Role for the pleckstrin homology domain-containing protein CKIP-1 in phosphatidylinositol 3-kinase-regulated muscle differentiation

In this work, we report the implication of the pleckstrin homology (PH) domain-containing protein CKIP-1 in phosphatidylinositol 3-kinase (PI3-K)-regulated muscle differentiation. CKIP-1 is upregulated during muscle differentiation in C2C12 cells. We show that CKIP-1 binds to phosphatidylinositol 3-phosphate through its PH domain and localizes to the plasma membrane in a PI3-K-dependent manner. Activation of PI3-K by insulin or expression of an active form of PI3-K p110 induces a rapid translocation of CKIP-1 to the plasma membrane. Conversely, expression of the 3-phosphoinositide phosphatase myotubularin or PI3-K inhibition by LY294002, wortmannin, or mutant p85 abolishes CKIP-1 binding to the membrane. Upon induction of differentiation in low-serum medium, CKIP-1 overexpression in C2C12 myoblasts first promotes proliferation and then stimulates the expression of myogenin and cell fusion in a manner reminiscent of the dual positive effect of insulin-like growth factors on muscle cells. Interference with the PI3-K pathway impedes the effect of CKIP-1 on C2C12 cell differentiation. Finally, silencing of CKIP-1 by RNA interference abolishes proliferation and delays myogenin expression. Altogether, these data strongly implicate CKIP-1 as a new component of PI3-K signaling in muscle differentiation.     

Identification and characterization of CKIP-1, a novel pleckstrin homology domain-containing protein that interacts with protein kinase CK2

The catalytic subunits of protein kinase CK2, CK2alpha and CK2alpha', are closely related to each other but exhibit functional specialization. To test the hypothesis that specific functions of CK2alpha and CK2alpha' are mediated by specific interaction partners, we used the yeast two-hybrid system to identify CK2alpha- or CK2alpha'-binding proteins. We report the identification and characterization of a novel CK2-interacting protein, designated CKIP-1, that interacts with CK2alpha, but not CK2alpha', in the yeast two-hybrid system. CKIP-1 also interacts with CK2alpha in vitro and is co-immunoprecipitated from cell extracts with epitope-tagged CK2alpha and an enhanced green fluorescent protein fusion protein encoding CKIP-1 (i.e. EGFP-CKIP-1) when they are co-expressed. CK2 activity is detected in anti-CKIP-1 immunoprecipitates performed with extracts from non-transfected cells indicating that CKIP-1 and CK2 interact under physiological conditions. The CKIP-1 cDNA is broadly expressed and encodes a protein with a predicted molecular weight of 46,000. EGFP-CKIP-1 is localized within the nucleus and at the plasma membrane. The plasma membrane localization is dependent on the presence of an amino-terminal pleckstrin homology domain. We postulate that CKIP-1 is a non-enzymatic regulator of one isoform of CK2 (i.e. CK2alpha) with a potential role in targeting CK2alpha to a particular cellular location.     

Plant actin-binding protein SCAB1 is dimeric actin cross-linker with atypical pleckstrin homology domain

SCAB1 is a novel plant-specific actin-binding protein that binds, bundles, and stabilizes actin filaments and regulates stomatal movement. Here, we dissected the structure and function of SCAB1 by structural and biochemical approaches. We show that SCAB1 is composed of an actin-binding domain, two coiled-coil (CC) domains, and a fused immunoglobulin and pleckstrin homology (Ig-PH) domain. We determined crystal structures for the CC1 and Ig-PH domains at 1.9 and 1.7 Å resolution, respectively. The CC1 domain adopts an antiparallel helical hairpin that further dimerizes into a four-helix bundle. The CC2 domain also mediates dimerization. At least one of the coiled coils is required for actin binding, indicating that SCAB1 is a bivalent actin cross-linker. The key residues required for actin binding were identified. The PH domain lacks a canonical basic phosphoinositide-binding pocket but can bind weakly to inositol phosphates via a basic surface patch, implying the involvement of inositol signaling in SCAB1 regulation. Our results provide novel insights into the functional organization of SCAB1.     

The role of CKIP-1 in cell morphology depends on its interaction with actin-capping protein

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.     

Translation elongation factor 1A is essential for regulation of the actin cytoskeleton and cell morphology

The binding of eukaryotic translation elongation factor 1A (eEF1A) to actin is a noncanonical function that may link two distinct cellular processes, cytoskeleton organization and gene expression. Using the yeast Saccharomyces cerevisiae, we have established an in vivo assay that directly identifies specific regions and residues of eEF1A responsible for actin interactions and bundling. Using a unique genetic screen, we isolated a series of eEF1A mutants with reduced actin bundling activity. These mutations alter actin cytoskeleton organization but not translation, indicating that these are separate functions of eEF1A. This demonstrates for the first time a direct consequence of eEF1A on cytoskeletal organization in vivo and the physiological significance of this interaction.     

WIP: a multifunctional protein involved in actin cytoskeleton regulation

Knowledge of the dynamics of actin-based structures is a major key to understanding how cells move and respond to their environment. The ability to reorganize actin filaments in a spatial and temporal manner to integrate extracellular signals is at the core of cell adhesion and cell migration. Several proteins have been described as regulators of actin polymerization: this review will focus on the role of WASP-interacting protein (WIP), an actin-binding protein that participates in actin polymerization regulation and signal transduction. WIP is widely expressed and interacts with Wiskott-Aldrich syndrome protein (WASP) (a hematopoietic-specific protein) and its more widely expressed homologue neural WASP (N-WASP), to regulate WASP/N-WASP function in Arp2/3-mediated actin polymerization. WIP also interacts with profilin, globular and filamentous actin (G- and F-actin, respectively) and stabilizes actin filaments. In vivo WIP participates in filopodia and lamellipodia formation, in T and B lymphocyte activation, in mast cell degranulation and signaling through the Fcepsilon receptor (FcepsilonR), in microbial motility and in Syk protein stability.     

The actin cytoskeleton is involved in the regulation of the plasmodesmal size exclusion limit

Plasmodesmata (PD) are the communication channels which allow the trafficking of macromolecules between neighboring cells. Such cell-to-cell movement of macromolecules is regulated during plant growth and development; however, little is known about the regulation mechanism of PD size exclusion limit (SEL). Plant viral movement proteins (MPs) enhance the invasion of viruses from cell to cell by increasing the SEL of the PD and are therefore a powerful means for the study of the plasmodesmal regulation mechanisms. In a recent study, we reported that the actin cytoskeleton is involved in the increase of the PD SEL induced by MPs. Microinjection experiments demonstrated that actin depolymerization was required for the Cucumber mosaic virus (CMV) MP-induced increase in the PD SEL. In vitro experiments showed that CMV MP severs actin filaments (F-actin). Furthermore, through the analyses of two CMV MP mutants, we demonstrated that the F-actin severing ability of CMV MP was required to increase the PD SEL. These results are similar to what has been found in Tobacco mosaic virus MP. Thus, our data suggest that actin dynamics may participate in the regulations of the PD SEL.Key words: plasmodesmata, size exclusion limit, movement protein, actin filaments, F-actin severing     

Citron-N is a neuronal Rho-associated protein involved in Golgi organization through actin cytoskeleton regulation

The actin cytoskeleton is best known for its role during cellular morphogenesis. However, other evidence suggests that actin is also crucial for the organization and dynamics of membrane organelles such as endosomes and the Golgi complex. As in morphogenesis, the Rho family of small GTPases are key mediators of organelle actin-driven events, although it is unclear how these ubiquitously distributed proteins are activated to regulate actin dynamics in an organelle-specific manner. Here we show that the brain-specific Rho-binding protein Citron-N is enriched at, and associates with, the Golgi apparatus of hippocampal neurons in culture. Suppression of the whole protein or expression of a mutant form lacking the Rho-binding activity results in dispersion of the Golgi apparatus. In contrast, high intracellular levels induce localized accumulation of RhoA and filamentous actin, protecting the Golgi from the rupture normally produced by actin depolymerization. Biochemical and functional analyses indicate that Citron-N controls actin locally by assembling together the Rho effector ROCK-II and the actin-binding, neuron-specific, protein Profilin-IIa (PIIa). Together with recent data on endosomal dynamics, our results highlight the importance of organelle-specific Rho modulators for actin-dependent organelle organization and dynamics.     

The Saccharomyces cerevisiae actin-related protein Arp2 is involved in the actin cytoskeleton

Arp2p is an essential yeast actin-related protein. Disruption of the corresponding ARP2 gene leads to a terminal phenotype characterized by the presence of a single large bud. Thus, Arp2p may be important for a late stage of the cell cycle (Schwob, E., and R.P. Martin, 1992. Nature (Lond.). 355:179-182). We have localized Arp2p by indirect immunofluorescence. Specific peptide antibodies revealed punctate staining under the plasma membrane, which partially colocalizes with actin. Temperature-sensitive arp2 mutations were created by PCR mutagenesis and selected by an ade2/SUP11 sectoring screen. One temperature-sensitive mutant that was characterized, arp2-H330L, was osmosensitive and had an altered actin cytoskeleton at a nonpermissive temperature, suggesting a role of Arp2p in the actin cytoskeleton. Random budding patterns were observed in both haploid and diploid arp2- H330L mutant cells. Endocytosis, as judged by Lucifer yellow uptake, was severely reduced in the mutant, at all temperatures. In addition, genetic interaction was observed between temperature-sensitive alleles arp2-H330L and cdc10-1. CDC10 is a gene encoding a neck filament- associated protein that is necessary for polarized growth and cytokinesis. Overall, the immunolocalization, mutant phenotypes, and genetic interaction suggest that the Arp2 protein is an essential component of the actin cytoskeleton that is involved in membrane growth and polarity, as well as in endocytosis.     

Tobacco WLIM1 is a novel F-actin binding protein involved in actin cytoskeleton remodeling

We used confocal microscopy and in vitro analyses to show that Nicotiana tabacum WLIM1, a LIM domain protein related to animal Cys-rich proteins, is a novel actin binding protein in plants. Green fluorescent protein (GFP)-tagged WLIM1 protein accumulated in the nucleus and cytoplasm of tobacco BY2 cells. It associated predominantly with actin cytoskeleton, as demonstrated by colabeling and treatment with actin-depolymerizing latrunculin B. High-speed cosedimentation assays revealed the ability of WLIM1 to bind directly to actin filaments with high affinity. Fluorescence recovery after photobleaching and fluorescence loss in photobleaching showed a highly dynamic in vivo interaction of WLIM1-GFP with actin filaments. Expression of WLIM1-GFP in BY2 cells significantly delayed depolymerization of the actin cytoskeleton induced by latrunculin B treatment. WLIM1 also stabilized actin filaments in vitro. Importantly, expression of WLIM1-GFP in Nicotiana benthamiana leaves induces significant changes in actin cytoskeleton organization, specifically, fewer and thicker actin bundles than in control cells, suggesting that WLIM1 functions as an actin bundling protein. This hypothesis was confirmed by low-speed cosedimentation assays and direct observation of F-actin bundles that formed in vitro in the presence of WLIM1. Taken together, these data identify WLIM1 as a novel actin binding protein that increases actin cytoskeleton stability by promoting bundling of actin filaments.     

The interaction of capping protein with the barbed end of the actin filament

The interaction of capping protein (CP) with actin filaments is an essential element of actin assembly and actin-based motility in nearly all eukaryotes. The dendritic nucleation model for Arp2/3-based lamellipodial assembly features capping of barbed ends by CP, and the formation of filopodia is proposed to involve inhibition of capping by formins and other proteins. To understand the molecular basis for how CP binds the barbed end of the actin filament, we have used a combination of computational and experimental approaches, primarily involving molecular docking and site-directed mutagenesis. We arrive at a model that supports all of our biochemical data and agrees very well with a cryo-electron microscopy structure of the capped filament. CP interacts with both actin protomers at the barbed end of the filament, and the amphipathic helix at the C-terminus of the β-subunit binds to the hydrophobic cleft on actin, in a manner similar to that of WH2 domains. These studies provide us with new molecular insight into how CP binds to the actin filament.     

hhLIM is a novel F-actin binding protein involved in actin cytoskeleton remodeling

Human heart LIM protein (hhLIM) is a newly cloned protein. In vitro analyses showed that green fluorescent protein (GFP)-tagged hhLIM protein accumulated in the cytoplasm of C2C12 cells and colocalized with F-actin, indicating that hhLIM is an actin-binding protein in C2C12 cells. Overexpression of hhLIM-GFP in C2C12 cells significantly stabilized actin filaments and delayed depolymerization of the actin cytoskeleton induced by cytochalasin B treatment. Expression of hhLIM-GFP in C2C12 cells also induced significant changes in the organization of the actin cytoskeleton, specifically, fewer and thicker actin bundles than in control cells, suggesting that hhLIM functions as an actin-bundling protein. This hypothesis was confirmed using low-speed co-sedimentation assays and direct observation of F-actin bundles that formed in vitro in the presence of hhLIM. hhLIM has two LIM domains. To identify the essential regions and sites for association, a series of truncated mutants was constructed which showed that LIM domain 2 has the same activity as full-length hhLIM. To further characterize the binding sites, the LIM domain was functionally destructed by replacing cysteine with serine in domain 2, and results showed that the second LIM domain plays a central role in bundling of F-actin. Taken together, these data identify hhLIM as an actin-binding protein that increases actin cytoskeleton stability by promoting bundling of actin filaments.     

The interaction between the adaptor protein APS and Enigma is involved in actin organisation

APS (adaptor protein with PH and SH2 domains) is an adaptor protein phosphorylated by several tyrosine kinase receptors including the insulin receptor. To identify novel binding partners of APS, we performed yeast two-hybrid screening. We identified Enigma, a PDZ and LIM domain-containing protein that was previously shown to be associated with the actin cytoskeleton. In HEK 293 cells, Enigma interacted specifically with APS, but not with the APS-related protein SH2-B. This interaction required the NPTY motif of APS and the LIM domains of Enigma. In NIH-3T3 cells that express the insulin receptor, Enigma and APS were partially co-localised with F-actin in small ruffling structures. Insulin increased the complex formation between APS and Enigma and their co-localisation in large F-actin containing ruffles. While in NIH-3T3 and HeLa cells the co-expression of both Enigma and APS did not modify the actin cytoskeleton organisation, expression of Enigma alone led to the formation of F-actin clusters. Similar alteration in actin cytoskeleton organisation was observed in cells expressing both Enigma and APS with a mutation in the NPTY motif. These results identify Enigma as a novel APS-binding protein and suggest that the APS/Enigma complex plays a critical role in actin cytoskeleton organisation.     

A new interaction between Abi.1 and βPIX involved in PDGF.activated actin cytoskeleton reorganisation

Members of the Rho family of GTPases are key regulators of the actin cytoskeleton. In particular, activated Racl stimulates membrane dorsal ruffle formation in response to platelet-derived growth factor （PDGF）. Abl-interactor （Abi）- 1 and βP1X, a guanine nucleotide exchange factor for Racl, localise at these Rac1-induced actin structures and play important roles in the induction of membrane dorsal ruffling in response to PDGF in fibroblasts. Here, we demonstrate a novel interaction between Abi-1 and βPIX using the yeast two-hybrid system, in vitro pull-down assays, and in vivo co-immunoprecipitation experiments. In vitro, the C-terminal fragment of βPIX interacted with Abi-1, while in vivo the N-terminal fragment of βPIX interacted with Abi-1. The biological function of this interaction was investigated in mouse fibroblasts in response to PDGF stimulation. Abi-1 and βPIX co-localised in the cytoplasm and to membrane dorsal ruffles after PDGF treatment. We show that the co-expression of Abi-1 and truncated forms of βPIX in mouse fibroblasts blocked PDGF-induced membrane dorsal ruffles. Together, these results show that the interaction between Abi-1 and βPIX is involved in the formation of growth factor-induced membrane dorsal ruffles.     

Distinct effects on the secretion of MTRAP and AMA1 in Plasmodium yoelii following deletion of acylated pleckstrin homology domain-containing protein

Plasmodium, the causative agents of malaria, are obligate intracellular organisms. In humans, pathogenesis is caused by the blood stage parasite, which multiplies within erythrocytes, thus erythrocyte invasion is an essential developmental step. Merozoite form parasites released into the blood stream coordinately secrets a panel of proteins from the microneme secretory organelles for gliding motility, establishment of a tight junction with a target naive erythrocyte, and subsequent internalization. A protein identified in Toxoplasma gondii facilitates microneme fusion with the plasma membrane for exocytosis; namely, acylated pleckstrin homology domain-containing protein (APH). To obtain insight into the differential microneme discharge by malaria parasites, in this study we analyzed the consequences of APH deletion in the rodent malaria model, Plasmodium yoelii, using a DiCre-based inducible knockout method. We found that APH deletion resulted in a reduction in parasite asexual growth and erythrocyte invasion, with some parasites retaining the ability to invade and grow without APH. APH deletion impaired the secretion of microneme proteins, MTRAP and AMA1, and upon contact with erythrocytes the secretion of MTRAP, but not AMA1, was observed. APH-deleted merozoites were able to attach to and deform erythrocytes, consistent with the observed MTRAP secretion. Tight junctions were formed, but echinocytosis after merozoite internalization into erythrocytes was significantly reduced, consistent with the observed absence of AMA1 secretion. Together with our observation that APH largely colocalized with MTRAP, but less with AMA1, we propose that APH is directly involved in MTRAP secretion; whereas any role of APH in AMA1 secretion is indirect in Plasmodium.     

B2-1, a Sec7- and pleckstrin homology domain-containing protein, localizes to the Golgi complex

B2-1 is a human protein that contains both a Sec7 and a pleckstrin homology domain. The yeast Sec7 protein was previously shown to be involved in vesicle formation in the Golgi and endoplasmic reticulum. Recently, several groups have shown that B2-1 and highly similar proteins (e.g., ARNO, ARNO3) have varied cellular functions and subcellular locations. One of these is an association of the B2-1 Sec7 domain with the plasma membrane, binding to the cytoplasmic portion of the integrin beta2 chain (CD18) and is postulated to be involved in inside-out signaling. Other groups have shown that B2-1 and these related proteins are guanine nucleotide-exchange factors that act upon ADP ribosylation factors (ARFs) and are localized to the Golgi or plasma membrane. Here we report the subcellular localization of B2-1 protein. Interestingly, B2-1 does not localize to the plasma membrane but rather associates with a distinct Golgi complex compartment. B2-1's distribution can be disrupted by brefeldin A, a drug that rapidly disrupts the Golgi apparatus by inhibiting ARF activity. Furthermore, transient transfection of GFP-tagged B2-1 shows Golgi complex targeting. Excessive overexpression of transfected B2-1 causes partial Golgi dispersion.     

Role of phosphatidylinositol 3' kinase and a downstream pleckstrin homology domain-containing protein in controlling chemotaxis in dictyostelium

We show that cells lacking two Dictyostelium class I phosphatidylinositol (PI) 3' kinases (PI3K and pi3k1/2-null cells) or wild-type cells treated with the PI3K inhibitor LY294002 are unable to properly polarize, are very defective in the temporal, spatial, and quantitative regulation of chemoattractant-mediated filamentous (F)-actin polymerization, and chemotax very slowly. PI3K is thought to produce membrane lipid-binding sites for localization of PH domain-containing proteins. We demonstrate that in response to chemoattractants three PH domain-containing proteins do not localize to the leading edge in pi3k1/2-null cells, and the translocation is blocked in wild-type cells by LY294002. Cells lacking one of these proteins, phdA-null cells, exhibit defects in the level and kinetics of actin polymerization at the leading edge and have chemotaxis phenotypes that are distinct from those described previously for protein kinase B (PKB) (pkbA)-null cells. Phenotypes of PhdA-dominant interfering mutations suggest that PhdA is an adaptor protein that regulates F-actin localization in response to chemoattractants and links PI3K to the control of F-actin polymerization at the leading edge during pseudopod formation. We suggest that PKB and PhdA lie downstream from PI3K and control different downstream effector pathways that are essential for proper chemotaxis.     

Unique sequence of a high molecular weight myosin light chain kinase is involved in interaction with actin cytoskeleton

Resolution of neutrophil mediated inflammation is achieved, in part, through induction of neutrophil apoptosis. This constitutively expressed programme can be delayed by inflammatory mediators and induced by ligation of the Fas receptor. However, functional activation of the neutrophil results in resistance to Fas signalled death. We evaluated the effects of Fas antibody engagement on caspase activation and mitochondrial permeability, and the impact of co-stimulation by lipopolysaccharide (LPS) or granulocyte macrophage-colony stimulating factor (GM-CSF) on these events. Fas engagement by an agonistic anti-Fas antibody resulted in enhanced caspase 3 and 8 activity and increased mitochondrial permeability. Studies with pharmacological inhibitors of caspase activity showed that activation of caspase 8 occurred before, and activation of caspase 3 occurred after mitochondrial disruption. The mitochondrial stabilising agent bongkrekic acid also inhibited caspase activation and apoptosis. LPS, GM-CSF and increased glutathione stabilised the mitochondria and inhibited caspase 3. Caspase 8 activity was also inhibited by co-stimulation through a mechanism independent of mitochondrial stabilisation. Glutathione directly inhibited caspase 3 and 8 activity. We conclude inhibition of Fas antibody induced apoptosis by inflammatory proteins is associated with augmented mitochondrial stability and reduced caspase 3 activity that may be glutathione mediated.