Conventional calpains are ubiquitous calcium-regulated cysteine proteases that have been implicated in cytoskeletal organization, cell proliferation, apoptosis, cell motility, and hemostasis. There are two forms of conventional calpains: the -calpain, or calpain I, which requires micromolar calcium for half-maximal activation, and the m-calpain, or calpain II, which functions at millimolar calcium concentrations. We evaluated the functional role of the 80-kDa catalytic subunit of -calpain by genetic inactivation using homologous recombination in embryonic stem cells. The -calpain-deficient mice are viable and fertile. The complete deficiency of -calpain causes significant reduction in platelet aggregation and clot retraction but surprisingly the mutant mice display normal bleeding times. No detectable differences were observed in the cleavage pattern and kinetics of calpain substrates such as the 3 subunit of ␣IIb3 integrin, talin, and ABP-280 (filamin). However, -calpain null platelets exhibit impaired tyrosine phosphorylation of several proteins including the 3 subunit of ␣IIb3 integrin, correlating with the agonist-induced reduction in platelet aggregation. These results provide the first direct evidence that -calpain is essential for normal platelet function, not by affecting the cleavage of cytoskeletal proteins but by potentially regulating the state of tyrosine phosphorylation of the platelet proteins.The calpains are a family of calcium-dependent neutral cysteine proteases present in essentially all tissues of higher animals (8,34,37). Calpain homologues distantly related to the catalytic subunits of conventional calpains are also found in lower organisms such as parasites, insects, nematodes, fungi, and yeast (34). They are believed to play functionally important roles in diverse biological processes such as reorganization of cortical cytoskeleton, cell motility, cell proliferation, apoptosis, and hemostasis (9,27,31,39). Calpains are divided into two broad classes, ubiquitous and tissue specific. Calpain I (also referred to as -calpain) and calpain II (also referred to as m-calpain) are expressed in all tissues in varying amounts and share ϳ61% sequence identity (20). Both the -and m-calpains contain an 80-kDa catalytic subunit that forms a heterodimer with the regulatory 30-kDa subunit (34). The 80-kDa catalytic subunits of the -and m-calpains are products of separate but closely related genes (referred to as Capn1 and Capn2, respectively), while the 30-kDa subunit (encoded by the Capn4 gene) is common to both (34). The -calpain is fully active in micromolar concentrations of calcium, while the mcalpain requires millimolar calcium concentrations for full activation. Larger tissue-specific calpains have been cloned from stomach and smooth muscle tissues (35, 37). Mutations of the muscle-specific Capn3 (calpain 3 gene) have been shown to cause one form of limb-girdle muscular dystrophy type 2A (30). More recently, several groups have identified CAPN10 (calpain 10) as the target gene for mutations in...
Dematin is an actin-binding and bundling protein of the erythrocyte membrane skeleton. Dematin is localized to the spectrin-actin junctions, and its actin-bundling activity is regulated by phosphorylation of cAMP-dependent protein kinase. The carboxyl terminus of dematin is homologous to the ''headpiece'' domain of villin, an actin-bundling protein of the microvillus cytoskeleton. The headpiece domain contains an actin-binding site, a cAMP-kinase phosphorylation site, plays an essential role in dematin self-assembly, and bundles F-actin in vitro. By using homologous recombination in mouse embryonic stem cells, the headpiece domain of dematin was deleted to evaluate its function in vivo. Dematin headpiece null mice were viable and born at the expected Mendelian ratio. Hematological evaluation revealed evidence of compensated anemia and spherocytosis in the dematin headpiece null mice. The headpiece null erythrocytes were osmotically fragile, and ektacytometry͞micropore filtration measurements demonstrated reduced deformability and filterability. In vitro membrane stability measurements indicated significantly greater membrane fragmentation of the dematin headpiece null erythrocytes. Finally, biochemical characterization, including the vesicle͞cytoskel-eton dissociation, spectrin self-association, and chemical crosslinking measurements, revealed a weakened membrane skeleton evidenced by reduced association of spectrin and actin to the plasma membrane. Together, these results provide evidence for the physiological significance of dematin and demonstrate a role for the headpiece domain in the maintenance of structural integrity and mechanical properties of erythrocytes in vivo.T he membrane bilayer and the network of membrane-associated proteins together regulate the characteristic shape and elastic properties of red blood cells (1, 2). When membrane skeletons are prepared in the presence of a high concentration of monovalent salt, the core of the membrane skeleton consists of spectrin, actin, protein 4.1, and dematin (3). Although the functions of spectrin, actin, and protein 4.1 have been extensively characterized (4, 5), virtually nothing is known about the physiological function in mature erythrocytes of dematin, a three-subunit protein that migrates in the protein 4.9 region during electrophoresis. The earliest evidence suggesting a membrane stabilizing role for dematin came from Holdstock and Ralston (6). They demonstrated that charged sulfhydryl compounds such as p-chloromercuribenzene sulfonateS preferentially attach to dematin and cause the disruption of erythrocyte cytoskeletons. Dematin is a substrate for multiple protein kinases, and phosphorylation of dematin by the cAMP-dependent protein kinase is known to regulate dematin's actin-bundling activity in vitro (7-9). The major phosphorylation site of the cAMPdependent protein kinase is located within the headpiece domain of dematin (10), but the physiological significance of dematin phosphorylation is not known.Siegel and Branton (11) conducted the first ...
DNA tumor viruses of the polyomavirus family have helped us understand important cellular processes, particularly those associated with the signal transduction of cell cycle regulation and transformation. For example, polyomavirus middle T antigen (PyMT) studies were crucial to the discovery of the importance of tyrosine phosphorylation (17) and phosphatidylinositol 3-kinase (PI3) kinase activity (70) in cellular signaling. Comparative studies of the individual members of the polyomavirus family have also been very informative. For example, p53 was discovered as a protein that associates with simian virus 40 (SV40) large T antigen (LT) (38, 39). Comparison of SV40 LT that binds p53 and transforms cells to polyomavirus LT (PyLT) that does neither of these focused attention on the role of p53 in cell cycle regulation.All members of the polyomavirus family produce a small T antigen (ST) as one of the early gene products. Polyomavirus small T antigen (POLST) and SV40 small T antigen (SVST) share many structural similarities with each other (Fig. 1). Both have an N-terminal J domain with a conserved HP DKGG motif that can bind heat shock proteins, and both possess zinc-binding cysteine motifs. Both POLST and SVST bind and perturb protein phosphatase 2A (PP2A) (51). PP2A functions as a trimeric ABC complex, where the scaffolding A subunit binds a catalytic subunit (C) and some regulatory B subunits (30). STs bind to the A and C subunit complexes (51, 64), displacing or preventing B subunits from binding. Since SVST binds to regions of the A subunit involved in B binding (12,14), the absence of B subunits in ST/PP2A complexes is not surprising.There is ample evidence suggesting that careful examination of ST is important. SVST contributes to the multioncoproteindirected transformation of human cells (24,59,76). Transgenic SVST contributes to mammary gland carcinogenesis (22). POLST complements MT for both transformation (4,45,49) and tumor induction (3). POLST effects on the cell cycle are well documented. POLST promotes cell cycle progression (46) and complements LT for S-phase induction (5, 51). Array analysis showed that SVST (43) and POLST (35) have large effects on cellular mRNA levels. SVST can transactivate (19,32,40,48,53,61,68) or repress (67) various promoters. POLST activates the fos (46) and myc (36) promoters. POLST is known to promote changes in viral chromatin structure that may underlie altered transcriptional activity (16). DNA viruses are very much concerned with issues of cell survival. It is therefore not surprising that both small Ts affect survival. Both POLST and SVST can be antiapoptotic. POLST can protect against serum starvation-induced apoptosis (2) and resists the effects of p53-induced apoptosis (54). SVST opposes apoptosis induced by LT (37) or CD95 (20). Both POLST (2) and SVST (21) can also be proapoptotic under some circumstances.Many ST functions are known to depend on their interactions with PP2A. Promotion of cell cycle by POLST (46), activation of promoters, such as myc (36), fos (4...
Murine polyomavirus small t antigen (PyST) regulates cell cycle, cell survival, apoptosis, and differentiation and cooperates with middle T antigen (MT) to transform primary cells in vitro and in vivo. Like all polyomavirus T antigens, PyST functions largely via its interactions with host cell proteins. Here, we show that PyST binds both Yes-associated protein 1 (YAP1) and YAP2, integral parts of the Hippo signaling pathway, which is a subject of increasing interest in human cancer. The transcription factor TEAD, which is a known target of YAP, is also found in PyST complexes. PyST enhanced YAP association with protein phosphatase 2A (PP2A), leading to decreased YAP phosphorylation. PyST increased YAP levels by decreasing its degradation. This effect was mediated by a reduction in YAP association with -transducin repeat protein (TRCP), which is known to regulate YAP turnover in a phosphorylation-dependent manner. Genetic analysis has identified PyST mutants defective in YAP binding. These mutants demonstrated that YAP binding is important for PyST to block myoblast differentiation and to synergize with the phosphodiesterase inhibitor isobutylmethylxanthine (IBMX) to promote cell death in 3T3-L1 preadipocytes placed under differentiation conditions. In addition to YAP binding, both of these phenotypes require PyST binding to PP2A. IMPORTANCEThe Hippo/YAP pathway is a highly conserved cascade important for tissue development and homeostasis. Defects in this pathway are increasingly being associated with cancer. Polyomavirus small t antigen is a viral oncogene that cooperates with middle T antigen in transformation. On its own, small t antigen controls cell survival and differentiation. By binding YAP, small t antigen brings it together with protein phosphatase 2A. This work shows how this association of small t antigen with YAP is important for its effects on cell phenotype. It also suggests that PyST can be used to characterize cellular processes that are regulated by YAP.
Polyoma small T antigen (PyST), an early gene product of the polyoma virus, has been shown to cause cell death in a number of mammalian cells in a protein phosphatase 2A (PP2A)-dependent manner. In the current study, using a cell line featuring regulated expression of PyST, we found that PyST arrests cells in mitosis. Live-cell and immunofluorescence studies showed that the majority of the PyST-expressing cells were arrested in prometaphase with almost no cells progressing beyond metaphase. These cells exhibited defects in chromosomal congression, sister chromatid cohesion and spindle positioning, resulting in the activation of the Spindle Assembly Checkpoint (SAC). Prolonged mitotic arrest then led to cell death via mitotic catastrophe. Cell cycle inhibitors that block cells in G1/S prevented PyST-induced death. PyST-induced cell death that occurs during M is not dependent on p53 status. These data suggested, and our results confirmed that, PP2A inhibition could be used to preferentially kill cancer cells with p53 mutations that proliferate normally in the presence of cell cycle inhibitors.
UNC5B is a dependence receptor that promotes survival in the presence of its ligand, netrin-1, while inducing cell death in its absence. The receptor has an important role in the development of the nervous and vascular systems. It is also involved in the normal turnover of intestinal epithelium. Netrin-1 and UNC5B are deregulated in multiple cancers, including colorectal, neuroblastoma, and breast tumors. However, the detailed mechanism of UNC5B function is not fully understood. We have utilized the murine polyomavirus small T antigen (PyST) as a tool to study UNC5B-mediated apoptosis. PyST is known to induce mitotic arrest followed by extensive cell death in mammalian cells. Our results show that the expression of PyST increases mRNA levels of UNC5B by approximately 3-fold in osteosarcoma cells (U2OS) and also stabilizes UNC5B at the posttranslational level. Furthermore, UNC5B is upregulated predominantly in those cells that undergo mitotic arrest upon PyST expression. Interestingly, although its expression was previously reported to be regulated by p53, our data show that the increase in UNC5B levels by PyST is p53 independent. The posttranslational stabilization of UNC5B by PyST is regulated by the interaction of PyST with PP2A. We also show that netrin-1 expression, which is known to inhibit UNC5B apoptotic activity, promotes survival of PyST-expressing cells. Our results thus suggest an important role of UNC5B in small-T antigen-induced mitotic catastrophe that also requires PP2A. IMPORTANCE UNC5B, PP2A, and netrin-1 are deregulated in a variety of cancers. UNC5B and PP2A are regarded as tumor suppressors, as they promote apoptosis and are deleted or mutated in many cancers. In contrast, netrin-1 promotes survival by inhibiting dependence receptors, including UNC5B, and is upregulated in many cancers. Here, we show that UNC5B-mediated apoptosis can occur independently of p53 but in a PP2A-dependent manner. A substantial percentage of cancers arise due to p53 mutations and are insensitive to chemotherapeutic treatments that activate p53. Unexpectedly, treatment of cancers having functional p53 with many conventional drugs leads to the upregulation of netrin-1 through activated p53, which is counterintuitive. Therefore, understanding the p53-independent mechanisms of the netrin-UNC5B axis, such as those involving PP2A, assumes greater clinical significance. Anticancer strategies utilizing anti-netrin-1 antibody treatment are already in clinical trials.
UNC-5 Homolog B (UNC5B) is a member of the dependence receptor family. This family of receptors can induce two opposite intracellular signaling cascades depending on the presence or absence of the ligand and is thus capable of driving two opposing processes. UNC5B signaling has been implicated in several cancers, where it induces cell death in the absence of its ligand Netrin-1 and promotes cell survival in its presence. In addition, inhibition of Netrin-1 ligand has been reported to decrease invasiveness and angiogenesis in tumors. UNC5B signaling pathway has also been reported to be involved in several processes such as neural development, developmental angiogenesis and inflammatory processes. However, literature pertaining to UNC5B signaling is scarce and scattered. Considering the importance of UNC5B signaling, we developed a resource of signaling events mediated by UNC5B. Using data mined from published literature, we compiled an integrated pathway map consisting of 88 UNC5B-mediated signaling events and 55 proteins. These signaling events include 27 protein-protein interaction events, 33 catalytic events involving various post-translational modifications, 9 events of UNC5B-mediated protein activation/inhibition, 27 gene regulation events and 2 events of translocation. This pathway resource has been made available to the research community through NetPath ( http://www.netpath.org /), a manually curated resource of signaling pathways (Database URL: http://www.netpath.org/pathways?path_id=NetPath_172 ). The current resource provides a foundation for the understanding of UNC5B-mediated cellular responses. The development of resource will serve researchers to explore the mechanisms of UNC-5B signaling in cancers.
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