FRINK Linked Data Fragments server

Matches in SemOpenAlex for { <https://semopenalex.org/work/W1996568154> ?p ?o ?g. }

• W1996568154 endingPage "20879" @default.
• W1996568154 startingPage "20873" @default.
• W1996568154 abstract "It is widely assumed that the members of the MARCKS protein family, MARCKS (an acronym for myristoylated alanine-rich C kinase substrate) and MARCKS-related protein (MRP), interact with actin via their effector domain, a highly basic segment composed of 24–25 amino acid residues. To clarify the mechanisms by which this interaction takes place, we have examined the effect of a peptide corresponding to the effector domain of MRP, the so-called effector peptide, on both the dynamic and the structural properties of actin. We show that in the absence of cations the effector peptide polymerizes monomeric actin and causes the alignment of the formed filaments into bundle-like structures. Moreover, we document that binding of calmodulin or phosphorylation by protein kinase C both inhibit the actin polymerizing activity of the MRP effector peptide. Finally, several effector peptides were synthesized in which positively charged or hydrophobic segments were deleted or replaced by alanines. Our data suggest that a group of six positively charged amino acid residues at the N-terminus of the peptide is crucial for its interaction with actin. While its actin polymerizing activity critically depends on the presence of all three positively charged segments of the peptide, hydrophobic amino acid residues rather modulate the polymerization velocity. It is widely assumed that the members of the MARCKS protein family, MARCKS (an acronym for myristoylated alanine-rich C kinase substrate) and MARCKS-related protein (MRP), interact with actin via their effector domain, a highly basic segment composed of 24–25 amino acid residues. To clarify the mechanisms by which this interaction takes place, we have examined the effect of a peptide corresponding to the effector domain of MRP, the so-called effector peptide, on both the dynamic and the structural properties of actin. We show that in the absence of cations the effector peptide polymerizes monomeric actin and causes the alignment of the formed filaments into bundle-like structures. Moreover, we document that binding of calmodulin or phosphorylation by protein kinase C both inhibit the actin polymerizing activity of the MRP effector peptide. Finally, several effector peptides were synthesized in which positively charged or hydrophobic segments were deleted or replaced by alanines. Our data suggest that a group of six positively charged amino acid residues at the N-terminus of the peptide is crucial for its interaction with actin. While its actin polymerizing activity critically depends on the presence of all three positively charged segments of the peptide, hydrophobic amino acid residues rather modulate the polymerization velocity. myristoylated alanine-rich C kinase substrate MARCKS-related protein calmodulin filamentous actin globular actin effector peptide with modifications belonging to group 1/2/3 protein kinase C catalytic subunit of PKC “wild type” MARCKS effector peptide Myristoylated alanine-rich C kinase substrate (MARCKS)1 and MARCKS-related protein (MRP) are the two members of the MARCKS protein family, a group of acidic rod-shaped proteins (1.Albert K.A. Nairn A.C. Greengard P. Proc. Natl. Acad. Sci. U. S. A. 1987; 84: 7046-7050Crossref PubMed Scopus (99) Google Scholar, 2.Manenti S. Sorokine O. Van Dorsselaer A. Taniguchi H. J. Biol. Chem. 1992; 267: 22310-22315Abstract Full Text PDF PubMed Google Scholar). MARCKS is a ubiquitous 32-kDa protein, whereas MRP (also known as F52 or MacMARCKS) has a molecular mass of 20 kDa and is mainly expressed in brain and reproductive tissues (3.Aderem A. Cell. 1992; 71: 713-716Abstract Full Text PDF PubMed Scopus (430) Google Scholar, 4.Blackshear P.J. J. Biol. Chem. 1993; 268: 1501-1504Abstract Full Text PDF PubMed Google Scholar). A comparison of their primary structures reveals three conserved domains: 1) the N-terminal domain contains the recognition site for myristoylation (5.McLaughlin S. Aderem A. Trends Biochem. Sci. 1995; 20: 272-276Abstract Full Text PDF PubMed Scopus (616) Google Scholar); 2) the MARCKS homology 2 domain, a 15-amino acid residue-long stretch whose function is unknown, shows sequence identity with the cytoplasmic tail of the mannose 6-phosphate receptor (3.Aderem A. Cell. 1992; 71: 713-716Abstract Full Text PDF PubMed Scopus (430) Google Scholar); 3) an extremely basic domain, known as the phosphorylation site domain or effector domain that is 24–25 amino acid residues long, is responsible for most of the interactions of MARCKS proteins with their common ligands (see below). Both MARCKS and MRP bind to calmodulin (CaM) with nanomolar affinity (6.Graff J.M. Young T.N. Johnson J.D. Blackshear P.J. J. Biol. Chem. 1989; 264: 21818-21823Abstract Full Text PDF PubMed Google Scholar, 7.Schleiff E. Schmitz A. McIlhinney R.A.J. Manenti S. Vergères G. J. Biol. Chem. 1996; 271: 26794-26802Abstract Full Text Full Text PDF PubMed Scopus (37) Google Scholar), are phosphorylated by protein kinase C (PKC) (8.Blackshear P.J. Verghese G.M. Johnson J.D. Haupt D.M. Stumpo D.J. J. Biol. Chem. 1992; 267: 13540-13546Abstract Full Text PDF PubMed Google Scholar, 9.Graff J.M. Stumpo D.J. Blackshear P.J. Mol. Endocrinol. 1989; 3: 1903-1906Crossref PubMed Scopus (45) Google Scholar), and bind to acidic lipid membranes (10.Vergères G. Manenti S. Weber T. Stürzinger C. J. Biol. Chem. 1995; 270: 19879-19887Abstract Full Text Full Text PDF PubMed Scopus (79) Google Scholar, 11.Taniguchi H. Manenti S. J. Biol. Chem. 1993; 268: 9960-9963Abstract Full Text PDF PubMed Google Scholar). Whereas binding of both MARCKS proteins to CaM is inhibited by phosphorylation in vitro (7.Schleiff E. Schmitz A. McIlhinney R.A.J. Manenti S. Vergères G. J. Biol. Chem. 1996; 271: 26794-26802Abstract Full Text Full Text PDF PubMed Scopus (37) Google Scholar, 12.McIlroy B.K. Walters J.D. Blackshear P.J. Johnson J.D. J. Biol. Chem. 1991; 266: 4959-4964Abstract Full Text PDF PubMed Google Scholar), phosphorylation-dependent inhibition of the binding to phospholipid vesicles has been reported for MARCKS (13.Kim J. Shishido T. Jiang X. Aderem A. McLaughlin S. J. Biol. Chem. 1994; 269: 28214-28219Abstract Full Text PDF PubMed Google Scholar), but not for MRP (14.Vergères G. Ramsden J.J. Biochem. J. 1998; 330: 5-11Crossref PubMed Scopus (31) Google Scholar, 15.Myat M.M. Chang S. Rodriguez-Boulan E. Aderem A. Curr. Biol. 1998; 8: 677-683Abstract Full Text Full Text PDF PubMed Scopus (24) Google Scholar). In cells, a connection between the subcellular localization of MARCKS and its phosphorylation state has been documented (16.Thelen M. Rosen A. Nairn A.C. Aderem A. Nature. 1991; 351: 320-322Crossref PubMed Scopus (308) Google Scholar). Whereas both the N-terminal myristoyl chain and the effector domain are involved in membrane binding (5.McLaughlin S. Aderem A. Trends Biochem. Sci. 1995; 20: 272-276Abstract Full Text PDF PubMed Scopus (616) Google Scholar, 10.Vergères G. Manenti S. Weber T. Stürzinger C. J. Biol. Chem. 1995; 270: 19879-19887Abstract Full Text Full Text PDF PubMed Scopus (79) Google Scholar, 14.Vergères G. Ramsden J.J. Biochem. J. 1998; 330: 5-11Crossref PubMed Scopus (31) Google Scholar), binding to CaM and phosphorylation by PKC mainly occur at the effector domain (17.Kim J. Blackshear P.J. Johnson J.D. McLaughlin S. Biophys. J. 1994; 67: 227-237Abstract Full Text PDF PubMed Scopus (145) Google Scholar, 18.Graff J.M. Rajan R.R. Randall R.R. Nairn A.C. Blackshear P.J. J. Biol. Chem. 1991; 266: 14390-14398Abstract Full Text PDF PubMed Google Scholar). Only little information is available on the cellular functions of MARCKS and even less on MRP. Evidently, both proteins are essential for brain development (19.Wu M. Chen D.F. Sasaoka T. Tonegawa S. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 2110-2115Crossref PubMed Scopus (84) Google Scholar, 20.Stumpo D.J. Bock C.B. Tuttle J.S. Blackshear P.J. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 944-948Crossref PubMed Scopus (265) Google Scholar, 21.Chen J. Chang S. Duncan S.A. Okano H.J. Fishell G. Aderem A. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 6275-6279Crossref PubMed Scopus (80) Google Scholar). It is well documented that at the cellular level MARCKS proteins mediate cross-talk between CaM and PKC (reviewed in Refs. 22.Schmitz A. Schleiff E. Vergères G. NATO ASI (Adv. Sci. Inst.) Ser. Ser. H Cell Biol. 1997; 101: 127-150Google Scholar and 23.Chakravarthy B. Morley P. Whitfield J. Trends Neurosci. 1999; 22: 12-16Abstract Full Text Full Text PDF PubMed Scopus (127) Google Scholar). Furthermore, these proteins colocalize with filamentous actin or actin-binding proteins in response to a number of cellular events during which a reorganization of the actin cytoskeleton is involved, such as cell spreading (24.Li J. Zhu Z. Bao Z. J. Biol. Chem. 1996; 271: 12985-12990Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar, 25.Manenti S. Malecaze F. Darbon J.M. FEBS Lett. 1997; 419: 95-98Crossref PubMed Scopus (24) Google Scholar), neurosecretion (26.Wu W.S. Walaas S.I. Nairn A.C. Greengard P. Proc. Natl. Acad. Sci. U. S. A. 1982; 79: 5249-5253Crossref PubMed Scopus (205) Google Scholar), or macrophage activation (27.Rosen A. Keenan K.F. Thelen M. Nairn A.C. Aderem A. J. Exp. Med. 1990; 172: 1211-1215Crossref PubMed Scopus (165) Google Scholar). In 1992, Hartwig et al. (28.Hartwig J.H. Thelen M. Rosen A. Janmey P.A. Nairn A.C. Aderem A. Nature. 1992; 356: 618-622Crossref PubMed Scopus (618) Google Scholar) found that MARCKS cross-links actin filaments in vitro. The findings that upon phosphorylation MARCKS is translocated from the membrane (16.Thelen M. Rosen A. Nairn A.C. Aderem A. Nature. 1991; 351: 320-322Crossref PubMed Scopus (308) Google Scholar) and that binding of CaM inhibits its cross-linking of actin filaments (28.Hartwig J.H. Thelen M. Rosen A. Janmey P.A. Nairn A.C. Aderem A. Nature. 1992; 356: 618-622Crossref PubMed Scopus (618) Google Scholar) led to the model that the primary function of MARCKS is to regulate the structure of the actin cytoskeleton by cross-linking actin filaments to networks or bundles at the membrane (28.Hartwig J.H. Thelen M. Rosen A. Janmey P.A. Nairn A.C. Aderem A. Nature. 1992; 356: 618-622Crossref PubMed Scopus (618) Google Scholar). To this end, it is assumed that this protein-protein interaction is mediated to a large extent by the effector domain of MARCKS (3.Aderem A. Cell. 1992; 71: 713-716Abstract Full Text PDF PubMed Scopus (430) Google Scholar). Since the effector domains of MARCKS and MRP exhibit very high sequence homology (96% identity), a similar function has been assumed for MRP. To gain insight into the role of the effector domain during the presumed interaction of MRP with actin, we investigated the effect of a peptide corresponding to the effector domain of MRP, the so-called effector peptide, on rabbit skeletal muscle actin. The 24-amino acid residue-long MRP effector peptide is highly basic and has the following primary sequence:KKKKK FSF KKPF K LSGLSF KRNRK, with the 12 positively charged residues (i.e. 10 Lys and 2 Arg) typed in bold letters and hydrophobic residues in italics. It differs from the MARCKS effector domain mainly in the lack of one arginine after the five N-terminal lysine residues and in the proline residue, which is replaced by a serine in MARCKS (3.Aderem A. Cell. 1992; 71: 713-716Abstract Full Text PDF PubMed Scopus (430) Google Scholar). Thus, both peptides are highly polycationic, but also possess hydrophobic regions which are probably involved in their binding to membranes (29.Qin Z. Cafiso D.S. Biochemistry. 1996; 35: 2917-2925Crossref PubMed Scopus (74) Google Scholar). Experimental evidence has been obtained that the MARCKS effector peptide assumes a random coil structure both in solution (30.Coburn C. Eisenberg J. Eisenberg M. McLaughlin S. Runnels L. Biophys. J. 1993; 64: A60Google Scholar) as well as when bound to CaM (31.Porumb T. Crivici A. Blackshear P.J. Ikura M. Eur. Biophys. J. 1997; 25: 239-247Crossref PubMed Scopus (42) Google Scholar) or membranes (29.Qin Z. Cafiso D.S. Biochemistry. 1996; 35: 2917-2925Crossref PubMed Scopus (74) Google Scholar), and similar properties can be assumed for the MRP effector peptide (31.Porumb T. Crivici A. Blackshear P.J. Ikura M. Eur. Biophys. J. 1997; 25: 239-247Crossref PubMed Scopus (42) Google Scholar). It has been shown that the structure of actin filament networks is strongly altered in the presence of very high concentrations of divalent cations (32.Hanson J. Proc. R. Soc. Lond. B Biol. Sci. 1973; 183: 39-58Crossref PubMed Scopus (76) Google Scholar) or by polycations (33.Oriol-Audit C. Eur. J. Biochem. 1978; 87: 371-376Crossref PubMed Scopus (87) Google Scholar). While high concentrations of divalent and polyvalent cations lead to the formation of paracrystalline arrays (34.Fowler W.E. Aebi U. J. Cell Biol. 1982; 93: 452-458Crossref PubMed Scopus (43) Google Scholar), lower concentrations of polycations like polylysine or [Co(NH3)6]3+ align actin filaments laterally to form bundles (35.Tang J.X. Janmey P.A. J. Biol. Chem. 1996; 271: 8556-8563Abstract Full Text Full Text PDF PubMed Scopus (360) Google Scholar). The effect of polycations can be explained by the condensation theory of Manning (36.Manning G.S. Q. Rev. Biophys. 1978; 11: 179-246Crossref PubMed Scopus (2630) Google Scholar), which assumes that (poly)cations are bound as counterions along the filaments and partially compensate the negative charges on actin, which normally prevent single filaments from forming bundles. In agreement with its polycationic nature, two studies have demonstrated that the effector domain of MARCKS or the corresponding effector peptide bundles actin filaments (28.Hartwig J.H. Thelen M. Rosen A. Janmey P.A. Nairn A.C. Aderem A. Nature. 1992; 356: 618-622Crossref PubMed Scopus (618) Google Scholar, 35.Tang J.X. Janmey P.A. J. Biol. Chem. 1996; 271: 8556-8563Abstract Full Text Full Text PDF PubMed Scopus (360) Google Scholar). The interaction of this peptide with actin was, however, not investigated from a kinetic point of view. This latter aspect is addressed in particular in this article. Moreover, the possibility for a regulatory function of CaM and PKC on the effector peptide activity is assessed by investigating their capability to affect the actin polymerization kinetics or the formation of higher order actin filament structures induced by the MRP effector peptide. Last but not least, to obtain some information on the role of the distinct sequence segments of the peptide during its interaction with actin, three groups of peptides were synthesized in which a particular amino acid segment was deleted or replaced by alanines: In group 1, some or all hydrophobic amino acid residues (i.e. phenylalanine and leucine) are replaced by alanines. In group 2, the N-terminal positively charged Lys residues are replaced by alanines or deleted, with or without replacement of the serine groups or the hydrophobic segments (for further details, see “Experimental Procedures”). In group 3, the central and/or the C-terminal positively charged Lys and Arg residues are replaced by alanines. These group 1 to group 3 “mutant” peptides are evaluated for their effect on both actin polymerization and/or actin filament reorganization into higher order structures, aimed at ultimately acquiring a more rational understanding of the interactions of the intact proteins MARCKS and MRP with actin. Actin was isolated and prepared from rabbit skeletal muscle according to the method of Spudich and Watt (37.Spudich J.A. Watt S. J. Biol. Chem. 1971; 246: 4866-4871Abstract Full Text PDF PubMed Google Scholar), as recently described by Steinmetz et al. (38.Steinmetz M.O. Goldie K.N. Aebi U. J. Cell Biol. 1997; 138: 559-574Crossref PubMed Scopus (106) Google Scholar), and stored at 4 °C in G-buffer (2.5 mm imidazole, pH 7.4, 0.2 mm CaCl2, 0.2 mm ATP) containing 0.001% NaN3. For fluorescence measurements, actin was labeled at cysteine 374 withN-(1-pyrene)iodoacetamide (Molecular Probes Europe B. V., Leiden, Netherlands) following the method of Kouyama and Mihashi (39.Kouyama T. Mihashi K. Eur. J. Biochem. 1981; 114: 33-38Crossref PubMed Scopus (719) Google Scholar). The extent of labeling was typically 80%. Actin concentration and its extent of pyrene labeling were determined as described by Cooperet al. (40.Cooper J.A. Walker S.B. Pollard T.D. J. Muscle Res. Cell Motil. 1983; 4: 253-262Crossref PubMed Scopus (367) Google Scholar). The absorption measurements were performed on a Kontron UVIKON spectrophotometer (Kontron Instruments, Zürich, Switzerland). A peptide corresponding to the effector domain of murine MRP (amino acid residues 86–109), the MRP effector peptide, was obtained from AMS Biotechnology (Lugano, Switzerland). The amino acid composition as well as the concentrations of the effector peptides were determined by quantitative amino acid analysis. CaM from bovine brain was purchased from Sigma (Division of Fluka Chemie AG, Buchs, Switzerland), and the catalytic subunit of rat brain protein kinase C (PKM) was obtained from Calbiochem. “Wild type” MARCKS effector peptide (wt peptide) and the 13 mutant peptides, which were a kind gift of Prof. Felix Althaus from the Tierspital, University of Zürich, Switzerland, were synthesized by Alexis Corp. (Läufelfingen, Switzerland). All other chemicals were purchased from Fluka (Fluka Chemie AG, Buchs, Switzerland) in the highest purity grade available. The following peptides were used (“mutations” are underlined). All peptides were of a purity grade >80% as determined by reversed-phase high performance liquid chromatography analysis (data sheet provided by the manufacturer). Actin polymerization was monitored by following the fluorescence increase of the pyrenated actin moiety, according to Kouyama and Mihashi (39.Kouyama T. Mihashi K. Eur. J. Biochem. 1981; 114: 33-38Crossref PubMed Scopus (719) Google Scholar). Unless indicated otherwise, pyrene-labeled actin was mixed with unlabeled actin to yield a final extent of 5%. 400 μl of actin solution was put into a 5 × 5-mm quartz glass cuvette (Hellma Suprasil, Hellma, Müllheim, Germany), and the fluorescence was recorded at an angle of 90°. The sample was excited at 365 nm with a bandwidth of 1.5 nm, and emission was measured at 407 nm with a bandwidth of 5 nm. To minimize scattering effects, a cut-off filter at 395 nm was used for the emitted light. The measurements were performed on a Jasco Spectrofluorometer FP-777 (Japan Spectroscopic Co., Ltd., Tokyo, Japan). In all experiments, the temperature was kept constant at (20 ± 1) °C. The data were evaluated with EasyPlot 3.0 (by Spiral Software, Brookline, MA and Massachusetts Institute of Technology, Cambridge, MA). Unless indicated otherwise, polymerization of actin was induced by adding MgCl2 to 2 mm and KCl to 50 mm to G-buffer (F-buffer). All polymerization experiments were performed with at least three different actin preparations to ensure that the observed results were not due to variations in the quality of a particular actin preparation. Phosphorylation of the MRP effector peptide at its Ser residues was achieved as described recently (7.Schleiff E. Schmitz A. McIlhinney R.A.J. Manenti S. Vergères G. J. Biol. Chem. 1996; 271: 26794-26802Abstract Full Text Full Text PDF PubMed Scopus (37) Google Scholar). Briefly, 5.4 μm MRP effector peptide was phosphorylated for 9 h at 30 °C in a total volume of 130 μl containing 6 mmMgCl2, 0.1 mm ATP, 0.001% Triton X-100, 2.5 mm imidazole, pH 7.4, and 20 nm PKM. Positive controls included the same reaction mixture except that PKM was substituted by the PKM storage buffer (Calbiochem data sheet). For negative controls, PKM was substituted by the PKM storage buffer, and the effector peptide was substituted by an equal amount of G-buffer. The stoichiometry of phosphorylation was determined by parallel incubation of aliquots with [γ-32P]ATP and by quantification of the incorporated radioactivity as described (7.Schleiff E. Schmitz A. McIlhinney R.A.J. Manenti S. Vergères G. J. Biol. Chem. 1996; 271: 26794-26802Abstract Full Text Full Text PDF PubMed Scopus (37) Google Scholar). A 5-μl aliquot of F-actin was adsorbed to a carbon-coated copper grid rendered hydrophilic by glow discharge at low air pressure. After 30 s, the sample drop was removed, and the grid briefly washed with water, before the sample was negatively stained with 2% uranyl acetate or 0.75% uranyl formiate. Specimens were inspected in a Philips 400 transmission electron microscope operated at 80 kV acceleration voltage, and micrographs were recorded on electron image film (SO-163, Eastman Kodak Co.) at a nominal magnification of × 55,000. For further details of specimen preparation and electron microscopy, see Steinmetz et al. (38.Steinmetz M.O. Goldie K.N. Aebi U. J. Cell Biol. 1997; 138: 559-574Crossref PubMed Scopus (106) Google Scholar). The effector domain of MRP is involved in most of the interactions with its reaction partners, including CaM, membranes, and PKC (5.McLaughlin S. Aderem A. Trends Biochem. Sci. 1995; 20: 272-276Abstract Full Text PDF PubMed Scopus (616) Google Scholar, 17.Kim J. Blackshear P.J. Johnson J.D. McLaughlin S. Biophys. J. 1994; 67: 227-237Abstract Full Text PDF PubMed Scopus (145) Google Scholar,18.Graff J.M. Rajan R.R. Randall R.R. Nairn A.C. Blackshear P.J. J. Biol. Chem. 1991; 266: 14390-14398Abstract Full Text PDF PubMed Google Scholar). Moreover, it has been proposed that the effector domain of MARCKS, which is almost identical to the effector domain of MRP, is essential for its actin cross-linking activity. Hence, we investigated the possibility that a peptide representing the effector domain of MRP might mediate an interaction of MRP with actin. In particular, with respect to the dynamic properties of the actin cytoskeleton, we have investigated these interactions from a kinetic point of view. The 24-amino acid-long effector domain of MRP contains 12 positively charged residues (i.e. 10 Lys and 2 Arg), so it is conceivable that its strong polycationic nature might influence actin polymerization. To assess this question, a peptide consisting of these 24 amino acids, the MRP effector peptide, was added to actin in G-buffer at different stoichiometries. Immediately upon addition, the effector peptide causes actin to polymerize in a concentration-dependent fashion (Fig.1 a, curves 3 and4). Upon reaching steady state, 2 mmMgCl2 and 50 mm KCl were added to polymerize the remaining actin (arrows). Comparison of the steady-state values in the absence and presence of Mg2+/K+clearly documents that the fraction of polymerized actin correlates with the concentration of effector peptide. This concentration dependence suggests the formation of a stoichiometric complex between actin and the effector peptide. These results can best be explained by assuming that the effector peptide binds to actin monomers and saturates the low affinity cation binding sites, which are normally occupied by mono- and divalent cations (41.Selden L.A. Estes J.E. Gershman L.C. J. Biol. Chem. 1989; 264: 9271-9277Abstract Full Text PDF PubMed Google Scholar, 42.Carlier M.-F. Pantaloni D. Korn E.D. J. Biol. Chem. 1986; 261: 10785-10792Abstract Full Text PDF PubMed Google Scholar, 43.Estes J.E. Selden L.A. Kinosian H.J. Gershman L.C. J. Muscle Res. Cell Motil. 1992; 13: 272-284Crossref PubMed Scopus (113) Google Scholar). In this way, the effector peptide could “mimic” the cations that lead to filament formation under physiological salt concentrations. Hence, it might be argued that the observed polymerization reaction is an unspecific event that could be initiated by many different polycations (34.Fowler W.E. Aebi U. J. Cell Biol. 1982; 93: 452-458Crossref PubMed Scopus (43) Google Scholar, 44.Brown S. Spudich J. J. Cell Biol. 1979; 80: 499-504Crossref PubMed Scopus (47) Google Scholar). To test this hypothesis, polymerization of G-actin was induced by addition of polylysine at an equimolar actin:Lys18 ratio. As can be gathered fromcurve 1 in Fig. 1 a, polylysine is indeed capable of inducing actin polymerization in the absence of any other mono- or multivalent cations, but the kinetics is about 2 orders of magnitude slower than when using MRP effector peptide. A comparably fast polymerization kinetics was only observed with polylysines of a very high degree of polymerization (for example, Lys38; data not shown). Evidently, the effector peptide interacts with actin monomers in a specific manner. We propose that the effector peptide has a distinct structure (e.g. amino acid sequence, secondary structure, aggregation state, or charge distribution), which, together with electrostatic interactions, leads to very fast association of the peptide with actin monomers in the absence of any other cations. Since the experiments described so far were performed in a rather unphysiological environment, it is necessary to also evaluate how the MRP effector peptide affects actin filament formation when polymerization is induced by addition of monovalent and divalent cationic salts; upon increasing the ionic strength I by a factor of 100–200 by adding 2 mm MgCl2, 50 mm KCl, the Debye-length λD will decrease approximately by a factor of 10 (since λD∝1/√I) (45.Atkins P.W. Physical Chemistry. 5th Ed. Oxford University Press, Oxford, Great Britain1997: A8-A10Google Scholar). Therefore, the interaction between actin monomers and the MRP peptide should be attenuated. Fig.1 b displays the fluorescence signal upon addition of an equimolar concentration of MRP effector peptide to 4 μmpyrene-labeled G-actin in the presence of 2 mmMgCl2 and 50 mm KCl. As expected, the actin polymerization velocity is still markedly increased (curve 2), however, at a considerably slower rate than at low salt concentration. The relatively long lag phase indicates that elongation rather than nucleation is enhanced by the peptide. It should be noted that more effector peptide is required to enhance actin polymerization when increasing the KCl concentration from 50 to 100 mm (data not shown). This suggests that the binding of the effector peptide to actin is, at least to some extent, governed by electrostatic interactions. However, the mechanism of the interaction is independent of the salt concentration. Only the effective concentration of effector peptide complexed with actin is lowered. A rather puzzling result is found when the amount of peptide is lowered to substoichiometric concentrations at a given salt concentration; compared with control experiments with actin, polymerized in the absence of the effector peptide, the polymerization rate is decreased (data not shown). We have recently developed a model approach that can qualitatively account for this interesting phenomenon (49.Wohnsland, F., Schmitz, A. A. P., Steinmetz, M. O., Aebi, U., and Vergères, G. (2000) Biophys. Chem., in pressGoogle Scholar). In 1982, Fowler and Aebi (34.Fowler W.E. Aebi U. J. Cell Biol. 1982; 93: 452-458Crossref PubMed Scopus (43) Google Scholar) documented the formation of F-actin bundles upon addition of polyvalent cations such as polylysine to F-actin. Similarly, bundling of F-actin filaments, which had previously been polymerized in F-buffer, has also been reported for a peptide corresponding to the MARCKS effector domain (35.Tang J.X. Janmey P.A. J. Biol. Chem. 1996; 271: 8556-8563Abstract Full Text Full Text PDF PubMed Scopus (360) Google Scholar). To test whether the MRP effector peptide exhibits a similar behavior, G-actin was incubated at a 1:1 molar ratio with the MRP effector peptide, and aliquots were prepared for and observed by transmission electron microscopy by negative staining after different incubation times. The micrographs displayed in Fig. 2, a–c, clearly document that, even in the absence of mono- or divalent cations, the MRP effector peptide induces the formation of actin filament bundles. Interestingly, after short incubation times (less than 5 min) occasionally very short bundles appeared that aligned along already existing filament bundles (Fig. 2 c) so as to anneal longitudinally into longer filament bundles, indicating that lateral association of filaments occurs before significant filament elongation takes place. The MARCKS effector peptide leads to the formation of actin bundles at a considerably lower stoichiometry than polycations of comparable charge (35.Tang J.X. Janmey P.A. J. Biol. Chem. 1996; 271: 8556-8563Abstract Full Text Full Text PDF PubMed Scopus (360) Google Scholar). This is in good agreement with the observed higher polymerizing activity of the MRP effector peptide, confirming a high specificity for actin. The electron micrographs recorded during the initial phase of filament formation (see Fig. 2 c) further document the assumption that two processes contribute to the drastic spontaneous polymerization in the absence of salt: 1) upon addition of the effector peptide to a G-actin solution, peptide molecules bind with very high affinity to actin monomers, thereby activating them by inducing a similar conformational change as is caused by mono- or divalent cations (as described, e.g. in Refs. 42.Carlier M.-F. Pantaloni D. Korn E.D. J. Biol. Chem. 1986; 261: 10785-10792Abstract Full Text PDF PubMed Google Scholar, 46.Carlier M.-F. Pantaloni D. Korn E.D. J. Biol. Chem. 1986; 261: 10778-10784Abstract Full Text PDF PubMed Google Scholar, and47.Selden L.A. Gershman L.C. Estes J.E. J. Muscle Res. Cell Motil. 1986; 7: 215-224Crossref PubMed Scopus (35) Google Scholar); 2) this monomer binding/activating step, in turn, greatly enhances polymerization into filaments. Last but not least, it is worth noting that the drastic effect of effector peptide on actin polymerization was also observed with the MARCKS effector peptide instead of the MRP effector peptide (data not shown), indicating that" @default.
• W1996568154 created "2016-06-24" @default.
• W1996568154 creator A5008287463 @default.
• W1996568154 creator A5011926747 @default.
• W1996568154 creator A5041503324 @default.
• W1996568154 creator A5058630619 @default.
• W1996568154 creator A5063261046 @default.
• W1996568154 date "2000-07-01" @default.
• W1996568154 modified "2023-10-13" @default.
• W1996568154 title "Interaction between Actin and the Effector Peptide of MARCKS-related Protein" @default.
• W1996568154 cites W1508801967 @default.
• W1996568154 cites W1517924156 @default.
• W1996568154 cites W1538692246 @default.
• W1996568154 cites W1565185374 @default.
• W1996568154 cites W1567986819 @default.
• W1996568154 cites W1568484052 @default.
• W1996568154 cites W1580075072 @default.
• W1996568154 cites W1593448218 @default.
• W1996568154 cites W1593795070 @default.
• W1996568154 cites W1593910029 @default.
• W1996568154 cites W1599345755 @default.
• W1996568154 cites W1600704552 @default.
• W1996568154 cites W1964199147 @default.
• W1996568154 cites W1966797972 @default.
• W1996568154 cites W1976319209 @default.
• W1996568154 cites W1979553346 @default.
• W1996568154 cites W1979607880 @default.
• W1996568154 cites W1992976076 @default.
• W1996568154 cites W1994045773 @default.
• W1996568154 cites W2009863318 @default.
• W1996568154 cites W2010955080 @default.
• W1996568154 cites W2019861301 @default.
• W1996568154 cites W2036728243 @default.
• W1996568154 cites W2037902728 @default.
• W1996568154 cites W2037943750 @default.
• W1996568154 cites W2038864295 @default.
• W1996568154 cites W2039448579 @default.
• W1996568154 cites W2042251760 @default.
• W1996568154 cites W2056629354 @default.
• W1996568154 cites W2061292285 @default.
• W1996568154 cites W2061834380 @default.
• W1996568154 cites W2062574001 @default.
• W1996568154 cites W2065609713 @default.
• W1996568154 cites W2066768102 @default.
• W1996568154 cites W2071354308 @default.
• W1996568154 cites W2071887382 @default.
• W1996568154 cites W2083643225 @default.
• W1996568154 cites W2102834112 @default.
• W1996568154 cites W21038042 @default.
• W1996568154 cites W2119091806 @default.
• W1996568154 cites W2134395967 @default.
• W1996568154 cites W2152173743 @default.
• W1996568154 cites W2159662555 @default.
• W1996568154 cites W2166157292 @default.
• W1996568154 cites W4297917728 @default.
• W1996568154 doi "https://doi.org/10.1074/jbc.m910298199" @default.
• W1996568154 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/10748210" @default.
• W1996568154 hasPublicationYear "2000" @default.
• W1996568154 type Work @default.
• W1996568154 sameAs 1996568154 @default.
• W1996568154 citedByCount "47" @default.
• W1996568154 countsByYear W19965681542012 @default.
• W1996568154 countsByYear W19965681542013 @default.
• W1996568154 countsByYear W19965681542014 @default.
• W1996568154 countsByYear W19965681542017 @default.
• W1996568154 countsByYear W19965681542018 @default.
• W1996568154 countsByYear W19965681542019 @default.
• W1996568154 countsByYear W19965681542020 @default.
• W1996568154 countsByYear W19965681542021 @default.
• W1996568154 crossrefType "journal-article" @default.
• W1996568154 hasAuthorship W1996568154A5008287463 @default.
• W1996568154 hasAuthorship W1996568154A5011926747 @default.
• W1996568154 hasAuthorship W1996568154A5041503324 @default.
• W1996568154 hasAuthorship W1996568154A5058630619 @default.
• W1996568154 hasAuthorship W1996568154A5063261046 @default.
• W1996568154 hasBestOaLocation W19965681541 @default.
• W1996568154 hasConcept C11960822 @default.
• W1996568154 hasConcept C125705527 @default.
• W1996568154 hasConcept C185592680 @default.
• W1996568154 hasConcept C2779281246 @default.
• W1996568154 hasConcept C2780780085 @default.
• W1996568154 hasConcept C51785407 @default.
• W1996568154 hasConcept C55493867 @default.
• W1996568154 hasConcept C77207865 @default.
• W1996568154 hasConcept C86803240 @default.
• W1996568154 hasConcept C95444343 @default.
• W1996568154 hasConceptScore W1996568154C11960822 @default.
• W1996568154 hasConceptScore W1996568154C125705527 @default.
• W1996568154 hasConceptScore W1996568154C185592680 @default.
• W1996568154 hasConceptScore W1996568154C2779281246 @default.
• W1996568154 hasConceptScore W1996568154C2780780085 @default.
• W1996568154 hasConceptScore W1996568154C51785407 @default.
• W1996568154 hasConceptScore W1996568154C55493867 @default.
• W1996568154 hasConceptScore W1996568154C77207865 @default.
• W1996568154 hasConceptScore W1996568154C86803240 @default.
• W1996568154 hasConceptScore W1996568154C95444343 @default.
• W1996568154 hasIssue "27" @default.
• W1996568154 hasLocation W19965681541 @default.

• next