Structure/Function Analysis of the Interaction of Phosphatidylinositol 4,5-Bisphosphate with Actin-capping Protein

Kim, Dong Kyu; McCully, Michelle E.; Bhattacharya, Nandini; Butler, Boyd; Sept, David; Cooper, John A.

doi:10.1074/jbc.m609850200

Cited by 77 publications

(99 citation statements)

References 37 publications

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“…Our conclusion that CAH3-driven uncapping involves interference in the function of the CP basic patch on and around its ␣ tentacle provides further evidence that this region of CP, which is known to drive the association of CP with the barbed end and much of its overall binding strength, is the crucial site for CP regulation, because it is also the binding site for V-1 7 and most likely the binding site for the anionic phospholipid phosphatidylinositol 4,5-bisphosphate (19,55). We note, however, that CAH3a/b is the first molecule identified whose effect on the function of the CP basic patch results from binding elsewhere on CP.…”

mentioning

confidence: 80%

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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mentioning

confidence: 80%

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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“…Kim et al (13) also investigated uncapping by PPIs using TIRF microscopy. They introduced capped filaments into flow cells, then flowed PIP 2 and fresh actin into the chambers, and counted the fraction of growing filaments at 5-min intervals.…”

Section: Discussionmentioning

confidence: 99%

“…High concentrations of negatively charged lipids could act as an efficient slide blocking agent, allowing barbed ends to grow as they broke or uncapped. As filaments were capped prior to their introduction into flow cells in the study of Kim et al (13), only uncapping would be affected by nonspecific adherence. In either case, ensuring that TIRF microscopy can accurately measure the same CP dissociation rates seen in bulk assays without PPIs is essential for determining the dissociation rates in the presence of PPIs.…”

Section: Discussionmentioning

confidence: 99%

“…This basic patch is adjacent to but distinct from a cluster of basic residues in the ␣-tentacle hinge region of many CPs, including fission yeast CP. Mutation of three of the basic residues in the hinge region reduce the affinity of chicken CP for PIP 2 about 4-fold (13).…”

Section: Homology Models To Identify Potential Ppi-binding Sites In Cp-mentioning

confidence: 96%

“…Steric interference would be the simplest way for PPIs to block interaction of CP with filament ends. Mutation of three residues surrounding a putative PIP 2 -binding site reduces the affinity of CP for PIP 2 5-60-fold and barbed ends 400-fold (13). Although the binding sites on CP for the barbed end and PIP 2 overlap, dissociation of CP from a barbed end by PIP 2 would presumably require an allosteric mechanism.…”

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confidence: 99%

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Single Molecule Kinetic Analysis of Actin Filament Capping

Kuhn

Pollard

2007

Journal of Biological Chemistry

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We investigated how heterodimeric capping proteins bind to and dissociate from the barbed ends of actin filaments by observing single muscle actin filaments by total internal reflection fluorescence microscopy. The barbed end rate constants for mouse capping protein (CP) association of 2.6 ؋ 10 6 M ؊1 s ؊1 and dissociation of 0.0003 s ؊1 agree with published values measured in bulk assays. The polyphosphoinositides (PPIs), phosphatidylinositol 3,4-bisphosphate (PI(3,4)P 2 ), PI(4,5)P 2 , and PI(3,4,5)P 3 , prevent CP from binding to barbed ends, but three different assays showed that none of these lipids dissociate CP from filaments at concentrations that block CP binding to barbed ends. The affinity of fission yeast CP for barbed ends is a thousandfold less than mouse CP, because of a slower association rate constant (1.1 ؋ 10 5 M ؊1 s ؊1 ) and a faster dissociation rate constant (0.004 s ؊1 ). PPIs do not inhibit binding of fission yeast CP to filament ends. Comparison of homology models revealed that fission yeast CP lacks a large patch of basic residues along the actin-binding surface on mouse CP. PPIs binding to this site might interfere sterically with capping, but this site would be inaccessible when CP is bound to the end of a filament.The motility of animal cells depends on the coordinated assembly of actin filaments at the leading edge. Arp2/3 complex promotes actin polymerization by nucleating filaments that grow as branches from a mother filament (1, 2). Capping protein (CP, 3 called CapZ in muscle) appears to play an important role in generating the zone of short, highly branched actin filaments at the leading edge. CP is a heterodimer of structurally similar ␣ and ␤ subunits that form a mushroom-like structure with one flexible and one immobile COOH-terminal "tentacle" that bind the two terminal subunits of an actin filament (3). CP binds the barbed end of actin filaments tightly with a K d of 0.1-1 nM. Given an association rate constant of 3-4 M Ϫ1 s Ϫ1 and a cytoplasmic concentration of 1 M (4), CP should terminate the growth of actin filaments with a half-time of about 0.2 s after their nucleation. Terminating growth quickly keeps the filaments short, so that they do not buckle under the force of polymerization as they push the membrane forward.Vertebrate CP dissociates from barbed ends with a half-time of 30 min (4), far too slow for simple, spontaneous dissociation to be involved with the conversion of the network of short, branched filaments at the leading edge to longer, unbranched filaments toward the cell body. As CP prevents both dissociation of subunits from the barbed end and end-to-end filament annealing, some mechanism must accelerate the removal of CP from filament ends.Both lipids and proteins, such as Ena/VASP (5-7) and CARMIL (8), can inhibit the interaction of CP with actin filaments. Polyphosphatidylinositides (PPIs) bind CP and block its interaction with filaments both in vivo (9) and in vitro (10). Addition of PIP 2 to mixtures of capped filaments, CP, and actin monomers in vi...

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Dynamic regulation of sarcomeric actin filaments in striated muscle

Ono

2010

Cytoskeleton

100

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In striated muscle, the actin cytoskeleton is differentiated into myofibrils. Actin and myosin filaments are organized in sarcomeres and specialized for producing contractile forces. Regular arrangement of actin filaments with uniform length and polarity is critical for the contractile function. However, the mechanisms of assembly and maintenance of sarcomeric actin filaments in striated muscle are not completely understood. Live imaging of actin in striated muscle has revealed that actin subunits within sarcomeric actin filaments are dynamically exchanged without altering overall sarcomeric structures. A number of regulators for actin dynamics have been identified, and malfunction of these regulators often result in disorganization of myofibril structures or muscle diseases. Therefore, proper regulation of actin dynamics in striated muscle is critical for assembly and maintenance of functional myofibrils. Recent studies have suggested that both enhancers of actin dynamics and stabilizers of actin filaments are important for sarcomeric actin organization. Further investigation of the regulatory mechanism of actin dynamics in striated muscle should be a key to understanding how myofibrils develop and operate. © 2010 Wiley-Liss, Inc.

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Structure/Function Analysis of the Interaction of Phosphatidylinositol 4,5-Bisphosphate with Actin-capping Protein

Cited by 77 publications

References 37 publications

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

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

Single Molecule Kinetic Analysis of Actin Filament Capping

Dynamic regulation of sarcomeric actin filaments in striated muscle

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