Identification of Acan125 as a Myosin-I-binding Protein Present with Myosin-I on Cellular Organelles of Acanthamoeba

Xu, Pin; Zot, Anita S.; Zot, Henry G.

doi:10.1074/jbc.270.43.25316

Cited by 55 publications

(46 citation statements)

References 34 publications

(30 reference statements)

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“…Homologs of CRML-1 in Acanthamoeba, Dictyostelium, mouse and human (24, 25, 32 and 33% identical to CRML-1, respectively) have been described (Jung et al, 2001;Xu et al, 1997;Yang et al, 2005). These homologs are known as CARMILs (Capping, Arp2/3, Myosin I Linker), and were first described in Acanthamoeba and Dictyostelium as proteins that bind to the SH3 domain of myosin I (Jung et al, 2001;Xu et al, 1997;Xu et al, 1995). Components of the Arp2/3 complex, which nucleates F-actin branching, also interact with CARMILs (Jung et al, 2001).…”

Section: Resultsmentioning

confidence: 99%

C. elegans CARMIL negatively regulates UNC-73/Trio function during neuronal development

et al. 2009

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Whereas many molecules that promote cell and axonal growth cone migrations have been identified, few are known to inhibit these processes. In genetic screens designed to identify molecules that negatively regulate such migrations, we identified CRML-1, the C. elegans homolog of CARMIL. Although mammalian CARMIL acts to promote the migration of glioblastoma cells, we found that CRML-1 acts as a negative regulator of neuronal cell and axon growth cone migrations. Genetic evidence indicates that CRML-1 regulates these migrations by inhibiting the Rac GEF activity of UNC-73, a homolog of the Rac and Rho GEF Trio. The antagonistic effects of CRML-1 and UNC-73 can control the direction of growth cone migration by regulating the levels of the SAX-3 (a Robo homolog) guidance receptor. Consistent with the hypothesis that CRML-1 negatively regulates UNC-73 activity, these two proteins form a complex in vivo. Based on these observations, we propose a role for CRML-1 as a novel regulator of cell and axon migrations that acts through inhibition of Rac signaling.

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Section: Resultsmentioning

confidence: 99%

C. elegans CARMIL negatively regulates UNC-73/Trio function during neuronal development

et al. 2009

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“…Shortly after its discovery, CARMIL was shown to bind CP very tightly both in vitro and in vivo (24,26,31,32). Two biochemical studies then pinpointed the CAH3 domain of CARMIL as containing its CP binding site (30,33), demonstrated the two anti-CP activities exhibited by the CAH3 domain, and identified several residues that are important for CAH3 domain function.…”

Section: Discussionmentioning

confidence: 99%

“…Genes encoding CARMIL proteins have been identified in Acanthamoeba, Dictyostelium, Caenorhabditis elegans, Drosophila, mice, and humans (24, 26 -30). The first CARMIL protein to be identified was Acan125 (31). This 125-kDa Acanthamoeba protein was discovered based on its ability to bind to the isolated Src homology 3 domain of Acanthamoeba myosin-IC, a monomeric unconventional myosin.…”

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confidence: 99%

Molecular Basis for Barbed End Uncapping by CARMIL Homology Domain 3 of Mouse CARMIL-1

Zwolak

Uruno

Piszczek

et al. 2010

Journal of Biological Chemistry

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Capping protein (CP) is a ubiquitously expressed, 62-kDa heterodimer that binds the barbed end of the actin filament with ϳ0.1 nM affinity to prevent further monomer addition. CARMIL is a multidomain protein, present from protozoa to mammals, that binds CP and is important for normal actin dynamics in vivo. The CARMIL CP binding site resides in its CAH3 domain (CARMIL homology domain 3) located at or near the protein's C terminus. CAH3 binds CP with ϳ1 nM affinity, resulting in a complex with weak capping activity (30 -200 nM). Solution assays and single-molecule imaging show that CAH3 binds CP already present on the barbed end, causing a 300-fold increase in the dissociation rate of CP from the end (i.e. uncapping). Here we used nuclear magnetic resonance (NMR) to define the molecular interaction between the minimal CAH3 domain (CAH3a/b) of mouse CARMIL-1 and CP. Specifically, we show that the highly basic CAH3a subdomain is required for the high affinity interaction of CAH3 with a complementary "acidic groove" on CP opposite its actin-binding surface. This CAH3a-CP interaction orients the CAH3b subdomain, which we show is also required for potent anti-CP activity, directly adjacent to the basic patch of CP, shown previously to be required for CP association to and high affinity interaction with the barbed end. The importance of specific residue interactions between CP and CAH3a/b was confirmed by site-directed mutagenesis of both proteins. Together, these results offer a mechanistic explanation for the barbed end uncapping activity of CARMIL, and they identify the basic patch on CP as a crucial regulatory site. Capping protein (CP)4 is a ubiquitously expressed, 62-kDa ␣/␤ heterodimer that binds the barbed end of the actin filament with high affinity (K d ϭ 0.1 nM) to prevent further actin monomer association and dissociation, thereby limiting the extent of filament elongation in vivo (1, 2). Consistent with such a central role in actin filament assembly, CP is one of only five proteins required for the reconstitution of actin-based motility in vitro (3-5), and cells lacking CP have profound deficiencies in actin cytoskeleton assembly (6 -10).Determination of the CP crystal structure led to the "tentacles" model of barbed end capping by CP (11). The two structurally homologous CP subunits form a central ␤-sheet, which comprises the bulk of the protein core, above which there are two antiparallel ␣-helices, one belonging to each subunit (11). At the end of these helices, each subunit contains a C-terminal "tentacle," which, on CP␣, is composed of an unstructured region punctuated in the middle by a short, 4-residue helix and, on CP␤, is composed of a longer amphipathic helix that protrudes from the protein core. Based on crystallographic evidence, it was proposed that these C-terminal tentacles are flexible in solution, allowing them to bind and cap the barbed end. Extensive mutational studies in yeast (12) and vertebrate (13) CP that focused on the tentacles provided strong support for the tentacles model of ca...

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“…In 1995, Zot (1) and colleagues identified a ϳ125-kDa protein from Acanthamoeba on the basis of its ability to bind to the isolated Src homology 3 (SH3) 1 domain of Acanthamoeba myosin IC (1). This protein, which they called Acan 125, coimmunoprecipitated with myosin IC and appeared to colocalize with the myosin in cellular surface projections involved in pinocytosis.…”

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confidence: 99%

CARMIL Is a Bona Fide Capping Protein Interactant

Remmert

Olszewski

Bowers

et al. 2004

Journal of Biological Chemistry

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CARMIL, also known as Acan 125, is a multidomain protein that was originally identified on the basis of its interaction with the Src homology 3 (SH3) domain of type I myosins from Acanthamoeba. In a subsequent study of CARMIL from Dictyostelium, pull-down assays indicated that the protein also bound capping protein and the Arp2/3 complex. Here we present biochemical evidence that Acanthamoeba CARMIL interacts tightly with capping protein. In biochemical preparations, CARMIL copurified extensively with two polypeptides that were shown by microsequencing to be the ␣-and ␤-subunits of Acanthamoeba capping protein. The complex between CARMIL and capping protein, which is readily demonstratable by chemical cross-linking, can be completely dissociated by size exclusion chromatography at pH 5.4. Analytical ultracentrifugation, surface plasmon resonance and SH3 domain pull-down assays indicate that the dissociation constant of capping protein for CARMIL is ϳ0.4 M or lower. Using CARMIL fusion proteins, the binding site for capping protein was shown to reside within the carboxyl-terminal, ϳ200 residue, proline-rich domain of CARMIL. Finally, chemical cross-linking, analytical ultracentrifugation, and rotary shadowed electron microscopy revealed that CARMIL is asymmetric and that it exists in a monomer 7 dimer equilibrium with an association constant of 1.0 ؋ 10 6 M ؊1 . Together, these results indicate that CARMIL selfassociates and interacts with capping protein with affinities that, given the cellular concentrations of the proteins (ϳ1 and 2 M for capping protein and CARMIL, respectively), indicate that both activities should be physiologically relevant.In 1995, Zot (1) and colleagues identified a ϳ125-kDa protein from Acanthamoeba on the basis of its ability to bind to the isolated Src homology 3 (SH3) 1 domain of Acanthamoeba myosin IC (1). This protein, which they called Acan 125, coimmunoprecipitated with myosin IC and appeared to colocalize with the myosin in cellular surface projections involved in pinocytosis. The subsequent cloning of the Acanthamoeba gene for Acan 125 (2) revealed a multidomain protein dominated by a central, ϳ460-residue leucine-rich repeat (LRR) domain. LRRs are ϳ29-residue sequences that contain a loose consensus dominated by leucines, form amphipathic ␣-␤-structural units and mediate protein-protein interactions, either by serving as the ligand binding sites themselves or by increasing the affinity and/or specificity of binding at a separate site (3). The second most striking structural feature of Acan 125 is its ϳ200-residue, proline-rich COOH-terminal domain. Consistent with the fact that SH3 domains mediate protein-protein interactions by binding to proline-rich target sequences containing the core element PXXP (4), Xu et al. (2) showed that a fusion protein containing the COOH-terminal 344 residues of Acan 125 bound to the isolated SH3 domain of myosin IC and that this interaction was abrogated by an 18-residue deletion spanning two PXXP motifs fitting the consensus for SH3 domain tar...

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Identification of Acan125 as a Myosin-I-binding Protein Present with Myosin-I on Cellular Organelles of Acanthamoeba

Cited by 55 publications

References 34 publications

C. elegans CARMIL negatively regulates UNC-73/Trio function during neuronal development

C. elegans CARMIL negatively regulates UNC-73/Trio function during neuronal development

Molecular Basis for Barbed End Uncapping by CARMIL Homology Domain 3 of Mouse CARMIL-1

CARMIL Is a Bona Fide Capping Protein Interactant

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