Abstract. The tyrosine kinase called pp125 FAK is believed to play an important role in integrin-mediated signal transduction, pp125 FAK is associated both functionally and spatially with integrins, which are the cell surface receptors for extracellular matrix components. Although the precise function of pp125 FAK is not known, two possibilities have been proposed: pp125 yAK may regulate the assembly of focal adhesions in spreading or migrating cells, or pp125 FAK may participate in a signal transduction cascade to inform the nucleus that the cell is anchored. To test these models in living cells, a peptide representing the focal adhesion kinase (FAK)-binding site of the 131 tail was coupled to carrier protein and injected into cultured cells to competitively inhibit the binding of pp125 FAK to endogenous integrin, thus inhibiting activation of pp125 FAK on a cell-by-cell basis. In addition, an antibody directed against an epitope adjacent to the focal adhesion targeting sequence on pp125 FAK was microinjected, as an alternative means of inhibiting pp125 FAK activation. It was observed that when rounded cells were injected with either the integrin peptide or the anti-FAK antibody, the cells rapidly began to apoptose, within 4 h after injection. These results indicate that pp125 FA~ may play a critical role in suppressing apoptosis in fibroblasts.

Integrin-mediated cell adhesion is necessary for the survival of many cell types, and loss of adhesion causes apoptosis. We have previously shown that the ␣ 5 ␤ 1 integrin supports cell survival on fibronectin and increases Bcl-2 protein expression. Here we show that bcl-2 transcription is elevated in cells that attach to fibronectin through ␣ v ␤ 1 or to vitronectin through ␣ v ␤ 3 but is not elevated in cells attaching through the ␣ v ␤ 1 integrin. Bcl-2 protein expression and protection from apoptosis under serum-free conditions correlated with bcl-2 transcription. This integrin-mediated regulation of bcl-2 is Shc-and FAK-dependent, and activation of Ras by FAK is required. Furthermore, Ras mediates this up-regulation of bcl-2 by activating the phosphatidylinositol 3-kinase-AKT pathway. Mitogen-activated protein kinase did not appear to be necessary for the activation of bcl-2 transcription. Therefore, our work characterizes the pathway that mediates the effect of integrins on bcl-2 transcription and cell survival.Cell-matrix interactions are important for promoting survival in a variety of cell types, and failure of cells to adhere to the extracellular matrix results in apoptosis, a phenomenon known as anoikis (1). These interactions and the resulting signaling events are mediated by integrins. The ability to prevent anoikis varies among integrins. Adhesion mediated by the ␣ 5 ␤ 1 integrin protects Chinese hamster ovary (CHO) 1 cells from apoptosis, whereas the ␣ v ␤ 1 integrin does not (2). ␣ 5 ␤ 1 -mediated adhesion has also been shown to protect cultured HT29 colon carcinoma cells from apoptosis caused by serum deprivation (3) and neuronal cells from ␤-amyloid-induced apoptosis (4). The ␣ 1 ␤ 1 and ␣ v ␤ 3 integrins are similar to ␣ 5 ␤ 1 in that they are able activate an anti-anoikis pathway (5). Mammary epithelial cells are protected from apoptosis when bound to laminin through the ␣ 6 ␤ 1 integrin (6). Furthermore, inhibiting ␣ v ␤ 3 signaling results in endothelial cell apoptosis (7,8). Therefore, at least the ␣ 5 ␤ 1 , ␣ v ␤ 3 , ␣ 1 ␤ 1 , and ␣ 6 ␤ 1 integrins can play a role in cell survival.Some of the downstream molecules involved in integrinmediated cell survival are known. Thus, activated or overexpressed focal adhesion kinase (FAK) can protect Madin-Darby canine kidney cells from anoikis (9, 10). PI3K can similarly render cells resistant to anoikis (11). Mammary epithelial cells use ␣ 6 ␤ 1 in conjunction with insulin signaling to activate PI3K and its downstream effector AKT (PKB) to promote cell survival (6). In intestinal epithelial cells, the ␣ 5 ␤ 1 pathway is AKT-dependent (12), whereas a FAK-Ras-JNK pathway has been found to be responsible for the survival effect of fibronectin attachment on primary rabbit synovial fibroblasts (13). Shc activation initiates an anti-anoikis signal from a subset of integrins including ␣ 5 ␤ 1 , ␣ 1 ␤ 1 , and ␣ v ␤ 3 (5). Therefore, integrins can mediate cell survival by activating a number of antiapoptotic pathways.Earlier studies by our laboratory have sh...

A delicate balance of signals regulates cell survival. One set of these signals is derived from integrin-mediated cell adhesion to the extracellular matrix (ECM). Loss of cell attachment to the ECM causes apoptosis, a process known as anoikis. In searching for proteins involved in cell adhesion-dependent regulation of anoikis, we identified Bit1, a mitochondrial protein that is released into the cytoplasm during apoptosis. Cytoplasmic Bit1 forms a complex with AES, a small Groucho/transducin-like enhancer of split (TLE) protein, and induces cell death with characteristics of caspase-independent apoptosis. Cell attachment to fibronectin counteracts the apoptotic effect of Bit1 and AES. Increasing Bit1 expression enhances anoikis, while suppressing the expression reduces it. Thus, we have elucidated an integrin-controlled pathway that is, at least in part, responsible for the cell survival effects of cell-ECM interactions.

ObjectiveTo identify the cause of a so-far unreported phenotype of infantile-onset multisystem neurologic, endocrine, and pancreatic disease (IMNEPD).MethodsWe characterized a consanguineous family of Yazidian-Turkish descent with IMNEPD. The two affected children suffer from intellectual disability, postnatal microcephaly, growth retardation, progressive ataxia, distal muscle weakness, peripheral demyelinating sensorimotor neuropathy, sensorineural deafness, exocrine pancreas insufficiency, hypothyroidism, and show signs of liver fibrosis. We performed whole-exome sequencing followed by bioinformatic analysis and Sanger sequencing on affected and unaffected family members. The effect of mutations in the candidate gene was studied in wild-type and mutant mice and in patient and control fibroblasts.ResultsIn a consanguineous family with two individuals with IMNEPD, we identified a homozygous frameshift mutation in the previously not disease-associated peptidyl-tRNA hydrolase 2 (PTRH2) gene. PTRH2 encodes a primarily mitochondrial protein involved in integrin-mediated cell survival and apoptosis signaling. We show that PTRH2 is highly expressed in the developing brain and is a key determinant in maintaining cell survival during human tissue development. Moreover, we link PTRH2 to the mTOR pathway and thus the control of cell size. The pathology suggested by the human phenotype and neuroimaging studies is supported by analysis of mutant mice and patient fibroblasts.InterpretationWe report a novel disease phenotype, show that the genetic cause is a homozygous mutation in the PTRH2 gene, and demonstrate functional effects in mouse and human tissues. Mutations in PTRH2 should be considered in patients with undiagnosed multisystem neurologic, endocrine, and pancreatic disease.

Among its interests, the company has initiated various programs to produce vaccines for Plasmodium falciparum malaria and Plasmodium vivax malaria.

The amyloid-β peptide (Aβ) can mediate cell attachment by binding to β1 integrins through an arg-his-asp sequence. We show here that the α5β1 integrin, a fibronectin receptor, is an efficient binder of Aβ, and mediates cell attachment to nonfibrillar Aβ. Cells engineered to express α5β1 internalized and degraded more added Aβ1-40 than did α5β1-negative control cells. Deposition of an insoluble Aβ1-40 matrix around the α5β1-expressing cells was reduced, and the cells showed less apoptosis than the control cells. Thus, the α5β1 integrin may protect against Aβ deposition and toxicity, which is a course of Alzheimer's disease lesions.

Abstract. Basement membrane-adherent type II alveolar cells isolated from lung assemble into lumencontaining cellular spheres which retain the correct polarity and thereby approximate the earliest fetal stage of alveolar morphogenesis. The molecular basis of this process, determined in initial experiments to be attributable mainly to the large heterotrimeric glycoprotein laminin, was probed with laminin proteolytic fragments, antibodies, and synthetic peptides. The carboxy-terminal fragment E8, but not equimolar amounts of fragment P1, blocked alveolar formation. To pursue this observation, we used several anti-E8 antibodies and identified one, prepared against A chain residues 2179-2198 ("SN-peptide') from the first loop of the G domain, as inhibitory. These results were confirmed by use of SN-peptide alone and further defined by trypsin digestion of SN-peptide to the sequence SINNNR. This conserved site promoted divalent cation dependent adhesion of both type II alveolar and HT1080 cells, was inhibitable with equimolar amounts of fragment E8 but not P1, and derives from a form of laminin present in fetal alveolar basement membranes. These studies point to an important novel cell adhesion site in the laminin E8 region with a key role in lung alveolar morphogenesis.

The ERK pathway responds to extracellular stimuli and oncogenes by modulating cellular processes, including transcription, adhesion, survival, and proliferation. ERK has diverse substrates that carry out these functions. The processes that are modulated are determined in part by the substrates that ERK phosphorylates. We demonstrate that PEA-15 (phosphoprotein enriched in astrocytes, 15 kDa) targets ERK to RSK2 and thereby enhances RSK2 activation. PEA-15 independently bound ERK and RSK2 and increased ERK association with RSK2 in a concentration-dependent manner. PEA-15 increased RSK2 activity and CREB-mediated transcription, and this process was regulated by phosphorylation of PEA-15. Finally, phorbol ester stimulation of PEA-15-null lymphocytes resulted in impaired RSK2 activation that was rescued by exogenous PEA-15 expression. Therefore, PEA-15 functions as a scaffold to enhance ERK activation of RSK2, and this activity is regulated by phosphorylation. Thus, PEA-15 can integrate signal transduction to provide a specific physiological outcome from activation of the multipotent ERK MAP kinase pathway.signal transduction ͉ scaffold ͉ transcription ͉ lymphocytes T he ERK MAPK signaling cascade has been implicated in diverse physiological processes, including differentiation, proliferation, migration, and adhesion (1). This ability to impact several processes is partly because ERK recognizes an array of substrates both cytoplasmic and nuclear to elicit specific responses. However, how such a multipotent pathway achieves specificity of response is an ongoing problem in understanding ERK function. ERK interaction with scaffolding proteins, regulation of the duration of the ERK signal, and restriction of ERK to specialized subcellular compartments have all been suggested to contribute to this specificity (2-4). However, thus far, the mammalian scaffolding proteins for ERK such as KSR are known to act only upstream of ERK to enhance ERK activation without directing ERK to a particular substrate (5, 6). PEA-15 (phosphoprotein enriched in astrocytes, 15 kDa) is a small death effector domain protein that binds ERK and RSK2 and sequesters them in the cytoplasm (7,8). RSK2 is a substrate of ERK and a kinase that can activate transcription, regulate apoptosis, and control cancer cell proliferation (9-11). Therefore, we investigated whether PEA-15 influences ERK activation of RSK2. We show that increasing expression of PEA-15 enhances ERK binding to RSK2, ERK phosphorylation of RSK2, and RSK2 activity. Moreover, genetic deletion of PEA-15 substantially abrogates ERK activation of RSK2 in astrocytes and lymphocytes. RSK2 activation can be rescued by ectopic expression of PEA-15 in these cells. Thus, PEA-15 functions as a scaffold to enhance ERK binding and phosphorylation of RSK2. It does not affect the phosphorylation of other ERK substrates, such as stathmin and Mnk1. This work provides a model to understand how scaffold proteins can integrate signal transduction and provide specificity in MAP kinase signaling.