Apoptosis commitment and activation of mitochondrial Bax during anoikis is regulated by p38MAPK.

Most cells undergo apoptosis through the intrinsic pathway. This is dependent on mitochondrial outer membrane permeabilisation (MOMP), which is mediated by the pro-apoptotic Bcl-2 family proteins, Bax and Bak. During apoptosis, Bax translocates from the cytosol to the outer mitochondrial membrane (OMM), wherein it contributes to the formation of pores to release cytochrome-c. However, it remains unclear whether Bax translocation is sufficient to bring about MOMP or whether Bax requires further signals on the OMM to be fully activated. We have previously shown that during mammary epithelial cell anoikis, Bax translocation does not commit cells to MOMP and detached cells are rescued if survival signals from the extracellular matrix (ECM) are restored. These findings implied that a second signal is required for mitochondrial Bax to fully activate and cause MOMP. We now identify p38MAPK (mitogen-activated protein kinase) as this necessary signal to activate Bax after its translocation to mitochondria. The inhibition of p38MAPK did not prevent Bax translocation, but its activity was required for mitochondrial Bax to bring about MOMP. p38MAPK was activated and recruited to a high molecular weight mitochondrial complex after loss of ECM attachment. Artificially targeting p38MAPK to the OMM increased the kinetics of anoikis, supporting a requirement for its mitochondrial localisation to regulate Bax activation and drive commitment to apoptosis.

Bax targeting to mitochondria occurs via both tail anchor-dependent and -independent mechanisms.

Bax is a member of the Bcl-2 family that, together with Bak, is required for permeabilisation of the outer mitochondrial membrane (OMM). Bax differs from Bak in that it is predominantly cytosolic in healthy cells and only associates with the OMM after an apoptotic signal. How Bax is targeted to the OMM is still a matter of debate, with both a C-terminal tail anchor and an N-terminal pre-sequence being implicated. We now show definitively that Bax does not contain an N-terminal import sequence, but does have a C-terminal anchor. The isolated N terminus of Bax cannot target a heterologous protein to the OMM, whereas the C terminus can. Furthermore, if the C terminus is blocked, Bax fails to target to mitochondria upon receipt of an apoptotic stimulus. Zebra fish Bax, which shows a high degree of amino-acid homology with mammalian Bax within the C terminus, but not in the N terminus, can rescue the defective cell-death phenotype of Bax/Bak-deficient cells. Interestingly, we find that Bax mutants, which themselves cannot target mitochondria or induce apoptosis, are recruited to clusters of activated wild-type Bax on the OMM of apoptotic cells. This appears to be an amplification of Bax activation during cell death that is independent of the normal tail anchor-mediated targeting.

The N-terminal conformation of Bax regulates cell commitment to apoptosis.

The Bcl-2 protein Bax normally resides in the cytosol, but during apoptosis it translocates to mitochondria where it is responsible for releasing apoptogenic factors. Using anoikis as a model, we have shown that Bax translocation does not commit cells to apoptosis, and they can be rescued by reattachment to extracellular matrix within a specific time. Bax undergoes an N-terminal conformational change during apoptosis that has been suggested to regulate conversion from its benign, cytosolic form to the active, membrane bound pore. We now show that the Bax N-terminus regulates commitment and mitochondrial permeabilisation, but not the translocation to mitochondria. We identify Proline 13 within the N-terminus of Bax as critical for this regulation. The subcellular distribution of Proline 13 mutant Bax was identical to wild-type Bax in both healthy and apoptotic cells. However, Proline 13 mutant Bax induced rapid progression to commitment, mitochondrial permeabilisation and death. Our data identify changes in Bax controlling commitment to apoptosis that are mechanistically distinct from those controlling its subcellular localisation. Together, they indicate that multiple regulatory steps are required to activate the proapoptotic function of Bax.

Anoikis.

Anoikis is apoptosis induced by loss of cell adhesion or inappropriate cell adhesion. Adhesion on the extracellular matrix is important to determine whether a cell is in the correct location and to delete displaced cells by apoptosis. The ability to overcome this requirement has important implications for metastatic cancer. However, how adhesion signals are interpreted by a cell into a life or death decision is complex. In this paper, we will examine this from the point of view of the apoptotic machinery of the cell, and discuss the various ways in which adhesion can influence this process.

Assessment of apoptosis in human breast tissue using an antibody against the active form of caspase 3: relation to tumour histopathological characteristics.

Apoptosis is of important significance in the pathogenesis of cancer. Many methods are available for the measurement of apoptosis but the 'gold standard' is to identify apoptotic cells by their morphological features using microscopy. Caspase 3 is a cytosolic enzyme that is activated only in cells committed to undergo apoptosis. The activation of caspase 3 precedes the development of the classical morphological features of apoptosis. Using immunohistochemistry with an antibody against the active form of caspase 3, the apoptotic index (AI) was measured in 116 samples of human breast tissue (22 normal/benign and 94 invasive carcinomas). The AI obtained by measuring caspase activation has a strong correlation with the AI derived by morphological assessment (r = 0.736, P < 0.01). The AI is higher in the invasive group than in the benign group (P = 0.008), and in invasive cancer high AI is associated with high tumour grade (P = 0.013), positive node status (P < 0.001) and negative steroid receptor status (P = 0.001 for ER; P = 0.004 for PR). No significant association is observed between AI and tumour size. Measurement of apoptosis by immunohistochemistry using an antibody against the active form of caspase 3 is therefore reliable and correlates strongly with morphological assessment.

Integrin-mediated survival signals regulate the apoptotic function of Bax through its conformation and subcellular localization.

Most normal cells require adhesion to extracellular matrix for survival, but the molecular mechanisms that link cell surface adhesion events to the intracellular apoptotic machinery are not understood. Bcl-2 family proteins regulate apoptosis induced by a variety of cellular insults through acting on internal membranes. A pro-apoptotic Bcl-2 family protein, Bax, is largely present in the cytosol of many cells, but redistributes to mitochondria after treatment with apoptosis-inducing drugs. Using mammary epithelial cells as a model for adhesion-regulated survival, we show that detachment from extracellular matrix induced a rapid translocation of Bax to mitochondria concurrent with a conformational change resulting in the exposure of its BH3 domain. Bax translocation and BH3 epitope exposure were reversible and occurred before caspase activation and apoptosis. Pp125FAK regulated the conformation of the Bax BH3 epitope, and PI 3-kinase and pp60src prevented apoptosis induced by defective pp125FAK signaling. Our results provide a mechanistic connection between integrin-mediated adhesion and apoptosis, through the kinase-regulated subcellular distribution of Bax.

Adhesion-mediated signaling in the regulation of mammary epithelial cell survival.

Tissue architecture in multicellular organisms is maintained through adhesive interactions between cells and their neighbors, and between cells and the underlying extracellular matrix. These interactions are important in the dynamic regulation of tissue organization as well as the control of cell proliferation, differentiation and apoptosis. The ultimate goal of this regulation is to promote cell growth and differentiation only when the cell is in the correct location, and to delete cells that have become displaced from their proper environment. It therefore plays an important role in development and tissue remodeling. In this review we consider the molecular mechanisms by which cell-matrix interactions contribute to cell survival, and discuss their role in mammary gland development and function.

Microinjection of protein tyrosine phosphatases into fibroblasts disrupts focal adhesions and stress fibers.

Microinjection and scrape-loading have been used to load cells in culture with soluble protein tyrosine phosphatases (PTPs). The introduction of protein tyrosine phosphatases into cells caused a rapid (within 5 minutes) decrease in tyrosine phosphorylation of major tyrosine phosphorylated substrates, including the focal adhesion kinase and paxillin. This decrease was detected both by blotting whole cell lysates with anti-phosphotyrosine antibodies and visualizing the phosphotyrosine in focal adhesions by immunofluorescence microscopy. After 30 minutes, many of the cells injected with tyrosine phosphatases revealed disruption of focal adhesions and stress fibers. To determine whether this disruption was due to the dephosphorylation of FAK and its substrates in focal adhesions, we have compared the effects of protein tyrosine phosphatase microinjection with the effects of displacing FAK from focal adhesions by microinjection of a dominant negative FAK construct. Although both procedures resulted in a marked decrease in the level of phosphotyrosine in focal adhesions, disruption of focal adhesions and stress fibers only occurred in cells loaded with exogenous protein tyrosine phosphatases. These results lead us to conclude that although tyrosine phosphorylation regulates focal adhesion and stress fiber stability, this does not involve FAK nor does it appear to involve tyrosine-phosphorylated proteins within focal adhesions. The critical tyrosine phosphorylation event is upstream of focal adhesions, a likely target being in the Rho pathway that regulates the formation of stress fibers and focal adhesions.

Talin contains three actin-binding sites each of which is adjacent to a vinculin-binding site.

We have determined the sequence of chicken talin (2,541 amino acids, M(r) 271,881) which is very similar (89% identity) to that of the mouse protein. Alignments with the Caenorhabditis elegans and Dictyostelium discoideum talin sequences show that the N- and C-terminal regions of the protein are conserved whereas the central part of the molecule is more divergent. By expressing overlapping talin polypeptides as fusion proteins, we have identified at least three regions of the protein which can bind F-actin: residues 102-497, 951-1,327 and 2,269-2,541. The N-terminal binding site contains a region with homology to the ERM family of actin-binding proteins, and the C-terminal site is homologous to the yeast actin-binding protein Sla2p. Each of the actin-binding sites is close to, but distinct from a binding site for vinculin, a protein which also binds actin. The Pro1176 to Thr substitution found in talin from Wistar-Furth rats does not destroy the capacity of this region of the protein to bind actin or vinculin. Microinjection studies showed that a fusion protein containing the N-terminal actin-binding site localised weakly to stress fibres, whereas one containing the C-terminal site initially localised predominantly to focal adhesions. The former was readily solubilised, and the latter was resistant to Triton extraction. The N-terminal talin polypeptide eventually disrupted actin stress fibres whereas the C-terminal polypeptide was without effect. However, a larger C-terminal fusion protein also containing a vinculin-binding site did disrupt stress fibres and focal adhesions. The results suggest that, although both the N- and C-terminal regions of talin bind actin, the properties of these two regions of the protein are distinct.

Inhibition of focal adhesion kinase (FAK) signaling in focal adhesions decreases cell motility and proliferation.

It has been proposed that the focal adhesion kinase (FAK) mediates focal adhesion formation through tyrosine phosphorylation during cell adhesion. We investigated the role of FAK in focal adhesion structure and function. Loading cells with a glutathione-S-transferase fusion protein (GST-Cterm) containing the FAK focal adhesion targeting sequence, but not the kinase domain, decreased the association of endogenous FAK with focal adhesions. This displacement of endogenous FAK in both BALB/c 3T3 cells and human umbilical vein endothelial cells loaded with GST-Cterm decreased focal adhesion phosphotyrosine content. Neither cell type, however, exhibited a reduction in focal adhesions after GST-Cterm loading. These results indicate that FAK mediates adhesion-associated tyrosine phosphorylation, but not the formation of focal adhesions. We then examined the effect of inhibiting FAK function on other adhesion-dependent cell behavior. Cells microinjected with GST-Cterm exhibited decreased migration. In addition, cells injected with GST-Cterm had decreased DNA synthesis compared with control-injected or noninjected cells. These findings suggest that FAK functions in the regulation of cell migration and cell proliferation.

Molecular mechanisms for focal adhesion assembly through regulation of protein-protein interactions.

Focal adhesions provide a useful model for studying cell/extracellular matrix interactions and the subsequent cytoskeletal reorganization. Recent advances have suggested potential mechanisms by which cells may regulate focal adhesion assembly following integrin-mediated cell adhesion.

Regulation of vinculin binding to talin and actin by phosphatidyl-inositol-4-5-bisphosphate.

Vinculin, a prominent cytoskeletal protein at cell-substrate adhesions (focal adhesions) and cell-cell adhesions (adherens junctions), interacts with other cytoskeletal proteins, including talin and actin. An intramolecular interaction between the head and tail domains of vinculin masks the binding sites for both proteins. The exposure of cryptic binding sites may be important for promoting focal adhesion assembly. Several agents that induce the formation of focal adhesions act through the GTP-binding protein Rho, which elevates phosphatidylinositol-4,5-bisphosphate (PtdInsP2) levels by activating phosphatidyl-inositol-4-phosphate-5-OH kinase (PtdIns-5-OH kinase). PtdInsP2 regulates several actin-binding proteins, including profilin, gelsolin and alpha-actinin, and interacts with vinculin. Here we report that PtdInsP2 dissociates vinculin's head-tail interaction, unmasking its talin- and actin-binding sites. Microinjection of antibodies against PtdInsP2 inhibit assembly of stress fibres and focal adhesions.

Association of structural repeats in the alpha-actinin rod domain. Alignment of inter-subunit interactions.

Fragments of the rod domain of chicken alpha-actinin, which comprises four spectrin-like repeat sequences, have been prepared by expression in Escherichia coli. Electron microscopy reveals that all products containing three or four complete repeats are rod-like. Self-association of fragments was detected by chemical cross-linking and analytical equilibrium sedimentation. The intact rod domain forms a stable dimmer, which does not dissociate measurably in the accessible concentration range. Elimination of either terminal repeat (repeat 1 or repeat 4) greatly diminishes the extent of dimerisation. The fragment comprising repeats 1-3 dimerises appreciably, with an association constant estimated from the sedimentation equilibrium distribution of approximately 5 x 10(5) M-1. The fragment made up of repeats 2-4 dimerises to a small extent, but also forms aggregates at high concentrations. The results are most easily reconciled with an aligned structure for the rod domain in solution, in which repeat 1 associates with repeat 4 of the partnering chain, and repeat 2 with repeat 3, rather than with a staggered structure, in which one of the terminal repeats does not participate in dimerisation. Possible explanations for the apparent difference observed between the alpha-actinin rod structure in solution and in two-dimensional crystalline arrays are examined.

Localisation of the human gene encoding the cytoskeletal protein talin to chromosome 9p.

The cytoskeletal protein talin is localised on the cytoplasmic face of the integrin family of adhesion receptors in cellular junctions with the extracellular matrix. Using polymerase chain reaction amplification and DNA from a panel of human-rodent somatic cell hybrids, we have assigned the talin gene to chromosome 9p. Deletions in 9p have been implicated in a variety of cancers, including malignant melanoma, and the concept that talin might be a candidate tumour suppressor gene is discussed.

Analysis of the phasing of four spectrin-like repeats in alpha-actinin.

Selected fragments of the central rod of chicken gizzard alpha-actinin were expressed as fusion proteins in Escherichia coli, with the aim of determining the positions in the sequence of the four successive spectrin-like repeats that make up this domain. The criteria for an independently folding unit were resistance to proteolysis and the high alpha helicity characteristic of the native protein. Sequences containing repeats 1-4, 2-4, 3-4 and 4 all generated stable fragments on digestion with trypsin and/or thermolysin and N-terminal sequencing gave the most probable starting position of each repeat. The sequences of all four inferred repeats and the sequences of the entire rod, were separately expressed and were shown to assume a stable, protease-resistant fold in solution. The repeat boundaries established in this way differed from those originally deduced from sequence alignments; the N-terminal boundaries of the repeats were 14-24 residues nearer the C-terminus than predicted. The ability to express individual repeats should facilitate identification of the binding sites for the cytoplasmic domains of beta 1 integrins and intercellular cell adhesion molecule-1 which have been localised to the rod domain of alpha-actinin.

The cytoskeletal protein talin contains at least two distinct vinculin binding domains.

We have mapped the vinculin-binding sites in the cytoskeletal protein talin as well as those sequences which target the talin molecule to focal contacts. Using a series of overlapping talin-fusion proteins expressed in E. coli and 125I-vinculin in both gel-overlay and microtitre well binding assays, we present evidence for three separable binding sites for vinculin. All three are in the tail segment of talin (residues 434-2541) and are recognized by the same fragment of vinculin (residues 1-258). Two sites are adjacent to each other and span residues 498-950, and the third site is more than 700 residues distant in the primary sequence. Scatchard analysis of 125I-vinculin binding to talin also indicates three sites, each with a similar affinity (Kd = 2-6 x 10(-7) M). We also detect a substoichiometric interaction of higher affinity (Kd = 3 x 10(-8) M) which remains unexplained. By expressing regions of the chicken talin molecule in heterologous cells, we have shown that the sequences required to target talin to focal contacts overlap those which bind vinculin.

Further characterisation of the talin-binding site in the cytoskeletal protein vinculin.

The cytoskeletal protein vinculin is a component of adherens-type junctions where it is one of a number of interacting proteins thought to link the cytoplasmic domain of adhesion receptors to F-actin. Vinculin has been shown to bind to at least three other cytoskeletal proteins, talin, paxillin and alpha-actinin. In this study, we further characterise the talin-binding domain in vinculin using a series of chick vinculin polypeptides expressed as glutathione-S-transferase fusion proteins in Escherichia coli. Thus 125I-talin bound to a fusion protein spanning residues 1-398, but not to those spanning residues 399-881 or 881-1066 in an SDS-PAGE gel-blot assay. We have previously characterised two chick vinculin cDNAs (2.89 kb cDNA and cVin5) which are identical in the region of overlap except that cVin5 lacks coding sequence for residues 167-207. Interestingly, a fusion protein spanning residues 1-398, but lacking residues 167-207, was unable to bind talin. However, further analysis showed that residues 167-207 are insufficient to support binding, and deletion of as few as 31 N-terminal residues abolished binding activity. The results of the gel-blot assay were essentially confirmed using purified fusion proteins adsorbed to glutathione-agarose beads. The smallest vinculin fusion protein able to bind talin contained residues 1-258. This fusion protein was as effective as whole vinculin in inhibiting the binding of 125I-vinculin to talin-coated microtitre wells. Interestingly, mutations which altered the charge characteristics of the highly conserved residues 178 and 181 abolished binding, whereas conservative substitutions were without effect. However, such mutations did not abolish the ability of mutant polypeptides spanning residues 1-398 to target to cell-matrix junctions in Cos cells. We have investigated the possible origin of the cDNA clone cVin5 by defining the structure of a 5' portion of the chicken vinculin gene, and by analysing vinculin transcripts in a variety of adult tissues and embryonic fibroblasts using reverse transcriptase and polymerase chain reaction. Although residues 167-207 are encoded on a separate exon, we have been unable to identify a tissue where this exon is alternatively spliced.