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The Arf GTPase-activating Protein, ASAP1, Binds Nonmuscle Myosin 2A to Control Remodeling of the Actomyosin Network*

Open AccessPublished:February 17, 2016DOI:https://doi.org/10.1074/jbc.M115.701292
      ASAP1 regulates F-actin-based structures and functions, including focal adhesions (FAs) and circular dorsal ruffles (CDRs), cell spreading and migration. ASAP1 function requires its N-terminal BAR domain. We discovered that nonmuscle myosin 2A (NM2A) directly bound the BAR-PH tandem of ASAP1 in vitro. ASAP1 and NM2A co-immunoprecipitated and colocalized in cells. Knockdown of ASAP1 reduced colocalization of NM2A and F-actin in cells. Knockdown of ASAP1 or NM2A recapitulated each other's effects on FAs, cell migration, cell spreading, and CDRs. The NM2A-interacting BAR domain contributed to ASAP1 control of cell spreading and CDRs. Exogenous expression of NM2A rescued the effect of ASAP1 knockdown on CDRs but ASAP1 did not rescue NM2A knockdown defect in CDRs. Our results support the hypothesis that ASAP1 is a positive regulator of NM2A. Given other binding partners of ASAP1, ASAP1 may directly link signaling and the mechanical machinery of cell migration.

      Introduction

      Arf GTPase-activating proteins (Arf GAPs)
      The abbreviations used are: ArfGAP, Arf GTPase-activating protein; Arf, ADP-ribosylation factor; BAR, Bin/Amphiphysin/Rvs; PH, pleckstrin-homology domain; ANK, ankyrin repeat; Pro-rich, proline-rich domain; SH3, Src homology3 domain; CDR, circular dorsal ruffle; ELC, essential light chain; RLC, regulatory light chain; S1, globular motor domain and lever arm of NM2A; FN, fibronectin; FA, focal adhesion; PI, PtdIns; LUV, large unilamellar vesicle.
      are a structurally diverse family of proteins encoded by 31 genes in humans (
      • Kahn R.A.
      • Bruford E.
      • Inoue H.
      • Logsdon J.M.
      • Nie Z.Z.
      • Premont R.T.
      • Randazzo P.A.
      • Satake M.
      • Theibert A.B.
      • Zapp M.L.
      • Cassel D.
      Consensus nomenclature for the human ArfGAP domain-containing proteins.
      ). The enzymatic function of Arf GAPs is to facilitate the hydrolysis of GTP on ADP-ribosylation factors (Arfs), which are members of the Ras superfamily. Both Arf GAPs and Arfs have been implicated as regulators of the actin cytoskeleton (
      • Myers K.R.
      • Casanova J.E.
      Regulation of actin cytoskeleton dynamics by Arf-family GTPases.
      ,
      • Randazzo P.A.
      • Inoue H.
      • Bharti S.
      Arf GAPs as regulators of the actin cytoskeleton.
      ). The role of Arf GAPs in actin-based structures has been primarily examined from the perspective of signaling. Two Arf GAPs are found to affect Arf6 and Rac1 activity (
      • Chen P.W.
      • Jian X.Y.
      • Yoon H.Y.
      • Randazzo P.A.
      ARAP2 signals through Arf6 and Rac1 to control focal adhesion morphology.
      ,
      • Nishiya N.
      • Kiosses W.B.
      • Han J.W.
      • Ginsberg M.H.
      An α(4) integrin-paxillin-Arf-GAP complex restricts Rac activation to the leading edge of migrating cells.
      ). The changes in Arf6 and Rac1, however, cannot completely account for the specific effects of particular Arf GAPs on the actin-based structures called focal adhesions (FAs) (
      • Chen P.W.
      • Jian X.Y.
      • Yoon H.Y.
      • Randazzo P.A.
      ARAP2 signals through Arf6 and Rac1 to control focal adhesion morphology.
      ). Structural domains outside the catalytic Arf GAP domain may also contribute to the control of the actin cytoskeleton.
      ASAP1 is an Arf GAP that associates with and regulates actin-based structures including FAs and circular dorsal ruffles (CDRs). It also affects cell spreading and cell migration, which involve actin remodeling (
      • Bharti S.
      • Inoue H.
      • Bharti K.
      • Hirsch D.S.
      • Nie Z.
      • Yoon H.Y.
      • Artym V.
      • Yamada K.M.
      • Mueller S.C.
      • Barr V.A.
      • Randazzo P.A.
      Src-dependent phosphorylation of ASAP1 regulates podosomes.
      ,
      • Liu Y.H.
      • Yerushalmi G.M.
      • Grigera P.R.
      • Parsons J.T.
      Mislocalization or reduced expression of arf GTPase-activating protein ASAP1 inhibits cell spreading and migration by influencing Arf1 GTPase cycling.
      ,
      • Onodera Y.
      • Hashimoto S.
      • Hashimoto A.
      • Morishige M.
      • Mazaki Y.
      • Yamada A.
      • Ogawa E.
      • Adachi M.
      • Sakurai T.
      • Manabe T.
      • Wada H.
      • Matsuura N.
      • Sabe H.
      Expression of AMAP1, an ArfGAP, provides novel targets to inhibit breast cancer invasive activities.
      ,
      • Randazzo P.A.
      • Andrade J.
      • Miura K.
      • Brown M.T.
      • Long Y.Q.
      • Stauffer S.
      • Roller P.
      • Cooper J.A.
      The Arf GTPase-activating protein ASAP1 regulates the actin cytoskeleton.
      ). ASAP1 is composed of BAR, PH, Arf GAP, Ank repeats, Proline-rich, E/DLPPKP repeat, and SH3 domains (Fig. 1A). ASAP1 binds to Src and CrkL through the proline-rich domain and focal adhesion kinase (FAK) via its SH3 domain (
      • Bharti S.
      • Inoue H.
      • Bharti K.
      • Hirsch D.S.
      • Nie Z.
      • Yoon H.Y.
      • Artym V.
      • Yamada K.M.
      • Mueller S.C.
      • Barr V.A.
      • Randazzo P.A.
      Src-dependent phosphorylation of ASAP1 regulates podosomes.
      ,
      • Liu Y.H.
      • Loijens J.C.
      • Martin K.H.
      • Karginov A.V.
      • Parsons J.T.
      The association of ASAP1, an ADP ribosylation factor-GTPase activating protein, with focal adhesion kinase contributes to the process of focal adhesion assembly.
      ,
      • Oda A.
      • Wada I.
      • Miura K.
      • Okawa K.
      • Kadoya T.
      • Kato T.
      • Nishihara H.
      • Maeda M.
      • Tanaka S.
      • Nagashima K.
      • Nishitani C.
      • Matsuno K.
      • Ishino M.
      • Machesky L.M.
      • Fujita H.
      • Randazzo P.
      CrkL directs ASAP1 to peripheral focal adhesions.
      ), which has been found to contribute to regulation of cell adhesions. The BAR domain of ASAP1 is also critical for its cellular function in regulation of actin-based structures (
      • Bharti S.
      • Inoue H.
      • Bharti K.
      • Hirsch D.S.
      • Nie Z.
      • Yoon H.Y.
      • Artym V.
      • Yamada K.M.
      • Mueller S.C.
      • Barr V.A.
      • Randazzo P.A.
      Src-dependent phosphorylation of ASAP1 regulates podosomes.
      ). Specific mechanisms by which the BAR domain contributes to ASAP1 control of actin-based structures have not been elucidated.
      Figure thumbnail gr1
      FIGURE 1ASAP1 directly interacts with NM2A. A, schematic representations of ASAP1 and NM2A domain structure and recombinant proteins used in this study. Targeting of ASAP1 to membranes is mediated primarily by the Pro-rich and SH3 domains. The PH domain regulates GAP activity of ASAP1. B, Coomassie Blue-stained SDS-PAGE gel of proteins sedimented with sucrose-loaded LUVs alone (no recombinant ASAP1) or coated with BAR-PH or PH. The image is a gel in which two central lanes were removed. The image of the gel is otherwise unaltered. C, co-sedimentation of ASAP1 and purified platelet NM2A. BAR-PH and NM2A were incubated either alone or together as indicated. Reaction mixtures were centrifuged and sedimented proteins were separated by SDS-PAGE and visualized with Coomassie Blue stain. Included in the gel were 25% of the NM2A in the reaction and the indicated percent of BAR-PH in the reaction mixture. Reactions were run in duplicate, and one experiment of three is shown. D, co-sedimentation of ASAP1 fragments (1 μm) with NM2A. The indicated fragments of ASAP1 were incubated with or without 0.6 μm NM2A and sedimented by centrifugation. Reactions were run in duplicate with the indicated proteins. The sedimented proteins and 25% input were separated by SDS-PAGE and visualized with Coomassie Blue stain. Asterisk indicates actin. Arrow indicates NM2A. Different fragments of ASAP1 were annotated in the schematic of ASAP1 domain structure. E and F, concentration-dependent ASAP1-NM2A association. NM2A was expressed in and purified from Sf9 cells. BAR-PH was titrated into a binding reaction containing 0.6 μm NM2A. At high concentrations of BAR-PH, some BAR-PH remains in the supernatant (S), indicating saturated binding. P, pellet. PH remains in the supernatant at all concentrations tested. The data of BAR-PH, after background corrections, were fit to an equation for one site binding. G, purified NM2A stimulates ASAP1 GAP activity dependent on BAR domain. GFP-NM2A purified from Sf9 cells was titrated into Arf GAP reactions containing the indicated recombinant ASAP1.
      Cytoplasmic nonmuscle myosin 2A (NM2A) is an F-actin binding ATPase that functions as a molecular motor in cellular events that involve force or translocation (
      • Vicente-Manzanares M.
      • Ma X.F.
      • Adelstein R.S.
      • Horwitz A.R.
      Non-muscle myosin II takes centre stage in cell adhesion and migration.
      ). NM2A is comprised of 2 heavy chains, 2 essential light chains (ECL), and 2 regulatory light chains (RLC) (Fig. 1A) and self-associates to form bipolar minifilaments. NM2A cross-links F-actin and the complex of NM2A and F-actin generates contractility necessary for cellular functions including regulation of FAs, cell spreading, and cell migration.
      Here, we set out to identify proteins that bind to and mediate the cellular functions of ASAP1. We found that NM2A directly binds to ASAP1 and associates with ASAP1 in cells. Knockdown of ASAP1 in cells reduced NM2A-F-actin colocalization. Knockdown of ASAP1 or NM2A had similar effects on FAs, cell spreading, cell migration, and CDRs. The effects of ASAP1 on CDRs require NM2A. Based on our results, we propose that ASAP1 positively regulates NM2A and speculate that ASAP1 integrates cell signals and links them to the machinery of actin remodeling.

      Discussion

      We set out to identify ASAP1-interacting protein(s) that mediate the effects of ASAP1 on actin-based structures. We found that ASAP1 directly bound to the actin-associated motor, NM2A. ASAP1 and NM2A affected a common set of actin structures and cell behaviors that depend on actin remodeling. The effect of ASAP1 on cell spreading and CDRs was dependent on the domain that bound directly to NM2A. The findings support the hypothesis that ASAP1 is a regulator of NM2A.
      ASAP1, a multi-domain Arf GAP, may represent a new class of NM2A regulators. ASAP1 itself is regulated by a unique set of signals including phosphorylation, and by multiple interactions that are relevant to the control of the cytoskeleton (
      • Bharti S.
      • Inoue H.
      • Bharti K.
      • Hirsch D.S.
      • Nie Z.
      • Yoon H.Y.
      • Artym V.
      • Yamada K.M.
      • Mueller S.C.
      • Barr V.A.
      • Randazzo P.A.
      Src-dependent phosphorylation of ASAP1 regulates podosomes.
      ,
      • Inoue H.
      • Randazzo P.A.
      Arf GAPs and their interacting proteins.
      ). Phosphoinositide binding to the PH domain may have a regulatory role, coordinating myosin activity with actin polymerization, which is also controlled by PI(4,5)P2 (
      • Campellone K.G.
      • Welch M.D.
      A nucleator arms race: cellular control of actin assembly.
      ). Also plausible, Arf·GTP binding to the ASAP1 Arf GAP domain might regulate the interaction with NM2A. This hypothesis is in line with the diminished binding of NM2A to BAR-PZA containing the Arf GAP domain compared with the isolated BAR-PH domain and with the effect of NM2A on ASAP1 GAP activity. Thus, ASAP1 may mediate effects of Arf on the actin cytoskeleton (
      • Myers K.R.
      • Casanova J.E.
      Regulation of actin cytoskeleton dynamics by Arf-family GTPases.
      ). ASAP1 is regulated by proteins known to affect actin remodeling including FAK, which binds to the SH3 domain of ASAP1, and Src family proteins, which bind to the proline-rich domain (
      • Bharti S.
      • Inoue H.
      • Bharti K.
      • Hirsch D.S.
      • Nie Z.
      • Yoon H.Y.
      • Artym V.
      • Yamada K.M.
      • Mueller S.C.
      • Barr V.A.
      • Randazzo P.A.
      Src-dependent phosphorylation of ASAP1 regulates podosomes.
      ,
      • Liu Y.H.
      • Loijens J.C.
      • Martin K.H.
      • Karginov A.V.
      • Parsons J.T.
      The association of ASAP1, an ADP ribosylation factor-GTPase activating protein, with focal adhesion kinase contributes to the process of focal adhesion assembly.
      ). The multiple binding partners regulate ASAP1 and target it to the plasma membrane, where it could localize NM2A function. Thus, ASAP1 could be the physical link between cellular signals and the mechanical machinery of actin remodeling in the context of cell movement and migration. Five other Arf GAPs may regulate NM2A. ASAP1–3 and ACAP1–3 all have an N-terminal BAR-PH tandem and the five tested affect the actin cytoskeleton (
      • Randazzo P.A.
      • Inoue H.
      • Bharti S.
      Arf GAPs as regulators of the actin cytoskeleton.
      ). Like ASAP1, all interact with signaling proteins and phosphoinositides. With unique responses to combined signals, each of these Arf GAPs could provide regulation specific to actin remodeling in disparate cellular behaviors that depend on NM2A.
      In testing for NM2A-ASAP1 association, we discovered NM2A stimulated ASAP1 GAP activity. Although NM2A was potent (half maximal effect at 10–50 nm NM2A), the effect was modest with a 2–5-fold stimulation, compared with the >10,000-fold stimulation observed with PI(4,5)P2 (
      • Kam J.L.
      • Miura K.
      • Jackson T.R.
      • Gruschus J.
      • Roller P.
      • Stauffer S.
      • Clark J.
      • Aneja R.
      • Randazzo P.A.
      Phosphoinositide-dependent activation of the ADP-ribosylation factor GTPase-activating protein ASAP1 - Evidence for the pleckstrin homology domain functioning as an allosteric site.
      ,
      • Jian X.
      • Tang W.K.
      • Zhai P.
      • Roy N.S.
      • Luo R.
      • Gruschus J.M.
      • Yohe M.E.
      • Chen P.W.
      • Li Y.
      • Byrd R.A.
      • Xia D.
      • Randazzo P.A.
      Molecular basis for cooperative binding of anionic phospholipids to the PH domain of the Arf GAP ASAP1.
      ). Although stimulated GAP activity may have a direct effector role, another plausible hypothesis, particularly given the modest effect, is that Arf·GTP binding to ASAP1 controls binding to NM2A. In this case, the GAP activity controls ASAP1-NM2A association.
      In the course of these studies, it was discovered that ASAP1 binds actin filaments.
      S. M. Heissler, P. W. Chen, K. Chinthalapudi, J. R. Sellers, and P. A. Randazzo, manuscript in preparation.
      The finding is consistent with the result presented in this report in Fig. 1B in which there was enrichment of actin in LUVs with BAR-PH. Though outside the scope of this current paper, we are investigating the possible mechanisms by which ASAP1, by binding both NM2A and actin, could regulate the dynamics of the actomyosin complex including stabilization by forming an ASAP1-actin-myosin ternary complex and by directly affecting the ATPase activity of myosin.
      In summary, we have tested the hypothesis that ASAP1 directly binds to NM2A to regulate actin-based structures and related cellular functions. We speculate that the interaction of ASAP1 with NM2A is controlled by ligands of ASAP1. Thus, PI(4,5)P2 binding, Arf·GTP binding, GTP hydrolysis, and Arf·GDP dissociation each could affect a specific transition along the cycle of binding to and dissociation from NM2A. Given that ASAP1 also binds to a number of signaling molecules, including PI(4,5)P2 and oncoproteins, it may integrate cellular signals and physically link the signals and cellular membrane to the machinery of cell movement important to normal physiology and pathological processes such as tumor cell invasion and metastasis.

      Author Contributions

      P. W. C. conceived the study, designed, performed experiments presented in Fig. 1B, Fig 2, C–E, Fig. 3, Fig. 4, and Fig. 5, prepared reagents for Fig. 1D and wrote the manuscript. X. J. prepared reagents, designed and implemented experiment presented in Fig. 1, C and F and edited the manuscript. S. M. H. designed and performed experiments presented in Fig. 1, D and E and edited the manuscript. K. L. designed and performed experiments presented in Fig. 2, A and B and edited the manuscript. R. L. designed and performed experiments presented in Fig 4, F–I and edited the manuscript. L. M. J. designed and performed the mass spectrometric analysis. A. N. conceived of initial experiments testing the effects of ASAP1 on NM2A and compiled preliminary data. J. M. conceived and directed the experiments presented in Fig. 2, A and B and edited the manuscript. J. R. S. conceived and directed the experiments presented in FIGURE 1, FIGURE 3 and wrote and edited the manuscript. P. A. R. conceived of the study, and wrote and edited the manuscript.

      Acknowledgments

      We thank Drs. Robert Adelstein and Mary Anne Conti for the GFP-NM2A plasmid and discussions and Dr. Kenneth Yamada for critical review of the manuscript.

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