling was assessed by a low speed pelleting assay. Filament aggregates of either ordered bundles or isotropic networks of cross-linked filaments sediment at low centrifugal forces. Proteins or peptides were added to Mg2+-F-actin (7 μm final concentration) to a final volume of 80 μl. After a 10-min incubation, samples were centrifuged at 8000 × g for 20 min to pellet actin filament aggregates and any associated proteins. Supernatants (30 μl) were removed and loaded onto 10% SDS-polyacrylamide gel. The presence of bundling or cross-linking is indicated by the depletion of actin from the supernatant. The effects of MARCKS on the time course of actin filament polymerization were measured by the fluorescence change associated with the polymerization of pyrenyl-actin (29Kouyama T. Mihashi K. Eur. J. Biochem. 1981; 114: 33-38Crossref PubMed Scopus (719) Google Scholar). Ca2+-G-actin (3 μm, 4% pyrenyl-actin) was converted to Mg2+-actin as described above, and polymerization was initiated by adjustment to 50 mm KCl and 2 mm MgCl2. (Experiments without KCl are specifically indicated below.) Seeded polymerization assays employed cross-linked oligomeric F-actin seeds (32Bubb M.R. Govindasamy L. Yarmola E.G. Vorobiev S.M. Almo S.C. Somasundaram T. Chapman M.S. Agbandje-McKenna M. McKenna R. J. Biol. Chem. 2002; 277: 20999-21006Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar) and 4% pyrenyl-labeled Mg2+-actin monomer (0.5 μm). The assay was shown to be linear in response to seed concentration and actin monomer concentration with variation from these conditions. Polymerization in the presence or absence of MARCKS was measured using pyrene fluorescence, and the initial polymerization rates were determined using time course data that could be fit with a line without systematic deviation. The time course of actin filament depolymerization was assayed by dilution of 10% pyrenyl-labeled F-actin (10 μm) polymerized with 2 mm MgCl2 to 0.1 μm in the same buffer containing 0 or 2.0 μm MARCKS. Calmodulin Binding Assays—Binding of the Rh-PSD peptide or MARCKS to CaM-Sepharose beads was determined in a pull-down assay as described previously (33Chuang T.T. Paolucci L. De Blasi A. J. Biol. Chem. 1996; 271: 28691-28696Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar). Varying amounts of CaM-Sepharose suspended beads (total CaM concentration of 0–2 μm) were pipetted into 0.5-ml Eppendorf tubes containing fixed amounts of Rh-PSD (0.6 or 0.06 μm) in 50 mm KCl, 0.1 mm CaCl2, and 10 mm Tris-HCl (pH 7.9) to a final volume of 400 μl. Tubes were centrifuged at 7840 × g for 1 min to pellet the CaM·Rh-PSD bead complex, and the amount of free Rh-PSD was quantified by fluorescence spectroscopy using excitation at 522 nm and emission at 575 nm. The concentration of available CaM on the Sepharose beads was calibrated based on data obtained at 0.6 μm Rh-PSD instead of using the manufacturer's estimate, which was ∼20% higher than the value we obtained (see “Results” for details). For full-length recombinant MARCKS, the amount of MARCKS in the supernatant was quantified by Coomassie Blue staining after SDS-PAGE. Because the fluorescence of the Rh-PSD peptide increased by a factor of 2.3 upon binding to CaM, the increment in fluorescence could also be used to measure the fraction of bound Rh-PSD at varying concentrations of CaM in solution. Also, the fluorescence anisotropy of the Rh-PSD peptide changed upon binding to CaM, providing another quantitative assay for binding, which is discussed more completely below. Finally, wheat germ MIANS-CaM, labeled specifically with a fluorophore at its single cysteine residue at position 27, has been used successfully to generate a binding isotherm for calmodulin-binding proteins based on a large increment in fluorescence intensity (34Kasturi R. Vasulka C. Johnson J.D. J. Biol. Chem. 1993; 268: 7958-7964Abstract Full Text PDF PubMed Google Scholar). Saturation of MIANS-CaM by recombinant MARCKS, but not by the PSD peptide, yielded a significant increase in fluorescence intensity with excitation at 322 nm and emission at 438 nm. Fluorescence Anisotropy—Data were collected on a Photon Technology International spectrofluorometer. The Rh-PSD was excited with vertically polarized light at 546 nm. The horizontal (Ih) and vertical (Iv) components of the emitted light were detected distal to a long-pass filter with a nominal cutoff of 570 nm (catalog no. 03 FCG 089; Melles Griot, Rochester, NY) for ∼20 s for each component. The total intensity of the Rh-PSD peptide fluorescence (Iv + 2GIh) was found to change proportionally upon MARCKS or CaM binding. The ratio (R) of the fluorescence of the bound species divided by that of the free species was calculated. The fluorescence anisotropy (r) was calculated using r = (Iv – GIh)/(Iv + 2GIh) (8Yarmola E.G. Edison A.R. Lenox R.H. Bubb M.R. J. Biol. Chem. 2001; 276: 22351-22358Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar). The G factor was determined for each experiment with the peptide in solution excited with horizontally polarized light and averaged over ∼10 measurements. The experiments were performed using 0.3-ml samples in glass cuvettes. For competition assays, the direct binding assay was used to estimate an amount of MARCKS that bound approximately two-thirds of the Rh-PSD peptide, and the anisotropy was measured at these concentrations of MARCKS and the Rh-PSD with varying concentrations of the competing unlabeled PSD. The fitting procedure for the fluorescence anisotropy data included correction for the fact that the total fluorescence intensity changed upon binding of the PSD to CaM (see Fig. 2, B and C) or MARCKS (see Fig. 4). Because bound MARCKS or CaM changed the fluorescence intensity of the Rh-PSD, the observed anisotropy (r) was a nonlinear function of the fraction of PSD that bound to MARCKS or CaM: r = (rf + (R·rb – rf)·b)/(1 + (R – 1)·b), where rf is the anisotropy of the free PSD, rb is the anisotropy of the PSD bound to MARCKS or CaM, b is the fraction of PSD bound to MARCKS or CaM, and the ratio R is defined above.Fig. 4Intramolecular interactions between the Rh-PSD and MARCKS.A, when MARCKS was added to the Rh-PSD (44 nm) in 50 mm KCl, 0.6 mm CaCl2, and 10 mm Tris-HCl (pH 7.9), the fluorescence of the Rh-PSD (not shown) decreased with increasing MARCKS concentrations, and at saturation, the fractional decrease was calculated as 0.53. Fluorescence anisotropy was measured in the same samples, with an observed increase in anisotropy consistent with a direct interaction between MARCKS and the smaller fluorescent ligand. Inset, the unlabeled PSD peptide was able to displace the Rh-PSD (44 nm) from MARCKS (1 μm), consistent with site-specific binding and a 1:1 stoichiometry of MARCKS to PSD or Rh-PSD. When saturating amounts of unlabeled PSD peptide were included in the mixture of MARCKS and the Rh-PSD, the anisotropy of the Rh-PSD decreased to a level near that expected for the free Rh-PSD (compare inset with origin of the main panel), and the level of fluorescence increased, also to base-line levels (not shown). A simultaneous fit to all of the data shown using the assumption that the intramolecular PSD of MARCKS competed equivalently with the intermolecular labeled and unlabeled PSD peptides for a single binding site on MARCKS gives a Kd in the range of 10 nm (solid lines), with good fits obtainable for any Kd ≤ 40 nm. Data consistent with site-specific binding were also obtained in the presence of increasing amounts of polylysine (▴). The apparent Kd for polylysine is ≤14 nm. When one polylysine occupies that site, Rh-PSD is excluded. B, shown are the results from SDS-PAGE of untreated MARCKS (lanes 1 and 4), EDC-cross-linked MARCKS (lanes 2 and 5), and EDC-cross-linked MARCKS with the rhodamine-labeled N-terminal PSD peptide (lanes 3 and 6) visualized by Coomassie Blue staining (lanes 1–3) or by indirect UV fluorescence of the rhodamine label (lanes 4–6). C, purified recombinant MARCKS (lanes 1, 4, 6, and 8) and two RAW 264.7 whole cell extracts (one in lanes 2, 5, 7, and 9 and the other in lanes 3 and 10) were stained with Coomassie Blue (lanes 1–3) or transferred to polyvinylidene difluoride membrane and Western-blotted for MARCKS (lanes 4–7) or for a gel overlay using the rhodamine-labeled N-terminal PSD peptide as a probe (lanes 8–10). The two Western blots are of identical samples transferred on different days and reveal that the major immunoreactive band in the cell extracts has slightly lower electrophoretic mobility compared with recombinant MARCKS. The gel overlay was imaged by indirect fluorescence, and the digital fluorescence image was electronically inverted. D, shown is the concentration dependence of pelleting for MARCKS after high speed centrifug" @default.
• W2023006281 created "2016-06-24" @default.
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• W2023006281 creator A5008851874 @default.
• W2023006281 creator A5016825542 @default.
• W2023006281 creator A5023816784 @default.
• W2023006281 creator A5040416711 @default.
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• W2023006281 date "2005-03-01" @default.
• W2023006281 modified "2023-09-27" @default.
• W2023006281 title "MARCKS Is a Natively Unfolded Protein with an Inaccessible Actin-binding Site" @default.
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