Binding of Myotrophin/V-1 to Actin-capping Protein

Myotrophin, a 12-kDa ankyrin repeat protein, stimulates protein synthesis and cardiomyocyte growth to initiate cardiac hypertrophy by activating the NF-B signaling cascade. We found that, after internalization into myocytes, myotrophin cotranslocates into the nucleus with p65 to stimulate myocyte growth. We used structure-based mutations on the hairpin loops of myotrophin to determine the effect of the loops on myotrophin and p65 localization, induction of protein synthesis, and cardiac hypertrophy. Loop mutants, most prominently glutamic acid 33 3 alanine (E33A), stimulated protein synthesis much less than wild type. Myotrophin-E33A internalized into myocytes but did not translocate into the nucleus and failed to promote nuclear translocation of p65. In addition, two cardiac hypertrophy marker genes, atrial natriuretic factor and ␤-myosin heavy chain, were not up-regulated in E33A-treated cells. Myotrophin-induced myocyte growth and initiation of hypertrophy thus require nuclear co-translocation of myotrophin and p65, in a manner that depends crucially on the myotrophin hairpin loops.Myotrophin, a 12-kDa protein, is the smallest known ankyrin (ANK) 2 repeat protein (1) and contains four ANK repeats (2, 3). Myotrophin, also known as V-1 protein, was identified in spontaneously hypertensive rat hearts, cardiomyopathic human hearts (4), and neonatal rat cerebella (5). Myotrophin expression is ubiquitous in mammalian tissues (6). It is known to participate in the catecholamine synthesis signaling pathway (7), in regulation of actin polymerization (8), and in regulation of capping and uncapping by actin capping protein at the barbed ends of actin filaments (9). Myotrophin plays an important role in stimulation of cardiac myocyte growth and cardiac hypertrophy (4, 10). Sil et al. (11) reported an increase in myotrophin levels in human cardiomyopathic heart compared with normal heart. Overexpression of myotrophin in transgenic mice initiates cardiac hypertrophy that progresses toward heart failure (12, 13). Significantly, an increase in myotrophin level in blood plasma is observed after acute myocardial infarction (14) and in patients with heart failure (15). These data have established myotrophin as an initiator of cardiac hypertrophy and eventual heart failure.Myotrophin has been shown to interact with NF-B proteins (6, 16), and myotrophin-induced myocyte growth appears to be mediated through activation of the NF-B pathway (10), although the details of these interactions are poorly characterized. Knuefermann et al. (16) showed that myotrophin physically associates with p65 and prevents the reassociation of the p65-p50 heterodimer, resulting in activation of the NF-B pathway. Separate studies have established the involvement of NF-B signaling in cardiac hypertrophy and dilated cardiomyopathy (17-19) and the importance of NF-B activation in the pathogenesis of cardiac remodeling and heart failure (20 -22). Recently, Gupta et al. (13) showed that progression of cardiac hypertrophy to heart failure is associated with...

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confidence: 99%

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confidence: 99%

Nuclear Co-translocation of Myotrophin and p65 Stimulates Myocyte Growth

Das

Gupta

Vasanji

et al. 2008

“…V-1 sequesters CP in a totally inactive complex) (23,25). Moreover, V-1 is unable to accelerate the dissociation of CP already present on the barbed end (23,25). These interactions represent a model for CP "sequestering" in which V-1 sequesters CP in an inactive complex and is completely incapable of uncapping CP-capped barbed ends.…”

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confidence: 99%

“…One possible cellular regulator of CP is V-1 (myotrophin), which binds CP in vitro with high affinity (K d ϳ40 nM) in a 1:1 complex that has no affinity for the barbed end (i.e. V-1 sequesters CP in a totally inactive complex) (23,25). Moreover, V-1 is unable to accelerate the dissociation of CP already present on the barbed end (23,25).…”

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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

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...

“…To date, proteins found to interact with CP include: V-1 (23,35,36), CARMIL (11,37,38), Twinfilin (39), CD2AP (40), CapZIP (41), and CKIP-1 (9). Like V-1 and CARMIL, our studies indicate that CKIP-1 inhibits the activity of CP in a dose-dependent manner (11,35).…”

Section: Discussionmentioning

confidence: 78%

The Role of CKIP-1 in Cell Morphology Depends on Its Interaction with Actin-capping Protein

Canton

Olsten

Niederstrasser

et al. 2006

Self Cite

CKIP-1 is a pleckstrin homology domain-containing protein that induces alterations of the actin cytoskeleton and cell morphology when expressed in human osteosarcoma cells. CKIP-1 interacts with the heterodimeric actin-capping protein in cells, so we postulated that this interaction was responsible for the observed cytoskeletal and morphological effects of CKIP-1. To test this postulate, we used peptide "walking arrays" and alignments of CKIP-1 with CARMIL, another CP-binding protein, to identify Arg-155 and Arg-157 of CKIP-1 as residues potentially required for its interactions with CP. CKIP-1 mutants harboring Arg-155 and Arg-157 substitutions exhibited greatly decreased CP binding, while retaining wild-type localization, the ability to interact with protein kinase CK2, and self-association. To examine the phenotype associated with expression of these mutants, we generated tetracycline-inducible human osteosarcoma cells lines expressing R155E,R157E mutants of CKIP-1. Examination of these cell lines reveals that CKIP-1 R155E,R157E did not induce the distinct changes in cell morphology and the actin cytoskeleton that are characteristic of wild-type CKIP-1 demonstrating that the interaction between CKIP-1 and CP is required for these cellular effects. CKIP-14 was identified in a yeast two-hybrid screen for novel interaction partners of protein kinase CK2 (1). The cDNA for CKIP-1 codes for a protein of ϳ46 kDa with an amino-terminal pleckstrin homology (PH) domain and a carboxyl-terminal leucine-rich region as well as five putative PXXP motifs. The PH domain of CKIP-1 is required for phospholipid binding in vitro and for plasma membrane localization in cells (1, 2). Furthermore, this domain is necessary for interactions with protein kinase CK2, because mutants lacking the PH domain fail to interact with the kinase. Additionally, we have demonstrated that a subpopulation of protein kinase CK2 is targeted to the plasma membrane by CKIP-1 in cells (2). This targeting of CK2 is lost when the PH domain of CKIP-1 is replaced by a myristoylation recognition sequence, even though the CKIP-1 mutant still localizes to the plasma membrane. These results suggest that CKIP-1 may function in an analogous manner to protein kinase A anchoring proteins, which target cAMP-dependent protein kinase A (3-6). In addition to this potential role as a CK2-targeting protein, CKIP-1 appears to have roles independent of CK2. Recent reports have shown that CKIP-1 functions in muscle cell differentiation (7) and AP-1 regulation and apoptosis (8).To investigate the cellular functions of CKIP-1, we generated cell lines with tetracycline-regulated expression of FLAG-CKIP-1. Induction of FLAG-CKIP-1 in these cells caused changes in cellular morphology, as well as increases in F-actin and total cellular levels of actin (9). To determine the mechanistic basis for these observations, we performed a proteomic screen using Tandem affinity purification (10) and large-scale immunoprecipitations to identify CKIP-1 interaction partners. We identified the h...