Protein kinase A-dependent increase in WAVE2 expression induced by the focal adhesion protein vinexin

The focal adhesion protein vinexin is a member of a family of adaptor proteins that are thought to participate in the regulation of cell adhesion, cytoskeletal reorganization, and growth factor signaling. Here, we show that vinexin beta increases the amount of and reduces the mobility on SDS-PAGE of Wiskott-Aldrich syndrome protein family verprolin-homologous protein (WAVE) 2 protein, which is a key factor modulating actin polymerization in migrating cells. This mobility retardation disappeared after in vitro phosphatase treatment. Co-immunoprecipitation assays revealed the interaction of vinexin beta with WAVE2 as well as WAVE1 and N-WASP. Vinexin beta interacts with the proline-rich region of WAVE2 through the first and second SH3 domains of vinexin beta. Mutations disrupting the interaction impaired the ability of vinexin beta to increase the amount of WAVE2 protein. Treatments with proteasome inhibitors increased the amount of WAVE2, but did not have an additive effect with vinexin beta. Inhibition of protein kinase A (PKA) activity suppressed the vinexin-induced increase in WAVE2 protein, while activation of PKA increased WAVE2 expression without vinexin beta. These results suggest that vinexin beta regulates the proteasome-dependent degradation of WAVE2 in a PKA-dependent manner.     

Recent advances in the biology of WASP and WIP

WASP, the product of the gene mutated in Wiskott–Aldrich syndrome, is expressed only in hematopoietic cells and is the archetype of a family of proteins that include N-WASP and Scar/WAVE. WASP plays a critical role in T cell activation and actin reorganization. WASP has multiple protein-interacting domains. Through its N-terminal EVH1 domain WASP binds to its partner WASP interacting protein (WIP) and through its C-terminal end it interacts with and activates the Arp2/3 complex. In lymphocytes, most of WASP is sequestered with WIP and binding to WIP is essential for the stability of WASP. The central proline-rich region of WASP serves as docking site to several adaptor proteins. Through these multiple interactions WASP integrates many cellular signals to actin cytoskeleton remodeling. In this review, we have summarized recent developments in the biology of WASP and the role of WIP in regulating WASP function. We also discuss WASP-independent functions of WIP.     

Effects of ectopically expressed neuronal Wiskott-Aldrich syndrome protein domains on Rickettsia rickettsii actin-based motility

Neuronal Wiskott-Aldrich syndrome protein (N-WASP) and the actin-related protein 2/3 (Arp2/3) complex have emerged as critical host proteins that regulate pathogen actin-based motility. Actin tail formation and motility in Listeria monocytogenes require the Arp2/3 complex but bypasses N-WASP signaling. Motility of Shigella flexneri and vaccinia virus requires both N-WASP and the Arp2/3 complex. Functional roles for these cytoskeletal regulatory proteins in actin-based motility of Rickettsia rickettsii have not been established. In this study, functional domains of N-WASP tagged with green fluorescent protein that have characterized effects on Shigella and vaccinia virus actin-based motility were ectopically expressed in HeLa cells infected with R. rickettsii to assess their effects on rickettsial motility. S. flexneri-infected cells were used as a control. Expressed N-WASP domains did not localize to R. rickettsii or their actin tails. Expression of N-WASP missing the VCA domain (for "verprolin homology, cofilin homology, and acidic domains"), which acts as a dominant-negative form of N-WASP, completely inhibited actin-based motility of S. flexneri while only moderately inhibiting motility of R. rickettsii. Similarly, expression of the VCA domain, which acts as a dominant-negative with respect to Arp2/3 complex function, severely inhibited actin-based motility of S. flexneri (no motility observed in the majority of expressing cells) but only moderately inhibited R. rickettsii motility. These results, taken together with the differential effects on motility observed upon expression of other N-WASP domains, suggest that actin-based motility of R. rickettsii is independent of N-WASP and the Arp2/3 complex.     

Junctional Rab13‐binding protein (JRAB) regulates cell spreading via filamins

We previously showed that Rab13 and its effector protein, junctional Rab13‐binding protein (JRAB)/molecules interacting with CasL‐like 2 (MICAL‐L2), regulate junctional development by modulating cell adhesion molecule transport and actin cytoskeletal reorganization in epithelial cells. Here, we investigated how JRAB regulates reorganization of the actin cytoskeleton in NIH3T3 fibroblasts, in an attempt to obtain novel insights into the mechanism of JRAB action. To this end, we expressed mutant proteins that adopt a constitutively open or closed state and then examined effect on cellular morphology of the resulting actin cytoskeletal reorganization. Expression of the JRABΔCT mutant (constitutively ‘closed’ state) induced stress fibers, whereas expression of the JRABΔCC mutant (constitutively ‘open’ state) caused cell spreading with membrane ruffles. Next, we identified the proteins involved in JRAB‐induced rearrangement of actin cytoskeleton leading to morphological changes. In NIH3T3 cells expressing HA‐JRABΔCC, filamin, an actin cross‐linking protein, coimmunoprecipitated with HA‐JRABΔCC. Expression of ASB2 induced degradation of all three filamin isoforms and inhibited the JRABΔCC‐induced cell spreading. Consistent with our previous results, actinin‐1/‐4 were also immunoprecipitated with HA‐JRABΔCC. However, actinin‐1/‐4 have no effect on the cell spreading regulated by JRABΔCC. These data suggest that JRAB contributes to the rearrangement of the actin cytoskeleton during cell spreading via filamins.     

HSP90 cross-links branched actin filaments induced by N-WASP and the Arp2/3 complex

N-WASP induces filopodial actin cytoskeleton through activation of the Arp2/3 complex. Here, we show that heat shock protein 90 (HSP90) regulates the structure of actin filaments induced by N-WASP and the Arp2/3 complex. HSP90 binds to N-WASP and to F-actin and bundles actin filaments. Bundling activity of HSP90 does not affect actin filament nucleation induced by N-WASP and the Arp2/3 complex. HSP90 is co-localized with N-WASP at branching points of actin filaments produced by the Arp2/3 complex and thereby bundles branched filaments; this bundled actin structure is inhibited by blocking direct binding between HSP90 and N-WASP. Furthermore, HSP90 converts branched actin filaments on N-WASP-coated beads to filopodia-like star-shaped bundles. These findings indicate that HSP90 promotes the formation of N-WASP/Arp2/3 complex-induced unbranched filopodial actin structures.     

A novel function of WAVE in lamellipodia: WAVE1 is required for stabilization of lamellipodial protrusions during cell spreading

When a cell spreads and moves, reorganization of the actin cytoskeleton pushes the cell membrane, and the resulting membrane protrusions create new points of contact with the substrate and generate the locomotive force. Membrane extension and adhesion to a substrate must be tightly coordinated for effective cell movement, but little is known about the mechanisms underlying these processes. WAVEs are critical regulators of Rac-induced actin reorganization. WAVE2 is essential for formation of lamellipodial structures at the cell periphery stimulated by growth factors, but it is thought that WAVE1 is dispensable for such processes in mouse embryonic fibroblasts (MEFs). Here we show a novel function of WAVE in lamellipodial protrusions during cell spreading. During spreading on fibronectin (FN), MEFs with knockouts (KOs) of WAVE1 and WAVE2 showed different membrane dynamics, suggesting that these molecules have distinct roles in lamellipodium formation. Formation of lamellipodial structures on FN was inhibited in WAVE2 KO MEFs. In contrast, WAVE1 is not essential for extension of lamellipodial protrusions but is required for stabilization of such structures. WAVE1-deficiency decreased the density of actin filaments and increased the speed of membrane extension, causing deformation of focal complex at the tip of spreading edges. Thus, at the tip of the lamellipodial protrusion, WAVE2 generates the membrane protrusive structures containing actin filaments, and modification by WAVE1 stabilizes these structures through cell-substrate adhesion. Coordination of WAVE1 and WAVE2 activities appears to be necessary for formation of proper actin structures in stable lamellipodia.     

Concerted upregulation of CLP36 and smooth muscle actin protein expression in human endometrium during decidualization

The human endometrium prepares for implantation of the blastocyst by reorganization of its whole cellular network. Endometrial stroma cells change their phenotype starting around the 23rd day of the menstrual cycle. These predecidual stroma cells first appear next to spiral arteries, and after implantation these cells further differentiate into decidual stroma cells. The phenotypical changes in these cells during decidualization are characterized by distinct changes in the actin filaments and filament-related proteins such as alpha-actinin. The carboxy-terminal LIM domain protein with a molecular weight of 36 kDa (CLP36) is a cytoskeletal component that has been shown to associate with contractile actin filaments and to bind to alpha-actinin supporting a role for CLP36 in cytoskeletal reorganization and signal transduction by binding to signaling proteins. The expression patterns of CLP36, alpha-actinin and actin were studied in endometrial stroma cells from different stages of the menstrual cycle and in decidual stroma cells from the 6th week of gestation until the end of pregnancy. During the menstrual cycle, CLP36 is only expressed in the luminal and glandular epithelium but not in endometrial stroma cells. During decidualization and throughout pregnancy, a parallel upregulation of CLP36 and smooth muscle actin, an early marker of decidualization in the baboon, was observed in endometrial decidual cells. Since both proteins maintain a high expression level throughout pregnancy, a role of both proteins is suggested in the stabilization of the cytoskeleton of these cells that come into close contact with invading trophoblast cells.     

Wiskott-Aldrich syndrome protein and the cytoskeletal dynamics of dendritic cells

The regulated migration and spatial localization of dendritic cells in response to environmental signals are critical events during the initiation of physiological immune responses and maintenance of tolerance. Cells deficient in the Wiskott-Aldrich syndrome protein (WASP) have been used to demonstrate the importance of the dynamic remodelling of the actin-based cytoskeleton during the selective adhesion and migration of these cells. Unlike most cell types, macrophages, dendritic cells, and osteoclasts utilize a specialized adhesive array termed the podosome in order to migrate. Podosomes are composed of many of the same structural and regulatory proteins as seen in the more commonly found focal adhesion, but are unique in their requirement for WASP. Without WASP, podosomes cannot form and the affected cells are obliged to use focal adhesions for their migratory activities. Once activated by a series of upstream regulatory proteins, WASP acts as a scaffold for the binding of the potent actin nucleating protein complex known as Arp2/3. This article reviews the available evidence that suggests that failures in the regulation of the actin cytoskeleton may contribute significantly to the immunopathology of the Wiskott-Aldrich syndrome.     

Enteropathogenic Escherichia coli and Vaccinia Virus Do Not Require the Family of WASP-Interacting Proteins for Pathogen-Induced Actin Assembly

The human pathogens enteropathogenic Escherichia coli (EPEC) and vaccinia virus trigger actin assembly in host cells by activating the host adaptor Nck and the actin nucleation promoter neural Wiskott-Aldrich syndrome protein (N-WASP). EPEC translocates effector molecules into host cells via type III secretion, and the interaction between the translocated intimin receptor (Tir) and the bacterial membrane protein intimin stimulates Nck and N-WASP recruitment, leading to the formation of actin pedestals beneath adherent bacteria. Vaccinia virus also recruits Nck and N-WASP to generate actin tails that promote cell-to-cell spread of the virus. In addition to Nck and N-WASP, WASP-interacting protein (WIP) localizes to vaccinia virus tails, and inhibition of actin tail formation upon ectopic expression of WIP mutants led to the suggestion that WIP is required for this process. Similar studies of WIP mutants, however, did not affect the ability of EPEC to form actin pedestals, arguing against an essential role for WIP in EPEC-induced actin assembly. In this study, we demonstrate that Nck and N-WASP are normally recruited by vaccinia virus and EPEC in the absence of WIP, and neither WIP nor the WIP family members CR16 and WIRE/WICH are essential for pathogen induced actin assembly. In addition, although Nck binds EPEC Tir directly, N-WASP is required for its localization during pedestal formation. Overall, these data highlight similar pathogenic strategies shared by EPEC and vaccinia virus by demonstrating a requirement for both Nck and N-WASP, but not WIP or WIP family members in pathogen-induced actin assembly.     

Actin binding LIM protein 3 (abLIM3)

LIM domain proteins were demonstrated to play key roles in various biological processes such as embryonic development, cell lineage determination, and cancer differentiation. Actin binding LIM protein 1 (abLIM1) was reported to be localized in a genomic region often deleted in human cancers and suggested to be involved in axon guidance. Recently, existence of a second family member was reported, actin binding LIM protein 2. By means of computational biology and comparative genomics, we now characterized an additional, third member of the actin binding LIM protein subgroup, actin binding LIM protein 3 (abLIM3). The human mRNA sequence was previously annotated as differentially regulated in hepatoblastoma compared to normal livers. Conservation of key structural features of abLIM1 and abLIM2, four LIM domains and a VHD domain, suggested comparable biological function of abLIM3 as a linker between actin cytoskeleton and cell signaling pathways. AbLIM3 was found to be conserved in vertebrates, as orthologous sequences were characterized for mouse, fish, and frog. In addition, we report the existence of abLIM2 orthologs in fish and frog, suggesting a similar degree of evolutionary conservation. The intracellular localization of the abLIM3 protein was predicted to be nuclear by means of Reinhardt's neural network and the k-nearest neighbor algorithm. The corresponding abLIM3 gene was localized to chromosome 5q32 and spanned 119 kb, organized in 24 exons. An RT-PCR based expression profile available from the human unidentified gene-encoded (HUGE) database demonstrated highest expression for abLIM3 in heart, lung, liver, and brain/cerebellum accompanied by lower expression in multiple other tissues. Furthermore, abLIM3 was expressed in fetal liver, CNS, and spinal cord.     

Neurofilament protein synthesis and phosphorylation

Neurofilament proteins, a major intermediate filament component of the neuronal cytoskeleton, are organized as 10 nm thick filaments in axons and dendrites. They are large, abundantly phosphorylated proteins with numerous phosphate acceptor sites, up to 100 in some cases, organized as numerous repeat motifs. Together with other cytoskeletal components such as microtubules, MAPs, actin and plectin-like linking molecules, they make up a dynamic lattice that sustains neuronal function from neuronal "birthday" to apoptotic cell death. The activity of the neuronal cytoskeleton is regulated by phosphorylation, dephosphorylation reactions mediated by numerous associated kinases, phosphatases and their regulators. Factors regulating multisite phosphorylation of NFs are topographically localized, with maximum phosphorylation of NF proteins consigned to axons. Phosphorylation defines the nature of NF interactions with one another and with other cytoskeletal components such as microtubules, MAPs and actin. To understand how these functional interactions are regulated by phosphorylation we attempt to identify the relevant kinases and phosphatases, their specific targets and the factors modulating their activity. As an initial working model we propose that NF phosphorylation is regulated topographically in neurons by compartment-specific macromolecular complexes of substrates, kinases and phosphatases. This implies that axonal complexes differ structurally and functionally from those in cell bodies and dendrites. Such protein assemblies, by virtue of conformational changes within proteins, facilitate ordered, sequential multisite phosphorylations that modulate dynamic cytoskeletal interactions.     

Abelson tyrosine kinase facilitates Salmonella enterica serovar Typhimurium entry into epithelial cells

The intracellular gram-negative bacterial pathogen Salmonella enterica serovar Typhimurium gains entry into nonphagocytic cells by manipulating the assembly of the host actin cytoskeleton. S. enterica serovar Typhimurium entry requires a functional type III secretion system, a conduit through which bacterial effector proteins are directly translocated into the host cytosol. We and others have previously reported the enhancement of tyrosine kinase activities during Salmonella serovar Typhimurium infection; however, neither specific kinases nor their targets have been well characterized. In this study, we investigated the roles of the cellular Abelson tyrosine kinase (c-Abl) and the related protein Arg in the context of serovar Typhimurium infection. We found that bacterial internalization was inhibited by more than 70% in cells lacking both c-Abl and Arg and that treatment of wild-type cells with a pharmaceutical inhibitor of the c-Abl kinase, STI571 (imatinib), reduced serovar Typhimurium invasion efficiency to a similar extent. Bacterial infection led to enhanced phosphorylation of two previously identified c-Abl substrates, the adaptor protein CT10 regulator of kinase (CrkII) and the Abelson-interacting protein Abi1, a component of the WAVE2 complex. Furthermore, overexpression of the nonphosphorylatable form of CrkII resulted in decreased invasion. Taken together, these findings indicate that c-Abl is activated during S. enterica serovar Typhimurium infection and that its phosphorylation of multiple downstream targets is functionally important in bacterial internalization.     

Regulation of WAVE1 expression in macrophages at multiple levels

M-CSF (or CSF-1) controls macrophage lineage development and function. A CSF-1-dependent culture system was established, which monitored the differentiation of CSF-1-responsive macrophage populations over time and upon adherence. Wiskott-Aldrich syndrome protein verprolin homologous (WAVE) proteins are involved in actin reorganization, a process critical to many cell functions. WAVE2 but not WAVE1 has been considered significant for macrophage function. Using the CSF-1-dependent differentiation system, we were able to demonstrate the contrasting regulation of the expression of WAVE1 and WAVE2; the levels of the latter rose over time and as the macrophage population became adherent, although those of the former increased over time but were down-regulated upon adherence. Evidence was obtained that WAVE1 was also cleaved to a novel, 60-kDa fragment by macrophage adherence and by another pathway involving calpain-mediated proteolysis. Mutagenesis studies indicated that cleavage of WAVE1 by calpain results in the removal of the verprolin-homology, cofilin-like, and acidic domain and thus, the loss of WAVE1 activity. We suggest that WAVE1 is also important for macrophage biology and that it could have separate functions to those of WAVE2.     

Cdc42 and the actin-related protein/neural Wiskott-Aldrich syndrome protein network mediate cellular invasion by Cryptosporidium parvum

Cryptosporidium parvum invasion of epithelial cells involves host cell membrane alterations which require a remodeling of the host cell actin cytoskeleton. In addition, an actin plaque, possibly associated with the dense-band region, forms within the host cytoplasm at the host-parasite interface. Here we show that Cdc42 and RhoA, but not Rac1, members of the Rho family of GTPases, are recruited to the host-parasite interface in an in vitro model of human biliary cryptosporidiosis. Interestingly, activation of Cdc42, but not RhoA, was detected in the infected cells. Neural Wiskott-Aldrich syndrome protein (N-WASP) and p34-Arc, actin-regulating downstream effectors of Cdc42, were also recruited to the host-parasite interface. Whereas cellular expression of a constitutively active mutant of Cdc42 promoted C. parvum invasion, overexpression of a dominant negative mutant of Cdc42, or depletion of Cdc42 mRNA by short interfering RNA-mediated gene silencing, inhibited C. parvum invasion. Expression of the WA fragment of N-WASP to block associated actin polymerization also inhibited C. parvum invasion. Moreover, inhibition of host cell Cdc42 activation by dominant negative mutation inhibited C. parvum-associated actin remodeling, membrane protrusion, and dense-band formation. In contrast, treatment of cells with a Rho inhibitor, exoenzyme C3, or cellular overexpression of dominant negative mutants of RhoA and Rac1 had no effect on C. parvum invasion. These data suggest that C. parvum invasion of target epithelia results from the organism's ability to activate a host cell Cdc42 GTPase signaling pathway to induce host cell actin remodeling at the attachment site.     

Signal transduction cascades underlying de novo protein synthesis required for neuronal morphogenesis in differentiating neurons

Differentiating neurons must acquire many unique morphological and functional characteristics in creating the precise neural circuits of the mature nervous system. The phenomenon of 'neuronal differentiation' includes a special set of simple, separate processes, that is, neuritogenesis, neurite outgrowth, pathfinding, targeting and synaptogenesis. All of these processes are critically dependent on the reorganization of actin cytoskeleton by many actin-binding proteins that function downstream of Rho-family GTPases. Furthermore, de novo synthesis of key proteins are critically involved in the reorganization of actin cytoskeleton during neuronal differentiation. In this article, we review recent progresses in the general mechanisms that control actin dynamics by various actin-binding proteins in differentiating neurons, including a series of recent studies from our laboratory on de novo synthesis of several key proteins that are essential for actin reorganization induced by second messengers. We demonstrated that dual regulation of cyclic AMP and Ca2+ determines cofilin (an actin-binding protein) phosphorylation states and LIM kinase 1 (a cofilin kinase) expression level during neuritogenesis.     

Hypoxia-induced cytoskeleton disruption in alveolar epithelial cells

Alveolar hypoxia, a common feature of many respiratory disorders, has been previously reported to induce functional changes, particularly a decrease of transepithelial Na and fluid transport. In polarized epithelia, cytoskeleton plays a regulatory role in transcellular and paracellular transport of ions and fluid. We hypothesized that exposure to hypoxia could damage cytoskeleton organization, which in turn, may adversely affect ion and fluid transport. Primary rat alveolar epithelial cells (AEC) were exposed to either mild (3% O(2)) or severe (0.5% O(2)) hypoxia for 18 h or to normoxia (21% O(2)). First, mild and severe hypoxia induced a disorganization of actin, a major protein of the cytoskeleton, reflected by disruption of F-actin filaments. Second, alpha-spectrin, an apical cytoskeleton protein, which binds to actin cytoskeleton and Na transport proteins, was cleaved by hypoxia. Pretreatment of AEC by a caspase inhibitor (z-VAD-fmk; 90 microM) blunted hypoxia-induced spectrin cleavage as well as hypoxia-induced decrease in surface membrane alpha-ENaC and concomitantly induced a partial recovery of hypoxia-induced decrease of amiloride-sensitive Na transport at 3% O(2). Finally, tight junctions (TJs) proteins, which are linked to actin and are a determinant of paracellular permeability, were altered by mild and severe hypoxia: hypoxia induced a mislocalization of occludin from the TJ to cytoplasm and a decrease in zonula occludens-1 protein level. These modifications were associated with modest changes in paracellular permeability at 0.5% O(2,) as assessed by small 4-kD dextran flux and transepithelial resistance measurements. Together, these findings indicate that hypoxia disrupted cytoskeleton and TJ organization in AEC and may participate, at least in part, to hypoxia-induced decrease in Na transport.     

Enteropathogenic Escherichia coli, Shigella flexneri, and Listeria monocytogenes recruit a junctional protein, zonula occludens-1, to actin tails and pedestals

Enteropathogenic Escherichia coli, Shigella flexneri, and Listeria monocytogenes induce localized actin polymerization at the cytoplasmic face of the plasma membrane or within the host cytoplasm, creating unique actin-rich structures termed pedestals or actin tails. The process is known to be mediated by the actin-related protein 2 and 3 (Arp2/3) complex, which in these cases acts downstream of neural Wiskott-Aldrich syndrome protein (N-WASP) or of a listerial functional homolog of WASP family proteins. Here, we show that zonula occludens-1 (ZO-1), a protein in the tight junctions of polarized epithelial cells, is recruited to actin tails and pedestals. Immunocytochemical analysis revealed that ZO-1 was stained most in the distal part of the actin-rich structures, and the incorporation was mediated by the proline-rich region of the ZO-1 molecule. The direct clustering of membrane-targeted Nck, which is known to activate the N-WASP-Arp2/3 pathway, triggered the formation of the ZO-1-associated actin tails. The results suggest that the activation of the Arp2/3 complex downstream of N-WASP or a WASP-related molecule is a key to the formation of the particular actin-rich structures that bind with ZO-1. We propose that an analysis of the recruitment on a molecular basis will lead to an understanding of how ZO-1 recognizes a distinctive actin-rich structure under pathophysiological conditions.     

Vaccine-induced modulation of gene expression in turbot peritoneal cells. A microarray approach

We used a microarray approach to examine changes in gene expression in turbot peritoneal cells after injection of the fish with vaccines containing the ciliate parasite Philasterides dicentrarchi as antigen and one of the following adjuvants: chitosan-PVMMA microspheres, Freund́s complete adjuvant, aluminium hydroxide gel or Matrix-Q (Isconova, Sweden). We identified 374 genes that were differentially expressed in all groups of fish. Forty-two genes related to tight junctions and focal adhesions and/or actin cytoskeleton were differentially expressed in free peritoneal cells. The profound changes in gene expression related to cell adherence and cytoskeleton may be associated with cell migration and also with the formation of cell-vaccine masses and their attachment to the peritoneal wall. Thirty-five genes related to apoptosis were differentially expressed. Although most of the proteins coded by these genes have a proapoptotic effect, others are antiapoptotic, indicating that both types of signals occur in peritoneal leukocytes of vaccinated fish. Interestingly, many of the genes related to lymphocytes and lymphocyte activity were downregulated in the groups injected with vaccine. We also observed decreased expression of genes related to antigen presentation, suggesting that macrophages (which were abundant in the peritoneal cavity after vaccination) did not express these during the early inflammatory response in the peritoneal cavity. Finally, several genes that participate in the inflammatory response were differentially expressed, and most participated in resolution of inflammation, indicating that an M2 macrophage response is generated in the peritoneal cavity of fish one day post vaccination.     

Ankycorbin: a novel actin cytoskeleton-associated protein

BACKGROUND: Actin cytoskeleton structures are essential for a wide variety of cell functions, including cell shape change, cell motility, cell adhesion, cell polarity and cytokinesis. Many actin filament (F-actin)-binding proteins have been isolated and implicated in the maintenance and reorganization of actin cytoskeleton structures. RESULTS: We purified here a novel protein with a molecular mass of about 125 kDa (p125) from rat liver. We cloned its cDNA from a mouse kidney cDNA library and determined its nucleotide and deduced amino acid sequences. p125 was a protein of 979 amino acids with a calculated Mr of 108 847. p125 contained six ankyrin repeats in the N-terminal region and a domain predicted to form a coiled-coil structure in the C-terminal region. We named p125 ankycorbin (ankyrin repeat- and coiled-coil structure-containing protein). Northern blot analysis indicated that ankycorbin was ubiquitously expressed in all the tissues examined. Immunofluorescence and immunoelectron microscope analyses revealed that ankycorbin was associated with the cortical actin cytoskeleton structures in terminal web and cell-cell adhesion sites and stress fibres. However, ankycorbin did not directly bind to F-actin as estimated by the F-actin co-sedimentation assay. CONCLUSIONS: These results indicate that ankycorbin is indirectly associated with the actin cytoskeleton structures, presumably through an unidentified factor and suggest that it is involved in their maintenance and/or reorganization.