Mammalian Abp1, a signal-responsive F-actin-binding protein, links the actin cytoskeleton to endocytosis via the GTPase dynamin

The actin cytoskeleton has been implicated in endocytosis, yet few molecular links to the endocytic machinery have been established. Here we show that the mammalian F-actin-binding protein Abp1 (SH3P7/HIP-55) can functionally link the actin cytoskeleton to dynamin, a GTPase that functions in endocytosis. Abp1 binds directly to dynamin in vitro through its SH3 domain. Coimmunoprecipitation and colocalization studies demonstrated the in vivo relevance of this interaction. In neurons, mammalian Abp1 and dynamin colocalized at actin-rich sites proximal to the cell body during synaptogenesis. In fibroblasts, mAbp1 appeared at dynamin-rich sites of endocytosis upon growth factor stimulation. To test whether Abp1 functions in endocytosis, we overexpressed several Abp1 constructs in Cos-7 cells and assayed receptor-mediated endocytosis. While overexpression of Abp1's actin-binding modules did not interfere with endocytosis, overexpression of the SH3 domain led to a potent block of transferrin uptake. This implicates the Abp1/dynamin interaction in endocytic function. The endocytosis block was rescued by cooverexpression of dynamin. Since the addition of the actin-binding modules of Abp1 to the SH3 domain construct also fully restored endocytosis, Abp1 may support endocytosis by combining its SH3 domain interactions with cytoskeletal functions in response to signaling cascades converging on this linker protein.     

Actin cytoskeleton and budding pattern are altered in the yeast rvs161 mutant: the Rvs161 protein shares common domains with the brain protein amphiphysin

The actin cytoskeleton cells is altered in rvs161 mutant yeast, with the defect becoming more pronounced under unfavorable growth conditions, as described for the rvs167 mutant. The cytoskeletal alteration has no apparent effect on invertase secretion and polarized growth. Mutations in RTVS161, just as in RI/S167, lead to a random budding pattern in a/ diploid cells. This behavior is not observed in a/a diploid cells homozygous for the rvs161-1 or rvs167-1 mutations. In addition, sequence comparisons revealed that amphiphysin, a protein first found in synaptic vesicles of chicken and shown to be the autoantigen of Stiff Man syndrome, presents similarity with both Rvs proteins. Furthermore, limited similarities with myosin heavy chain and tropomyosin alpha chain from higher eukaryotic cells allow for the definition of a possible consensus sequence. The finding of related sequences suggests the existence of a function for these proteins that is conserved among eukaryotic organisms.     

Actin cytoskeleton and budding pattern are altered in the yeast rvs161 mutant: the Rvs161 protein shares common domains with the brain protein amphiphysin

The actin cytoskeleton cells is altered in rvs161 mutant yeast, with the defect becoming more pronounced under unfavorable growth conditions, as described for the rvs167 mutant. The cytoskeletal alteration has no apparent effect on invertase secretion and polarized growth. Mutations in RTVS161, just as in RI/S167, lead to a random budding pattern in a/α diploid cells. This behavior is not observed in a/a diploid cells homozygous for the rvs161-1 or rvs167-1 mutations. In addition, sequence comparisons revealed that amphiphysin, a protein first found in synaptic vesicles of chicken and shown to be the autoantigen of Stiff Man syndrome, presents similarity with both Rvs proteins. Furthermore, limited similarities with myosin heavy chain and tropomyosin alpha chain from higher eukaryotic cells allow for the definition of a possible consensus sequence. The finding of related sequences suggests the existence of a function for these proteins that is conserved among eukaryotic organisms.     

A novel link between a rab GTPase and Rvs proteins: the yeast amphiphysin homologues

The BAR proteins are a well-conserved family of proteins including Rvsp in yeast, amphiphysins and Bin proteins in mammals. In yeast, as in mammals, BAR proteins are known to be implicated in vesicular traffic. The Gyp5p (Ypl249p) and Ymr192p proteins interact in two-hybrid tests with both Rvs161p and Rvs167p. Gyp5p is a Ypt/Rab-specific GAP and Ymr192p is highly similar to Gyp5p. To specify the interaction between Rvsp and Gyp5p, we used two-hybrid tests to determine the domains necessary for these interactions. The specific SH3 domain of Rvs167p interacted with the N-terminal domain of Gyp5p. Moreover, Gyp5p could form a homodimer. Fus2 protein is a specific partner of Rvs161p in two-hybrid tests. To characterize the functional relationships between these five proteins, we have studied cellular phenotypes in single, double and triple mutant strains for which rvs mutants present defects, such as polarity, cell fusion and meiosis. Phenotypic analysis showed that Gyp5p, Ymr192p and Fus2p were involved in bipolar budding pattern and in meiosis. Specific epistasis or suppressive phenomena were found between the five mutations. Finally, The Gyp5p-GFP fusion protein was localized at the bud tip during apical growth and at the mother-bud neck during cytokinesis. Moreover, Rvs167p and Rvs161p were shown to be essential for the correct localization of Gyp5p. Altogether, these data support the hypothesis that both Rvsp proteins act in vesicular traffic through physical and functional interactions with Ypt/Rab regulators.     

The pleckstrin homology domain-containing protein CKIP-1 is involved in regulation of cell morphology and the actin cytoskeleton and interaction with actin capping protein

CKIP-1 is a pleckstrin homology domain-containing protein that interacts with protein kinase CK2. To elucidate the functions of CKIP-1, we generated human osteosarcoma cell lines with tetracycline-regulated expression of Flag-CKIP-1. Flag-CKIP-1 expression resulted in distinct changes in cellular morphology. Therefore, we examined the actin profile by immunofluorescence, quantitative measurement of phalloidin binding, and immunoblot analysis. These studies demonstrate that Flag-CKIP-1 expression resulted in increases in F-actin staining and protein levels of beta-actin. To elucidate the mechanisms behind the observed phenotype, we utilized tandem affinity purification to isolate CKIP-1 interacting proteins. Mass spectrometry analysis led to the identification of the actin capping protein subunits, CPalpha and CPbeta, as novel CKIP-1 interaction partners. Interactions were confirmed by coimmunoprecipitation and by colocalization. Furthermore, we demonstrate that Ser9 of CPalpha is phosphorylated by protein kinase CK2 in vitro, that CPalpha is phosphorylated in vivo, and that treatment with a CK2-specific inhibitor results in a decrease in CPalpha phosphorylation. Finally, we demonstrate that CKIP-1 and CK2 inhibit the activity of actin capping protein at the barbed ends of actin filaments. Overall, our results are consistent with CKIP-1 playing a role in the regulation of the actin cytoskeleton through its interactions with actin capping protein.     

Characterization of a novel GTPase-activating protein associated with focal adhesions and the actin cytoskeleton

In the present study we characterize a novel RhoGAP protein (RC-GAP72) that interacts with actin stress fibers, focal adhesions, and cell-cell adherens junctions via its 185-amino acid C-terminal region. Overexpression of RC-GAP72 in fibroblasts induces cell rounding with partial or complete disruption of actin stress fibers and formation of membrane ruffles, lamellipodia, and filopodia. RC-GAP72 mutant truncated downstream of the GTPase-activating protein (GAP) domain retains the ability to stimulate membrane protrusions but fails to affect stress fiber integrity or induce cell retraction. A mutant protein consisting of the C terminus of RC-GAP72 and lacking the GAP domain does not exert any visible effect on cellular morphology. Inactivation of the GAP domain by a point mutation does not abolish the effect of RC-GAP72 on actin stress fibers but moderates its capability to induce membrane protrusions. Our data imply that the cytoskeletal localization of RC-GAP72 and its interaction with GTPases are essential for its effect on the integrity of actin stress fibers, whereas the induction of lamellipodia and filopodia depends on the activity of the GAP domain irrespective of binding to the actin cytoskeleton. We propose that RC-GAP72 affects cellular morphology by targeting activated Cdc42 and Rac1 GTPases to specific subcellular sites, triggering local morphological changes. The overall physiological functions of RC-GAP72 are presently unknown, yet our data suggest that RC-GAP72 plays a role in regulating cell morphology and cytoskeletal organization.     

Phosphorylation of a novel myosin binding subunit of protein phosphatase 1 reveals a conserved mechanism in the regulation of actin cytoskeleton

The myotonic dystrophy kinase-related kinases RhoA binding kinase and myotonic dystrophy kinase-related Cdc42 binding kinase (MRCK) are effectors of RhoA and Cdc42, respectively, for actin reorganization. Using substrate screening in various tissues, we uncovered two major substrates, p130 and p85, for MRCKalpha-kinase. p130 is identified as myosin binding subunit p130, whereas p85 is a novel related protein. p85 contains N-terminal ankyrin repeats, an alpha-helical C terminus with leucine repeats, and a centrally located conserved motif with the MRCKalpha-kinase phosphorylation site. Like MBS130, p85 is specifically associated with protein phosphatase 1delta (PP1delta), and this requires the N terminus, including the ankyrin repeats. This association is required for the regulation of both the catalytic activities and the assembly of actin cytoskeleton. The N terminus, in association with PP1delta, is essential for actin depolymerization, whereas the C terminus antagonizes this action. The C-terminal effects consist of two independent events that involved both the conserved phosphorylation inhibitory motif and the alpha-helical leucine repeats. The former was able to interact with PP1delta only in the phosphorylated state and result in inactivation of PP1delta activity. This provides further evidence that phosphorylation of a myosin binding subunit protein by specific kinases confers conformational changes in a highly conserved region that plays an essential role in the regulation of its catalytic subunit activities.     

Distinct domains of paracingulin are involved in its targeting to the actin cytoskeleton and regulation of apical junction assembly

Paracingulin is an M(r) 150-160 kDa cytoplasmic protein of vertebrate epithelial tight and adherens junctions and comprises globular head, coiled-coil rod, and globular tail domains. Unlike its homologous tight junction protein cingulin, paracingulin has been implicated in the control of junction assembly and has been localized at extrajunctional sites in association with actin filaments. Here we analyze the role of paracingulin domains, and specific regions within the head and rod domains, in the function and localization of paracingulin by inducible overexpression of exogenous proteins in epithelial Madin Darby canine kidney (MDCK) cells and by expression of mutated and chimeric constructs in Rat1 fibroblasts and MDCK cells. The overexpression of the rod + tail domains of paracingulin perturbs the development of the tight junction barrier and Rac1 activation during junction assembly by the calcium switch, indicating that regulation of junction assembly by paracingulin is mediated by these domains. Conversely, only constructs containing the head domain target to junctions in MDCK cells and Rat1 fibroblasts. Furthermore, expression of chimeric cingulin and paracingulin constructs in Rat1 fibroblasts and MDCK cells identifies specific sequences within the head and rod domains of paracingulin as critical for targeting to actin filaments and regulation of junction assembly, respectively. In summary, we characterize the functionally important domains of paracingulin that distinguish it from cingulin.     

Control of intramolecular interactions between the pleckstrin homology and Dbl homology domains of Vav and Sos1 regulates Rac binding

Vav and Sos1 are Dbl family guanine nucleotide exchange factors, which activate Rho family GTPases in response to phosphatidylinositol 3-kinase products. A pleckstrin homology domain adjacent to the catalytic Dbl homology domain via an unknown mechanism mediates the effects of phosphoinositides on guanine nucleotide exchange activity. Here we tested the possibility that phosphatidylinositol 3-kinase substrates and products control an interaction between the pleckstrin homology domain and the Dbl homology domain, thereby explaining the inhibitory effects of phosphatidylinositol 3-kinase substrates and stimulatory effects of the products. Binding studies using isolated fragments of Vav and Sos indicate phosphatidylinositol 3-kinase substrate promotes the binding of the pleckstrin homology domain to the Dbl homology domain and blocks Rac binding to the DH domain, whereas phosphatidylinositol 3-kinase products disrupt the Dbl homology/pleckstrin homology interactions and permit Rac binding. Additionally, Lck phosphorylation of Vav, a known activating event, reduces the affinities between the Vav Dbl homology and pleckstrin homology domains and permits Rac binding. We also show Vav activation in cells, as monitored by phosphorylation of Vav, Vav association with phosphatidylinositol 3,4,5-trisphosphate, and Vav guanine nucleotide exchange activity, is blocked by the phosphatidylinositol 3-kinase inhibitor wortmannin. These results suggest the molecular mechanisms for activation of Vav and Sos1 require disruption of inhibitory intramolecular interactions involving the pleckstrin homology and Dbl homology domains.     

Identification and characterization of a protein containing formin homology (FH1/FH2) domains.

A novel member of the Formin/Diaphanous family of proteins was cloned and characterized. A 4kB mRNA is ubiquitously expressed but is found in abundance in the spleen. FHOS (Formin Homologue Overexpressed in Spleen) contains a 3414bp open reading frame and encodes for an approximately 128kDa protein. FHOS has sequence homology to Diaphanous and Formin proteins within the Formin Homology (FH)1 and FH2 domains. FHOS also contains a coiled-coil, a collagen-like domain, two nuclear localization signals, and several potential PKC and PKA phosphorylation sites. FHOS-specific antiserum was generated and used to determine that FHOS is a predominantly cytoplasmic protein and is expressed in a variety of human cell lines. FHOS was mapped to chromosome 16q22 between framework markers WI-5594 and WI-9392.     

Adenosine-oligoarginine conjugate, a novel bisubstrate inhibitor, effectively dissociates the actin cytoskeleton

Aberrant regulation of protein kinases impairs normal cellular functioning and may lead to disease. The protein kinase involved in the regulation of the dynamics of the actin cytoskeleton, Rho-kinase (ROCK), phosphorylates various substrates (e.g. myosin light chain, myosin phosphatase), causing the formation of actin fibers and tension inside cells. Hyperactivation of ROCK, for example, causes hypertension and cardiovascular disorders. Thus, the design of highly specific protein kinase inhibitors is of the utmost importance. To date, the majority of inhibitors investigated have been found to mimic and compete with ATP. However, in the present study we characterized the cellular effects of a novel bisubstrate inhibitor -- adenosine-oligoarginine conjugate (ARC) -- designed to interfere simultaneously with the ATP site and the substrate-binding pocket of basophilic kinases. ARC effectively pulled down ROCK from cell lysates, showed no cytotoxicity and suppressed the assembly of the actin cytoskeleton (especially central actin bundles) as the result of interference with the activity of the kinase. Combination of ARC with chloroquine yielded a stronger inhibitory effect and gave results similar to treatment with Y-27632. However, treatment with ARC produced more actin fragments and yielded a longer-lasting effect than treatment with Y-27632. Additionally, quantification of phosphorylated myosin light chain levels in ARC-treated or Y-27632-treated cells implies that ARC is more effective than Y-27632 in suppressing the phosphorylation of at least one of the substrates of ROCK. We believe that the described bisubstrate strategy could be a useful lead for designing novel, highly specific inhibitors for different protein kinases.     

MAP-1, a novel proapoptotic protein containing a BH3-like motif that associates with Bax through its Bcl-2 homology domains

A novel Bax-associating protein, named MAP-1 (Modulator of Apoptosis), has been identified in a yeast two-hybrid screen. MAP-1 contains a BH3-like (BH: Bcl-2 homology) motif and mediates caspase-dependent apoptosis in mammalian cells when overexpressed. MAP-1 homodimerizes and associates with the proapoptotic Bax and the prosurvival Bcl-2 and Bcl-X(L) of the Bcl-2 family in vitro and in vivo in mammalian cells. Mutagenesis analyses revealed that the BH3-like domain in MAP-1 is not required for its association with Bcl-X(L) but is required for association with Bax and for mediating apoptosis. Interestingly, in contrast to other Bax-associating proteins such as Bcl-X(L) and Bid, which require the BH3 and BH1 domains of Bax, respectively, for binding, the binding of MAP-1 to Bax appears to require all three BH domains (BH1, BH2, and BH3) of Bax, because point mutation of the critical amino acid in any one of these domains is sufficient to abolish its binding to MAP-1. These data suggest that MAP-1 mediates apoptosis through a mechanism that involves binding to Bax.     

CIPER, a novel NF kappaB-activating protein containing a caspase recruitment domain with homology to Herpesvirus-2 protein E10

We have identified and characterized CIPER, a novel protein containing a caspase recruitment domain (CARD) in its N terminus and a C-terminal region rich in serine and threonine residues. The CARD of CIPER showed striking similarity to E10, a product of the equine herpesvirus-2. CIPER formed homodimers via its CARD and interacted with viral E10 but not with several apoptosis regulators containing CARDs including ARC, RAIDD, RICK, caspase-2, caspase-9, or Apaf-1. Expression of CIPER induced NF-kappaB activation, which was inhibited by dominant-negative NIK and a nonphosphorylable IkappaB-alpha mutant but not by dominant-negative RIP. Mutational analysis revealed that the N-terminal region of CIPER containing the CARD was sufficient and necessary for NF-kappaB-inducing activity. Point mutations in highly conserved residues in the CARD of CIPER disrupted the ability of CIPER to activate NF-kappaB and to form homodimers, indicating that the CARD is essential for NF-kappaB activation and dimerization. We propose that CIPER acts in a NIK-dependent pathway of NF-kappaB activation.     

Concerted regulation of cell dynamics by BNIP-2 and Cdc42GAP homology/Sec14p-like,proline-rich,and GTPase-activating protein domains of a novel Rho GTPase-activating protein,BPGAP1

RhoA, Cdc42, and Rac1 are small GTPases that regulate cytoskeletal reorganization leading to changes in cell morphology and cell motility. Their signaling pathways are activated by guanine nucleotide exchange factors and inactivated by GTPase-activating proteins (GAPs). We have identified a novel RhoGAP, BPGAP1 (for BNIP-2 and Cdc42GAP Homology (BCH) domain-containing, Proline-rich and Cdc42GAP-like protein subtype-1), that is ubiquitously expressed and shares 54% sequence identity to Cdc42GAP/p50RhoGAP. BP-GAP1 selectively enhanced RhoA GTPase activity in vivo although it also interacted strongly with Cdc42 and Rac1. "Pull-down" and co-immunoprecipitation studies indicated that it formed homophilic or heterophilic complexes with other BCH domain-containing proteins. Fluorescence studies of epitope-tagged BPGAP1 revealed that it induced pseudopodia and increased migration of MCF7 cells. Formation of pseudopodia required its BCH and GAP domains but not the proline-rich region, and was differentially inhibited by coexpression of the constitutively active mutant of RhoA, or dominant negative mutants of Cdc42 and Rac1. However, the mutant without the proline-rich region failed to confer any increase in cell migration despite the induction of pseudopodia. Our findings provide evidence that cell morphology changes and migration are coordinated via multiple domains in BPGAP1 and present a novel mode of regulation for cell dynamics by a RhoGAP protein.     

Protein kinase C and the regulation of the actin cytoskeleton

Protein kinase C (PKC) isoforms are central components in intracellular networks that regulate a vast number of cellular processes. It has long been known that in most cell types, one or more PKC isoforms influences the morphology of the F-actin cytoskeleton and thereby regulates processes that are affected by remodelling of the microfilaments. These include cellular migration and neurite outgrowth. This review focuses on the role of classical and novel PKC isoforms in migration and neurite outgrowth, and highlights some regulatory steps that may be of importance in the regulation by PKC of migration and neurite outgrowth. Many studies indicate that integrins are crucial mediators both upstream and downstream of PKC in inducing morphological changes. Furthermore, a number of PKC substrates, directly associated with the microfilaments, such as MARCKS, GAP43, adducin, fascin, ERM proteins and others have been identified. Their potential role in PKC effects on the cytoskeleton is discussed.     

EHD2 and the novel EH domain binding protein EHBP1 couple endocytosis to the actin cytoskeleton

Here we identified two novel proteins denoted EH domain protein 2 (EHD2) and EHD2-binding protein 1 (EHBP1) that link clathrin-mediated endocytosis to the actin cytoskeleton. EHD2 contains an N-terminal P-loop and a C-terminal EH domain that interacts with NPF repeats in EHBP1. Disruption of EHD2 or EHBP1 function by small interfering RNA-mediated gene silencing inhibits endocytosis of transferrin into EEA1-positive endosomes as well as GLUT4 endocytosis into cultured adipocytes. EHD2 localizes with cortical actin filaments, whereas EHBP1 contains a putative actin-binding calponin homology domain. High expression of EHD2 or EHBP1 in intact cells mediates extensive actin reorganization. Thus EHD2 appears to connect endocytosis to the actin cytoskeleton through interactions of its N-terminal domain with membranes and its C-terminal EH domain with the novel EHBP1 protein.     

Endocytosis of GPI-anchored proteins in human lymphocytes: role of glycolipid-based domains, actin cytoskeleton, and protein kinases

GPI-anchored surface proteins mediate many important functions, including transport, signal transduction, adhesion, and protection against complement. They cluster into glycolipid-based membrane domains and caveolae, plasmalemmal vesicles involved in the transcytosis and endocytosis of these surface proteins. However, in lymphocytes, neither the characteristic flask shaped caveolae nor caveolin, a transmembrane protein typical of caveolae, have been observed. Here, we show that the GPI-anchored CD59 molecule on Jurkat T cells is internalized after cross-linking, a process inhibited by nystatin, a sterol chelating agent. Clustered CD59 molecules mostly accumulate in non-coated invaginations of the lymphocyte membrane before endocytosis, in marked contrast with the pattern of CD3-TCR internalization. Cytochalasin H blocked CD59 internalization in lymphocytes, but neither CD3 internalization nor transferrin uptake. Confocal microscopy analysis of F-actin distribution within lymphocytes showed that CD59 clusters were associated with patches of polymerized actin. Also, we found that internalization of CD59 was prevented by the protein kinase C inhibitor staurosporine and by the protein kinase A activator forskolin. Thus, in lymphocytes, as in other cell types, glycolipid-based domains provide sites of integration of signaling pathways involved in GPI-anchored protein endocytosis. This process, which is regulated by both protein kinase C and A activity, is tightly controlled by the dynamic organization of actin cytoskeleton, and may be critical for polarized contacts of circulating cells.     

VASP-dependent regulation of actin cytoskeleton rigidity, cell adhesion, and detachment

Enabled/vasodilator-stimulated phosphoprotein (Ena/VASP) proteins are established regulators of actin-based motility, platelet aggregation, and growth cone guidance. However, the molecular mechanisms involved essentially remain elusive. Here we report on a novel mechanism of VASP action, namely the regulation of tensile strength, contractility, and rigidity of the actin cytoskeleton. Compared to wild-type cells fibroblasts derived from VASP-deficient mice have thicker and more stable actin stress fibres. Furthermore focal adhesions are enlarged, myosin light chain phosphorylation is increased, and the rigidity of the filament-supported plasma membrane is elevated about three- to fourfold, as is evident from atomic force microscopy. Moreover, fibronectin-coated beads adhere stronger to the surface of VASP-deficient cells. The resistance of these beads to mechanical displacement by laser tweezers is dramatically increased in an F-actin-dependent mode. Cytoskeletal stabilization coincides with slower cell adhesion and detachment, while overall adhesion is increased. Interestingly, many of these effects observed in VASP (−/−) cells are recapitulated in VASP-overexpressing cells, hinting towards a balanced stoichiometry necessary for appropriate VASP function. Taken together, our results suggest that VASP regulates surface protrusion formation and cell adhesion through modulation of the mechanical properties of the actin cytoskeleton.Annette B. Galler, Maísa I. García Arguinzonis these authors contributed equally to this work