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Functional Characterization of Human Myosin-18A and Its Interaction with F-actin and GOLPH3*

  • Manuel H. Taft
    Correspondence
    To whom correspondence should be addressed: Institut für Biophysikalische Chemie, Medizinische Hochschule Hannover, Carl-Neuberg-Str. 1, 30625 Hannover, Germany. Tel.: 49-511-5328657; Fax: 49-511-5322909;
    Affiliations
    From the Institute for Biophysical Chemistry, Hannover Medical School, OE 4350, Carl-Neuberg-Strasse 1, 30625 Hannover, Germany and
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  • Elmar Behrmann
    Footnotes
    Affiliations
    the Department of Physical Biochemistry, Max-Planck-Institute of Molecular Physiology, 44227 Dortmund, Germany
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  • Lena-Christin Munske-Weidemann
    Affiliations
    From the Institute for Biophysical Chemistry, Hannover Medical School, OE 4350, Carl-Neuberg-Strasse 1, 30625 Hannover, Germany and
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  • Claudia Thiel
    Affiliations
    From the Institute for Biophysical Chemistry, Hannover Medical School, OE 4350, Carl-Neuberg-Strasse 1, 30625 Hannover, Germany and
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  • Stefan Raunser
    Affiliations
    the Department of Physical Biochemistry, Max-Planck-Institute of Molecular Physiology, 44227 Dortmund, Germany
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  • Dietmar J. Manstein
    Affiliations
    From the Institute for Biophysical Chemistry, Hannover Medical School, OE 4350, Carl-Neuberg-Strasse 1, 30625 Hannover, Germany and
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  • Author Footnotes
    * This work was supported by Deutsche Forschungsgemeinschaft Grants MA 1081/19–1 (to D. J. M.) and RA 1781/1–1 (to S. R.), Fonds der Chemischen Industrie Grant 684052 (to E. B.), and the Max Planck Society (to S. R. and E. B.).
    2 Present address: Institute of Medical Physics and Biophysics, Charité, Universitätsmedizin Berlin, 10117 Berlin, Germany.
Open AccessPublished:August 29, 2013DOI:https://doi.org/10.1074/jbc.M113.497180
      Molecular motors of the myosin superfamily share a generic motor domain region. They commonly bind actin in an ATP-sensitive manner, exhibit actin-activated ATPase activity, and generate force and movement in this interaction. Class-18 myosins form heavy chain dimers and contain protein interaction domains located at their unique N-terminal extension. Here, we characterized human myosin-18A molecular function in the interaction with nucleotides, F-actin, and its putative binding partner, the Golgi-associated phosphoprotein GOLPH3. We show that myosin-18A comprises two actin binding sites. One is located in the KE-rich region at the start of the N-terminal extension and appears to mediate ATP-independent binding to F-actin. The second actin-binding site resides in the generic motor domain and is regulated by nucleotide binding in the absence of intrinsic ATP hydrolysis competence. This core motor domain displays its highest actin affinity in the ADP state. Electron micrographs of myosin-18A motor domain-decorated F-actin filaments show a periodic binding pattern independent of the nucleotide state. We show that the PDZ module mediates direct binding of myosin-18A to GOLPH3, and this interaction in turn modulates the actin binding properties of the N-terminal extension. Thus, myosin-18A can act as an actin cross-linker with multiple regulatory modulators that targets interacting proteins or complexes to the actin-based cytoskeleton.
      Background: Class-18A myosins share a unique N-terminal extension comprising a PDZ module and a KE-rich region.
      Results: Human myosin-18A binds F-actin via its motor domain in a nucleotide-dependent manner and via the KE-rich region, modulated by direct interaction between the PDZ module and GOLPH3.
      Conclusion: Myosin-18A binds F-actin and recruits interaction partners to the cytoskeleton.
      Significance: This work establishes a molecular basis for myosin-18A mediated membrane-cytoskeleton interplay.

      Introduction

      Myosins constitute a large superfamily of molecular motors that use the chemical energy provided by ATP hydrolysis to cyclically interact with filamentous F-actin and generate force and movement (
      • Bloemink M.J.
      • Geeves M.A.
      Shaking the myosin family tree. Biochemical kinetics defines four types of myosin motor.
      ). All myosins share a generic motor domain that harbors the binding sites for ATP and F-actin. Based on sequence alignments of the motor domain, myosins can be grouped into 35 classes (
      • Odronitz F.
      • Kollmar M.
      Drawing the tree of eukaryotic life based on the analysis of 2,269 manually annotated myosins from 328 species.
      ). In humans, 40 genes are found that encode for myosins from 13 of these classes. The molecular details of the mechanochemical transduction of energy by myosins from different classes have been unraveled with great accuracy. Nevertheless, most myosins have not been characterized in depth, in particular members of the myosin family, such as class-18 myosins, which show distinct structural features setting them apart. Class-18 myosins are found in various species, from vertebrates to arthropods. They contain protein interaction domains that are located at their N terminus outside the motor domain (
      • Furusawa T.
      • Ikawa S.
      • Yanai N.
      • Obinata M.
      Isolation of a novel PDZ-containing myosin from hematopoietic supportive bone marrow stromal cell lines.
      ). Like the founding member of this myosin class, MysPDZ (now termed mouse myosin-18A), human myosin-18A comprises a region rich in lysine and glutamate residues (KE) and a PDZ
      The abbreviations used are: PDZ
      PSD-95/Discs-Large/ZO-1
      TRITC
      tetramethylrhodamine isothiocyanate
      M18A-MD
      myosin-18A motor domain
      mant
      2′/3′-O-(N-methyl-anthraniloyl)
      SH2 and SH3
      Src homology 2 and 3, respectively
      TCEP
      tris(2-carboxyethyl)phosphine
      CM-loop
      cardiomyopathy loop.
      module in its N-terminal extension. This domain is followed by a generic motor domain with an adjacent neck domain that can bind essential and regulatory light chains (
      • Guzik-Lendrum S.
      • Heissler S.M.
      • Billington N.
      • Takagi Y.
      • Yang Y.
      • Knight P.J.
      • Homsher E.
      • Sellers J.R.
      Mammalian myosin-18A, a highly divergent myosin.
      ). The tail domain contains long stretches of coiled-coils that support heavy chain dimerization (
      • Isogawa Y.
      • Kon T.
      • Inoue T.
      • Ohkura R.
      • Yamakawa H.
      • Ohara O.
      • Sutoh K.
      The N-terminal domain of MYO18A has an ATP-insensitive actin-binding site.
      ). The molecular mass of the protein varies between 180 kDa for the shortest isoform, which lacks the N-terminal extension (myosin-18Aβ), and 233 kDa for the longest isoform, termed myosin-18Aα. A recent study identified the gene encoding human myosin-18A to be alternatively spliced in non-small cell lung cancer, leading to in-frame variations in the protein sequence (
      • Langer W.
      • Sohler F.
      • Leder G.
      • Beckmann G.
      • Seidel H.
      • Gröne J.
      • Hummel M.
      • Sommer A.
      Exon array analysis using re-defined probe sets results in reliable identification of alternatively spliced genes in non-small cell lung cancer.
      ). Furthermore, the gene was identified as a partner in the three-way chromosomal translocation of stem cell leukemia-lymphoma syndrome (
      • Walz C.
      • Chase A.
      • Schoch C.
      • Weisser A.
      • Schlegel F.
      • Hochhaus A.
      • Fuchs R.
      • Schmitt-Gräff A.
      • Hehlmann R.
      • Cross N.C.
      • Reiter A.
      The t(8;17)(p11;q23) in the 8p11 myeloproliferative syndrome fuses MYO18A to FGFR1.
      ) and forms the fusion gene MYO18A-PDGFRB in eosinophilia-associated atypical myeloproliferative neoplasms (
      • Walz C.
      • Haferlach C.
      • Hänel A.
      • Metzgeroth G.
      • Erben P.
      • Gosenca D.
      • Hochhaus A.
      • Cross N.C.
      • Reiter A.
      Identification of a MYO18A-PDGFRB fusion gene in an eosinophilia-associated atypical myeloproliferative neoplasm with a t(5;17)(q33–34;q11.2).
      ). A three-way translocation of the highly promiscuous oncogene MLL, a histone methyltransferase, and the reciprocal partner gene MYO18A was described in acute myeloid leukemia (
      • Ussowicz M.
      • Jaśkowiec A.
      • Meyer C.
      • Marschalek R.
      • Chybicka A.
      • Szczepański T.
      • Haus O.
      A three-way translocation of MLL, MLLT11, and the novel reciprocal partner gene MYO18A in a child with acute myeloid leukemia.
      ). These studies suggest that functional myosin-18A protein is required for the normal regulation of the cell cycle and the suppression of key processes involved in cancer progression.
      Up to now, information on the biochemical properties of class-18 myosins is scarce and in parts controversial. The function of the unique N-terminal extension is only poorly understood (
      • Mori K.
      • Furusawa T.
      • Okubo T.
      • Inoue T.
      • Ikawa S.
      • Yanai N.
      • Mori K.J.
      • Obinata M.
      Genome structure and differential expression of two isoforms of a novel PDZ-containing myosin (MysPDZ) (Myo18A).
      ,
      • Mori K.
      • Matsuda K.
      • Furusawa T.
      • Kawata M.
      • Inoue T.
      • Obinata M.
      Subcellular localization and dynamics of MysPDZ (Myo18A) in live mammalian cells.
      ). In a recent study, Drosophila myosin-18 has been found to be an actin-binding protein that does not bind nucleotide, has no ATPase activity, and cannot actively translocate over actin filaments (
      • Guzik-Lendrum S.
      • Nagy A.
      • Takagi Y.
      • Houdusse A.
      • Sellers J.R.
      Drosophila melanogaster myosin-18 represents a highly divergent motor with actin tethering properties.
      ). A current publication of the same group on functional features of mouse myosin-18A reports actin and nucleotide binding properties but no significant ATPase activity and suggests that this myosin is not a traditional motor (
      • Guzik-Lendrum S.
      • Heissler S.M.
      • Billington N.
      • Takagi Y.
      • Yang Y.
      • Knight P.J.
      • Homsher E.
      • Sellers J.R.
      Mammalian myosin-18A, a highly divergent myosin.
      ). Moreover, the amino acid sequence of active site elements of the myosin-18 motor domain exhibits changes in highly conserved regions, which can prevent myosin-18 from productively interacting with ATP in the same way as other myosins.
      Furthermore, it has been shown that the N-terminal extension of human myosin-18A has an ATP-insensitive actin-binding site outside the PDZ module (
      • Isogawa Y.
      • Kon T.
      • Inoue T.
      • Ohkura R.
      • Yamakawa H.
      • Ohara O.
      • Sutoh K.
      The N-terminal domain of MYO18A has an ATP-insensitive actin-binding site.
      ). It was suggested that the motor domain of human myosin-18A does not bind to actin, because YFP-tagged motor domain constructs obtained from cell lysates do not cosediment with actin. This observation is in contrast to the studies on Drosophila myosin-18 and mouse myosin-18A that attribute actin binding properties to the motor domain.
      Recently, it has been shown that myosin-18Aα is a novel binding partner of the PAK2·βPIX·GIT1 complex (
      • Hsu R.-M.
      • Tsai M.-H.
      • Hsieh Y.-J.
      • Lyu P.-C.
      • Yu J.-S.
      Identification of MYO18A as a novel interacting partner of the PAK2/βPIX/GIT1 complex and its potential function in modulating epithelial cell migration.
      ). This suggests that myosin-18A may play an important role in regulating epithelial cell migration. The Rac/Cdc42-binding kinase MRCK (myotonic dystrophy kinase-related Cdc42-binding kinase) has been shown to associate with the myosin-18Aα PDZ domain via a linker protein, LRAP35a (
      • Tan I.
      • Yong J.
      • Dong J.M.
      • Lim L.
      • Leung T.
      A tripartite complex containing MRCK modulates lamellar actomyosin retrograde flow.
      ). The resulting phosphorylation of the non-muscle RLC2A (myosin-2A regulatory light chain) suggests an association of myosin-18Aα with RLC2A. This hypothesis is supported by the fact that in vitro mouse myosin-18A binds essential and regulatory light chains via its neck region (
      • Guzik-Lendrum S.
      • Heissler S.M.
      • Billington N.
      • Takagi Y.
      • Yang Y.
      • Knight P.J.
      • Homsher E.
      • Sellers J.R.
      Mammalian myosin-18A, a highly divergent myosin.
      ). The tripartite MRCK·LRAP35a·myosin-18Aα complex localizes to lamellar actomyosin bundles, where non-muscle myosin-2A drives the retrograde flow (
      • Tan I.
      • Yong J.
      • Dong J.M.
      • Lim L.
      • Leung T.
      A tripartite complex containing MRCK modulates lamellar actomyosin retrograde flow.
      ,
      • Heissler S.M.
      • Manstein D.J.
      Nonmuscle myosin-2. Mix and match.
      ). Therefore, myosin-18A can play a role the regulation and organization of the actin cytoskeleton within lamellipodia.
      Additionally, Dippold et al. (
      • Dippold H.C.
      • Ng M.M.
      • Farber-Katz S.E.
      • Lee S.-K.
      • Kerr M.L.
      • Peterman M.C.
      • Sim R.
      • Wiharto P.A.
      • Galbraith K.A.
      • Madhavarapu S.
      • Fuchs G.J.
      • Meerloo T.
      • Farquhar M.G.
      • Zhou H.
      • Field S.J.
      GOLPH3 bridges phosphatidylinositol-4-phosphate and actomyosin to stretch and shape the Golgi to promote budding.
      ) have shown that GOLPH3 binds to myosin-18A and connects the Golgi apparatus to F-actin to provide a tensile force required for efficient tubule and vesicle formation. However, this function would presumably implicate active motor properties for myosin-18A.
      Here, we show that human myosin-18A contains two distinct actin binding sites per heavy chain (four per dimer), one of which is regulated by nucleotide binding and is capable of targeting interacting proteins to the actin cytoskeleton, where it can function as an efficient and adjustable actin cross-linker. The PDZ module is shown to mediate direct binding of myosin-18A to GOLPH3, and this interaction modulates the actin binding properties of the unique N-terminal extension of myosin-18A.

      DISCUSSION

      In the present study, we aimed to elucidate the molecular properties of three fundamental human myosin-18A functional domains: the KE-rich region and the PDZ module, which together constitute the N-terminal extension, as well as the generic myosin motor domain. We established the soluble expression and purification of different constructs, using two expression systems; a minimal construct encompassing the core motor domain (M18A-MD) and a motor domain construct including the N-terminally located PDZ module (PDZ-M18A-MD) were successfully produced in the Sf9/insect cell system. The N-terminal extension construct (KEPDZ) as well as the separate KE-rich region (KE) and the PDZ module (PDZ) were produced in E. coli and purified to homogeneity. In addition, we expressed the phosphoprotein GOLPH3 as a GST fusion protein in E. coli and utilized protease cleavage to obtain a “tag-free” version for interaction studies. Using these constructs, we studied the biochemical properties of the individual myosin-18A domains to assemble an overall picture of the protein's molecular mechanism.
      We show that the core myosin-18A motor domain binds mant-labeled ATP and ADP nucleotides but does not exhibit intrinsic basal or actin-activated ATPase activity. Nucleotide binding modulates the actin affinity of the motor domain and regulates its partitioning between an actin binding-competent and -incompetent state. The ADP-bound state has the highest actin affinity, and complete fractional binding of the myosin-18A motor domain to the actin filament is only observed in this state. These results are supported by the fact that in electron micrographs, we observe complete decoration of F-actin filaments with the typical arrowhead appearance of the myosin motor domain in all nucleotide states. Like generic myosin motors, the myosin-18A motor domain appears to dock with similar orientation and employing conserved surface contacts to the actin filament. Accordingly, the myosin-18A motor domain has preserved the myosin-inherent ability to bind nucleotide and to couple the conformational information from the nucleotide binding pocket to the actin binding site.
      The core motor domain of myosin-18A, omitting the N-terminal extension, comprises 786 amino acids. It is considerably extended compared with human skeletal muscle myosin-2 (701 amino acids without the N-terminal SH3-like subdomain) or D. discoideum myosin-2 (685 amino acids without SH3). The underlying insertions are distributed over the nucleotide binding pocket elements (switch-2 region), the actin binding regions (cardiomyopathy loop (CM-loop) and activation loop), and prominent surface loops (near the SH2 domain) of the motor domain. We prepared a structural model of the motor domain using the I-TASSER protein structure and function prediction server (
      • Roy A.
      • Kucukural A.
      • Zhang Y.
      I-TASSER. A unified platform for automated protein structure and function prediction.
      ,
      • Wu S.
      • Skolnick J.
      • Zhang Y.
      Ab initio modeling of small proteins by iterative TASSER simulations.
      ). The model shows the location of these inserts and their likely effect on conserved structural elements in the myosin motor domain (Fig. 8, A and B; alignment depicted in Fig. 8C).
      Figure thumbnail gr8
      FIGURE 8Structural model of the H. sapiens myosin-18A motor domain. The D. discoideum myosin-2 motor domain structure (Protein Data Bank code 1G8X) was used as a template to generate a homology model of the human myosin-18A motor domain (residues 399–1185 of the full-length sequence). A, the overall fold of the motor domain of human myosin-18A displays high similarity with generic myosin motor domains. Nevertheless, H. sapiens myosin-18A contains four major insertions in the motor domain sequence, which are located near switch-2 (pink; 6 residues), at the CM-loop (blue; 14 residues), at the activation loop (orange; 13 residues), and preceding the SH2 helix (yellow; 29 residues). ADP is shown in a stick representation with black carbon atoms; the orange sphere designates the location of the Mg2+ ion. B, close-up view of the nucleotide binding pocket. The molecule was subjected to a left-handed rotation of about 45º around a vertical axis through the Mg2+ ion. Important features of the binding pocket are colored as follows: cyan, P-loop (GSSGSGKT); red, switch-1 (NGNATR); light blue, switch-2 (DTPGFQ). C, multiple-sequence alignment of the motor domain (MD) of D. discoideum (Dd) myosin-2 and the motor domains of human (Hs) myosin-18A, mouse (Mm) myosin-18A, and D. melanogaster (Dm) myosin-18. Important myosin motor domain features are indicated and labeled. The gray shaded box marks Glu-459 (D. discoideum), which constitutes the salt bridge with Arg-238 in D. discoideum myosin-2.
      The switch-2 region of myosin-18A displays an insertion of six residues, and the conserved glutamic acid in switch-2 is changed to glutamine (pink in Fig. 8, A and B). Previously, it has been shown that this residue plays a key role in the mechanism of chemo-mechanical coupling (
      • Onishi H.
      • Kojima S.
      • Katoh K.
      • Fujiwara K.
      • Martinez H.M.
      • Morales M.F.
      Functional transitions in myosin. Formation of a critical salt-bridge and transmission of effect to the sensitive tryptophan.
      ) and that mutation of this residue abolishes the cellular functions of myosin-2 in D. discoideum cells (
      • Furch M.
      • Fujita-Becker S.
      • Geeves M.A.
      • Holmes K.C.
      • Manstein D.J.
      Role of the salt-bridge between switch-1 and switch-2 of Dictyostelium myosin.
      ). Accordingly, the elongation of the switch-2 region in myosin-18A by a flexible linker (GGSARGA) and the disturbed salt bridge between switch-1 and switch-2 are possible structural determinants for the absent intrinsic ATPase activity of the motor domain.
      Furthermore, the region preceding the SH2 domain contains a 29-residue surface loop extension (yellow in Fig. 8, A and B). This flexible loop is located near the entrance of the nucleotide binding pocket and comprises multiple serine residues. We observed biphasic ATP and ADP binding kinetics that may possibly be attributed to the existence of two structural states of this extended SH2 loop modulating nucleotide interactions and triggering slow and fast binding and release kinetics. This interpretation is supported by the fact that both slow and fast rates for ATP and ADP binding are dependent on nucleotide concentration. The biphasic nucleotide binding behavior has not been observed for the mouse myosin-18A S1 construct (
      • Guzik-Lendrum S.
      • Heissler S.M.
      • Billington N.
      • Takagi Y.
      • Yang Y.
      • Knight P.J.
      • Homsher E.
      • Sellers J.R.
      Mammalian myosin-18A, a highly divergent myosin.
      ). Also, the overall nucleotide affinities of human M18A-MD are about 30 times higher than for the mouse isoform, and the ADP-mediated switching to full binding ability has not been described before. Because mouse and human isoforms of myosin-18A share complete amino acid sequence conservation in the SH2 loop extension, this is not the only determinant for the nucleotide binding properties of myosin-18A.
      The CM-loop is located at the tip of the motor domain and involved in the actomyosin binding interface. Remarkably, in myosin-18A, the loop is considerably modified because it contains 14 additional amino acids (blue in Fig. 8A). This enables higher flexibility of the loop and alters the actin binding properties in comparison with generic myosin motor domains. A more flexible and elongated CM-loop might render the motor's actin affinity more independent from cleft closure and thereby permit high actin affinity that is less influenced by the nucleotide state of the active site than in other myosins.
      The activation loop, which is part of the actin-myosin interface, is located within helix HR in the helix-loop-helix actin binding motif (orange in Fig. 8A) (
      • Várkuti B.H.
      • Yang Z.
      • Kintses B.
      • Erdélyi P.
      • Bárdos-Nagy I.
      • Kovács A.L.
      • Hári P.
      • Kellermayer M.
      • Vellai T.
      • Málnási-Csizmadia A.
      A novel actin binding site of myosin required for effective muscle contraction.
      ,
      • Behrmann E.
      • Müller M.
      • Penczek P.A.
      • Mannherz H.G.
      • Manstein D.J.
      • Raunser S.
      Structure of the rigor actin-tropomyosin-myosin complex.
      ). In human myosin-18A, this loop is elongated by 13 extra amino acids that introduce additional positive charges (three arginine residues instead of one in D. discoideum myosin-2), which interferes with regular activation by this loop (cf. Fig. 8C).
      The actin affinities of the human myosin-18A motor domain in the absence and presence of nucleotide are 2 orders of magnitude stronger than for mouse myosin-18A S1 (Table 1). Accordingly, for the mouse isoform, no decoration of F-actin filaments could be observed by electron microscopic investigation. In contrast, M18A-MD shows the classic arrowhead decoration of F-actin filaments. The sequence comparison (Fig. 8C) of the actin binding regions (CM-loop and activation loop) displays only subtle differences between the two myosin isoforms. Nevertheless, in their study, Guzik-Lendrum et al. (
      • Guzik-Lendrum S.
      • Heissler S.M.
      • Billington N.
      • Takagi Y.
      • Yang Y.
      • Knight P.J.
      • Homsher E.
      • Sellers J.R.
      Mammalian myosin-18A, a highly divergent myosin.
      ) utilized an S1 construct comprising a short N-terminal sequence preceding the motor domain and a neck region with bound essential and regulatory light chains. A previous study revealed differences for chicken skeletal muscle myosin S1 with wild-type versus truncated essential light chains in actin binding and ATPase activity, suggesting a direct interaction of the essential light chain N terminus with actin that is regulated by the SH3-like subdomain of myosin (
      • Lowey S.
      • Saraswat L.D.
      • Liu H.
      • Volkmann N.
      • Hanein D.
      Evidence for an interaction between the SH3 domain and the N-terminal extension of the essential light chain in class II myosins.
      ). Accordingly, one could speculate about a similar mechanism for myosin-18A, where the short N-terminal sequence of the mouse myosin-18A S1 construct used in the study of Guzik-Lendrum et al. (
      • Guzik-Lendrum S.
      • Heissler S.M.
      • Billington N.
      • Takagi Y.
      • Yang Y.
      • Knight P.J.
      • Homsher E.
      • Sellers J.R.
      Mammalian myosin-18A, a highly divergent myosin.
      ) and the bound light chains modulate the actin (and maybe also nucleotide) affinity of myosin-18A.
      The biochemical characterization of modules that reside in the large N-terminal extension of human myosin-18Aα provides information on the molecular function of this myosin in the cellular context. Two previous studies proposed ATP-independent actin binding for the N-terminal subdomain of myosin-18Aα (
      • Isogawa Y.
      • Kon T.
      • Inoue T.
      • Ohkura R.
      • Yamakawa H.
      • Ohara O.
      • Sutoh K.
      The N-terminal domain of MYO18A has an ATP-insensitive actin-binding site.
      ,
      • Mori K.
      • Matsuda K.
      • Furusawa T.
      • Kawata M.
      • Inoue T.
      • Obinata M.
      Subcellular localization and dynamics of MysPDZ (Myo18A) in live mammalian cells.
      ). In both studies, immunoprecipitation from human cell line-derived cell lysates showed an interaction of the N-terminal extension of myosin-18A with actin filaments. In expanding these studies, we utilized purified bacterially expressed N-terminal subdomain constructs to define binding affinities and saturation ratios for F-actin. We confirm that the actin binding site resides within the KE-rich subdomain (amino acids 1–219 of human myosin-18Aα) with intermediate affinity and full binding ability. Although the isolated PDZ module does not interact with actin, it significantly strengthens the actin affinity of the N-terminal extension. Remarkably, the direct interaction of GOLPH3 with the PDZ module leads to changes in the actin binding properties, and this interplay acts as a modulator for the KE motif-mediated cytoskeleton interaction. Likewise, the affinity of the PDZ module with GOLPH3 is significantly enhanced by the presence of the KE-rich region (Fig. 7, C and D). This further confirms an interdependent connection between the neighboring functional domains KE and PDZ. The observed independence of the binding affinities from ATP confirms the assumption of Isogawa et al. (
      • Isogawa Y.
      • Kon T.
      • Inoue T.
      • Ohkura R.
      • Yamakawa H.
      • Ohara O.
      • Sutoh K.
      The N-terminal domain of MYO18A has an ATP-insensitive actin-binding site.
      ) that this interaction is ATP-insensitive. The N-terminal extension of myosin-18A could therefore be used by the protein to cross-link F-actin filaments to higher order complexes in a GOLPH3 binding-regulated fashion.
      We determined the ADP affinity of myosin-18A in the actin-bound state (KAD) to be ∼12 μm, which is in the range of reported physiological concentrations: 12 μm MgADP in brain and 6 μm MgADP in resting muscle or 43 μm MgADP in muscle after heavy exercise (
      • Roth K.
      • Weiner M.W.
      Determination of cytosolic ADP and AMP concentrations and the free energy of ATP hydrolysis in human muscle and brain tissues with 31P NMR spectroscopy.
      ); 40 to 140 μm (relaxed/contracted) free ADP in the cytosol of smooth muscle cells (
      • Krisanda J.M.
      • Paul R.J.
      Phosphagen and metabolite content during contraction in porcine carotid artery.
      ,
      • Khromov A.
      • Somlyo A.V.
      • Somlyo A.P.
      MgADP promotes a catch-like state developed through force-calcium hysteresis in tonic smooth muscle.
      ). Within the cytosol, local high ADP concentrations are expected in areas of high ATP turnover, such as myosin filaments or near membranes were ion pumps are located. Transiently increased ADP levels may facilitate strong actin binding of all four binding sites of a dimeric myosin-18A molecule, resulting in stable cross-linking of F-actin filaments or an attenuation of myosin filament contractility. However, in all possible nucleotide states, a fraction of the motor domain is strongly bound to the actin filaments. It is therefore likely that in cellular conditions, a release factor is needed to detach myosin-18A from the actin cytoskeleton to allow redistribution.
      When feeding all equilibrium binding constants of M18A-MD for actin and ADP into Scheme 1, the product should equal 1. Using the values determined in this study, we obtain a product of 0.029. This discrepancy suggests the presence of at least one additional step in the interaction of M18A-MD with actin and/or ADP that is not resolved by our experiments.
      For human myosin-18A, a number of confirmed or putative binding partners have been described (
      • Hsu R.-M.
      • Tsai M.-H.
      • Hsieh Y.-J.
      • Lyu P.-C.
      • Yu J.-S.
      Identification of MYO18A as a novel interacting partner of the PAK2/βPIX/GIT1 complex and its potential function in modulating epithelial cell migration.
      ,
      • Tan I.
      • Yong J.
      • Dong J.M.
      • Lim L.
      • Leung T.
      A tripartite complex containing MRCK modulates lamellar actomyosin retrograde flow.
      ,
      • Dippold H.C.
      • Ng M.M.
      • Farber-Katz S.E.
      • Lee S.-K.
      • Kerr M.L.
      • Peterman M.C.
      • Sim R.
      • Wiharto P.A.
      • Galbraith K.A.
      • Madhavarapu S.
      • Fuchs G.J.
      • Meerloo T.
      • Farquhar M.G.
      • Zhou H.
      • Field S.J.
      GOLPH3 bridges phosphatidylinositol-4-phosphate and actomyosin to stretch and shape the Golgi to promote budding.
      ,
      • Yang C.-H.
      • Szeliga J.
      • Jordan J.
      • Faske S.
      • Sever-Chroneos Z.
      • Dorsett B.
      • Christian R.E.
      • Settlage R.E.
      • Shabanowitz J.
      • Hunt D.F.
      • Whitsett J.A.
      • Chroneos Z.C.
      Identification of the surfactant protein A receptor 210 as the unconventional myosin 18A.
      ,
      • Matsui K.
      • Parameswaran N.
      • Bagheri N.
      • Willard B.
      • Gupta N.
      Proteomics analysis of the ezrin interactome in B cells reveals a novel association with Myo18aα.
      ). They can in principle act as modulators of myosin-18A function or stimulate enzymatic activity when bound to the protein. Furthermore, the presence of the complete N-terminal extension may be necessary for motor domain catalytic function as for example in myosin-3A the presence of the N-terminal kinase domain happens to modulate the kinetics of the motor domain (
      • Dosé A.C.
      • Ananthanarayanan S.
      • Moore J.E.
      • Burnside B.
      • Yengo C.M.
      Kinetic mechanism of human myosin IIIA.
      ,
      • Dosé A.C.
      • Ananthanarayanan S.
      • Moore J.E.
      • Corsa A.C.
      • Burnside B.
      • Yengo C.M.
      The kinase domain alters the kinetic properties of the myosin IIIA motor.
      ). Nevertheless, Guzik-Lendrum et al. (
      • Guzik-Lendrum S.
      • Heissler S.M.
      • Billington N.
      • Takagi Y.
      • Yang Y.
      • Knight P.J.
      • Homsher E.
      • Sellers J.R.
      Mammalian myosin-18A, a highly divergent myosin.
      ) report nucleotide and actin binding but no significant ATPase activity for a recombinant mouse myosin-18Aα motor construct, which contains the complete N-terminal domain.
      In an attempt to characterize the interaction of myosin-18A with GOLPH3, we were able to prove the direct binding of the phosphoprotein to the N-terminal extension of myosin-18A. Because the affinity is ionic strength-dependent, we suppose that charge-charge interactions are responsible for high affinity binding. We could show a direct effect of GOLPH3 on myosin-18A function because its binding to the PDZ module enhances the actin affinity of the N-terminal extension. Moreover, the GOLPH3·myosin-18A complex serves as a junction between the Golgi membrane and the cytoskeleton and may thus be highly regulated, presumably by other associated proteins.
      The motor domain of myosin-18A appears to have evolved to serve as an actin cross-linker, whose activity is modulated in a nucleotide- and cargo-dependent manner. Direct actin binding to the motor domain involves generic actin binding motifs and conserved albeit attenuated communication pathways between nucleotide and actin binding regions. Actin binding to the N-terminal extension is modulated by binding of GOLPH3 and potentially other cargo molecules to the PDZ module. Non-muscle myosin-2A and myosin-18A have been shown to share the same essential and regulatory light chains (
      • Guzik-Lendrum S.
      • Heissler S.M.
      • Billington N.
      • Takagi Y.
      • Yang Y.
      • Knight P.J.
      • Homsher E.
      • Sellers J.R.
      Mammalian myosin-18A, a highly divergent myosin.
      ). Moreover, they colocalize near the cell periphery in lamellar actomyosin bundles (
      • Tan I.
      • Yong J.
      • Dong J.M.
      • Lim L.
      • Leung T.
      A tripartite complex containing MRCK modulates lamellar actomyosin retrograde flow.
      ). Therefore, it is tempting to speculate that myosin-18A is part of these bipolar filaments acting as mediator between membrane and cytoskeleton components. Other potential roles of myosin-18A include a ratchet-like function, where the positive strain resulting from the productive interaction of non-muscle myosin-2A with actin pushes the molecule forward to the next actin binding site, where it snaps into place. In the same manner, strain-induced conformational changes can affect nucleotide and actin affinity, enabling myosin-18A to work as an efficient strain sensor within the contractile machinery.

      Acknowledgments

      We thank Michael Radke, Henning Grosskopf, Georg Adler-Gunzelmann, and Michal Stanczak for help and discussions.

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