Dianne Watters

PKC-delta is an apoptotic lamin kinase.

Protein kinase C-δ is activated during apoptosis, following proteolytic cleavage by caspase 3. Furthermore, overexpression of the catalytic kinase fragment of PKC-δ induces the nuclear phenotype associated with apoptosis, though the molecular basis of this effect has not been determined. In these studies we have examined the role of PKC-δ in the disassembly of the nuclear lamina at apoptosis. The nuclear lamina is disassembled during mitosis and apoptosis and mitotic disassembly involves hyperphosphorylation of lamin proteins by mitotic lamin kinases. During apoptosis, lamin proteins are degraded by caspase 6 and the contribution made by phosphorylation has not been proven. We show here that protein kinase C-δ co-localized with lamin B during apoptosis and activation of PKC-δ by caspase 3 was concomitant with lamin B phosphorylation and proteolysis. Inhibition of PKC-δ delayed lamin proteolysis, even in the presence of active caspase 6, whilst inhibitors of mitotic lamin kinases were without effect. In addition recombinant human PKC-δ was able to phosphorylate lamin B in vitro suggesting that its actions are direct and not via an intermediary kinase. We propose that PKC-δ is an apoptotic lamin kinase and that efficient lamina disassembly at apoptosis requires both lamin hyperphosphorylation and caspase mediated proteolysis.

Cellular localisation of the ataxia-telangiectasia (ATM) gene product and discrimination between mutated and normal forms.

The recently cloned gene (ATM) mutated in the human genetic disorder ataxia-telangiectasia (A-T) is involved in DNA damage response at different cell cycle checkpoints and also appears to have a wider role in signal transduction. Antibodies prepared against peptides from the predicted protein sequence detected a ∼ 350 kDa protein corresponding to the open reading frame, which was absent in 13/23 A-T homozygotes. Subcellular fractionation, immunoelectronmicroscopy and immunofluorescence showed that the ATM protein is present in the nucleus and cytoplasmic vesicles. This distribution did not change after irradiation. We also provide evidence that ATM protein binds to p53 and this association is defective in A-T cells compatible with the defective p53 response in these cells. These results provide further support for a role for the ATM protein as a sensor of DNA damage and in a more general role in cell signalling, compatible with the broader phenotype of the syndrome.

Localization of a Portion of Extranuclear ATM to Peroxisomes

The gene mutated in the human genetic disorder ataxia-telangiectasia codes for a protein, ATM, the known functions of which include response to DNA damage, cell cycle control, and meiotic recombination. Consistent with these functions, ATM is predominantly present in the nucleus of proliferating cells; however, a significant proportion of the protein has also been detected outside the nucleus in cytoplasmic vesicles. To understand the possible role of extra-nuclear ATM, we initially investigated the nature of these vesicles. In this report we demonstrate that a portion of ATM co-localizes with catalase, that ATM is present in purified mouse peroxisomes, and that there are reduced levels of ATM in the post-mitochondrial membrane fraction of cells from a patient with a peroxisome biogenesis disorder. Furthermore the use of the yeast two-hybrid system demonstrated that ATM interacts directly with a protein involved in the import of proteins into the peroxisome matrix. Because peroxisomes are major sites of oxidative metabolism, we investigated catalase activity and lipid hydroperoxide levels in normal and A-T fibroblasts. Significantly decreased catalase activity and increased lipid peroxidation was observed in several A-T cell lines. The localization of ATM to peroxisomes may contribute to the pleiotropic nature of A-T.

HETERONUCLEAR RIBONUCLEOPROTEINS C1 AND C2, COMPONENTS OF THE SPLICEOSOME,ARE SPECIFIC TARGETS OF INTERLEUKIN 1BETA -CONVERTING ENZYME-LIKE PROTEASES IN APOPTOSIS

Apoptosis induced by a variety of agents results in the proteolytic cleavage of a number of cellular substrates by enzymes related to interleukin 1β-converting enzyme (ICE). A small number of substrates for these enzymes have been identified to date, including enzymes involved in DNA repair processes: poly(ADP-ribose) polymerase and DNA-dependent protein kinase. We describe here for the first time the specific cleavage of the heteronuclear ribonucleoproteins (hnRNPs) C1 and C2 in apoptotic cells induced to undergo apoptosis by a variety of stimuli, including ionizing radiation, etoposide, and ceramide. No cleavage was observed in cells that are resistant to apoptosis induced by ionizing radiation. Protease inhibitor data implicate the involvement of an ICE-like protease in the cleavage of hnRNP C. Using recombinant ICE-like proteases and purified hnRNP C proteins in vitro, we show that the C proteins are cleaved by Mch3α and CPP32 and, to a lesser extent, by Mch2α, but not by ICE, Nedd2, Tx, or the cytotoxic T-cell protease granzyme B. The results described here demonstrate that the hnRNP C proteins, abundant nuclear proteins thought to be involved in RNA splicing, belong to a critical set of protein substrates that are cleaved by ICE-like proteases during apoptosis.

Oxidative Stress Is Responsible for Deficient Survival and Dendritogenesis in Purkinje Neurons from Ataxia-Telangiectasia Mutated Mutant Mice

Atm gene-disrupted mice recapitulate the majority of characteristics observed in patients with the genetic disorder ataxia-telangiectasia (A-T). However, although they exhibit defects in neuromotor function and a distinct neurological phenotype, they do not show the progressive neurodegeneration seen in human patients, but there is evidence that ataxia-telangiectasia mutated (Atm)-deficient animals have elevated levels of oxidized macromolecules and some neuropathology. We report here that in vitro survival of cerebellar Purkinje cells from both Atm “knock-out” and Atm “knock-in” mice was significantly reduced compared with their wild-type littermates. Although most of the Purkinje neurons from wild-type mice exhibited extensive dendritic elongation and branching under these conditions, most neurons from Atm-deficient mice had dramatically reduced dendritic branching. An antioxidant (isoindoline nitroxide) prevented Purkinje cell death in Atm-deficient mice and enhanced dendritogenesis to wild-type levels. Furthermore, administration of the antioxidant throughout pregnancy had a small enhancing effect on Purkinje neuron survival in Atm gene-disrupted animals and protected against oxidative stress in older animals. These data provide strong evidence for a defect in the cerebellum of Atm-deficient mice and suggest that oxidative stress contributes to this phenotype.

Textilinins from Pseudonaja textilis textilis. Characterization of two plasmin inhibitors that reduce bleeding in an animal model.

The incidence of vein-graft occlusion associated with myocardial infarction and thrombosis following the use of the plasmin inhibitor, aprotinin, to reduce blood loss during vascular surgery has prompted the isolation of an alternative kinetically distinct inhibitor of plasmin from the venom ofPseudonaja textilis. This inhibitor has been called textilinin (Txln) and two distinct forms have been isolated from the Brown-snake venom (molecular weight, 6688 and 6692). A comparison of plasmin inhibitor constants for aprotinin and the Txlns 1 and 2 indicated that the former bound very tightly (inhibitor constant,Ki ≈ 10−11 mol/l), while both of the latter bound less tightly (Ki ≈ 10−9 mol/l). Homogeneity of Txlns 1 and 2 was confirmed by sodium dodecyl sulphate-polyacrylamide gel electrophoresis and mass spectrometry. A sequence difference of six amino acids was observed between the two forms of Txln. Txln 1 and 2 showed, respectively, 45 and 43% homology with aprotinin, while there was 58 and 55% homology, respectively, with a plasmin inhibitor from the venom of eastern Taipan,Oxyuranus scutellatus. Both Txlns have six cysteines, like other inhibitors of this group, and homology was determined by alignment of these cysteines. Both have been shown to reduce blood loss by about 60% in a murine tail vein bleeding model. It is proposed that the kinetic profiles of Txln 1 and 2 for plasmin allow the arrest of haemorrhage without the possible threat of thrombosis.

Role of protein kinase activity in apoptosis

The transmission of signals from the plasma membrane to the nucleus involves a number of different pathways all of which have in common protein modification. The modification is primarily in the form of phosphorylation which leads to the activation of a series of protein kinases. It is now evident that these pathways are common to stimuli that lead to mitogenic and apoptotic responses. Even the same stimuli under different physiological conditions can cause either cell proliferation or apoptosis. Activation of specific protein kinases can in some circumstances protect against cell death, while in others it protects the cell against apoptosis. Some of the pathways involved lead to activation of transcription factors and the subsequent induction of genes involved in the process of cell death or proliferation. In other cases, such as for the tumour suppressor gene product p53, activation may be initiated both at the level of gene expression or through pre-existing proteins. Yet in others, while the initial steps in the pathway are ill-defined, it is clear that downstream activation of a series of cysteine proteases is instrumental in pushing the cell towards apoptosis. In this report we review the involvement of protein kinases at several different levels in the control of cell behavior.

Caspase-mediated Cleavage of the Ubiquitin-protein Ligase Nedd4 during Apoptosis

The onset of apoptosis is coupled to the proteolytic activation of a family of cysteine proteases, termed caspases. These proteases cleave their target proteins after an aspartate residue. Following caspase activation during apoptosis, a number of specific proteins have been shown to be cleaved. Here we show that Nedd4, a ubiquitin-protein ligase containing multiple WW domains and a calcium/lipid-binding domain, is also cleaved during apoptosis induced by a variety of stimuli including Fas-ligation, γ-radiation, tumor necrosis factor-α, C-8 ceramide, and etoposide treatment. Extracts from apoptotic cells also generated cleavage patterns similar to that seen in vivo, and this cleavage was inhibited by an inhibitor of caspase-3-like proteases. In vitro, Nedd4 was cleaved by a number of caspases, including caspase-1, -3, -6, and -7. By site-directed mutagenesis, one of the in vitro caspase cleavage sites in mouse Nedd4 was mapped to a DQPD237↓ sequence, which is conserved between mouse, rat, and human proteins. This is the first report demonstrating that an enzyme of the ubiquitin pathway is cleaved by caspases during apoptosis.

Epidermal Growth Factor Sensitizes Cells to Ionizing Radiation by Down-regulating Protein Mutated in Ataxia-Telangiectasia

Epidermal growth factor (EGF) has been reported to either sensitize or protect cells against ionizing radiation. We report here that EGF increases radiosensitivity in both human fibroblasts and lymphoblasts and down-regulates both ATM (mutated in ataxia-telangiectasia (A-T)) and the catalytic subunit of DNA-dependent protein kinase (DNA-PKcs). No further radiosensitization was observed in A-T cells after pretreatment with EGF. The down-regulation of ATM occurs at the transcriptional level. Concomitant with the down-regulation of ATM, the DNA binding activity of the transcription factor Sp1 decreased. A causal relationship was established between these observations by demonstrating that up-regulation of Sp1 DNA binding activity by granulocyte/macrophage colony-stimulating factor rapidly reversed the EGF-induced decrease in ATM protein and restored radiosensitivity to normal levels. Failure to radiosensitize EGF-treated cells to the same extent as observed for A-T cells can be explained by induction of ATM protein and kinase activity with time post-irradiation. Although ionizing radiation damage to DNA rapidly activates ATM kinase and cell cycle checkpoints, we have provided evidence for the first time that alteration in the amount of ATM protein occurs in response to both EGF and radiation exposure. Taken together these data support complex control of ATM function that has important repercussions for targeting ATM to improve radiotherapeutic benefit.

Critical targets of protein kinase C in differentiation of tumour cells

The ultimate target of pharmacological research is to find new drugs for treating human diseases such as cancer. Agents causing differentiation and thus growth arrest should be particularly useful in this regard. A potential target for such anticancer therapy is the enzyme family protein kinase C (PKC), which is involved in the transduction of signals for cell proliferation, differentiation, and apoptosis. Our recent work showing the induction of differentiation in melanoma cells by an activator of one PKC isoform, PKCdelta, touches on several important areas of investigation, which will form the basis of this review: the role of individual isoforms of PKC, their downstream targets and their specific substrates, the mechanism of activation of specific genes involved in the differentiation process, and the molecular basis for the morphological changes associated with differentiation. The central role that PKC plays in these processes points to the need for a greater understanding of the signalling pathways utilized by individual isoforms of this family of enzymes.