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G Shani

Publications and source records attributed to G Shani.

10 recordsLinked to original sources
PubMed2001

The pro-apoptotic function of death-associated protein kinase is controlled by a unique inhibitory autophosphorylation-based mechanism.

Death-associated protein kinase is a calcium/calmodulin serine/threonine kinase, which positively mediates programmed cell death in a variety of systems. Here we addressed its mode of regulation and identified a mechanism that restrains its apoptotic function in growing cells and enables its activation during cell death. It involves autophosphorylation of Ser(308) within the calmodulin (CaM)-regulatory domain, which occurs at basal state, in the absence of Ca(2+)/CaM, and is inversely correlated with substrate phosphorylation. This type of phosphorylation takes place in growing cells and is strongly reduced upon their exposure to the apoptotic stimulus of C(6)-ceramide. The substitution of Ser(308) to alanine, which mimics the ceramide-induced dephosphorylation at this site, increases Ca(2+)/CaM-independent substrate phosphorylation as well as binding and overall sensitivity of the kinase to CaM. At the cellular level, it strongly enhances the death-promoting activity of the kinase. Conversely, mutation to aspartic acid reduces the binding of the protein to CaM and abrogates almost completely the death-promoting function of the protein. These results are consistent with a molecular model in which phosphorylation on Ser(308) stabilizes a locked conformation of the CaM-regulatory domain within the catalytic cleft and simultaneously also interferes with CaM binding. We propose that this unique mechanism of auto-inhibition evolved to impose a locking device, which keeps death-associated protein kinase silent in healthy cells and ensures its activation only in response to apoptotic signals.

Amino Acid Motifs↗
PubMed2001

Autophosphorylation restrains the apoptotic activity of DRP-1 kinase by controlling dimerization and calmodulin binding.

DRP-1 is a pro-apoptotic Ca2+/calmodulin (CaM)-regulated serine/threonine kinase, recently isolated as a novel member of the DAP-kinase family of proteins. It contains a short extra-catalytic tail required for homodimerization. Here we identify a novel regulatory mechanism that controls its pro-apoptotic functions. It comprises a single autophosphorylation event mapped to Ser308 within the CaM regulatory domain. A negative charge at this site reduces both the binding to CaM and the formation of DRP-1 homodimers. Conversely, the dephosphorylation of Ser308, which takes place in response to activated Fas or tumour necrosis factor-alpha death receptors, increases the formation of DRP-1 dimers, facilitates the binding to CaM and activates the pro-apoptotic effects of the protein. Thus, the process of enzyme activation is controlled by two unlocking steps that must work in concert, i.e. dephosphorylation, which probably weakens the electrostatic interactions between the CaM regulatory domain and the catalytic cleft, and homodimerization. This mechanism of negative autophosphorylation provides a safety barrier that restrains the killing effects of DRP-1, and a target for efficient activation of the kinase by various apoptotic stimuli.

Amino Acid Sequence↗
PubMed2000

Death-associated protein kinase-related protein 1, a novel serine/threonine kinase involved in apoptosis.

In this study we describe the identification and structure-function analysis of a novel death-associated protein (DAP) kinase-related protein, DRP-1. DRP-1 is a 42-kDa Ca(2+)/calmodulin (CaM)-regulated serine threonine kinase which shows high degree of homology to DAP kinase. The region of homology spans the catalytic domain and the CaM-regulatory region, whereas the remaining C-terminal part of the protein differs completely from DAP kinase and displays no homology to any known protein. The catalytic domain is also homologous to the recently identified ZIP kinase and to a lesser extent to the catalytic domains of DRAK1 and -2. Thus, DAP kinase DRP-1, ZIP kinase, and DRAK1/2 together form a novel subfamily of serine/threonine kinases. DRP-1 is localized to the cytoplasm, as shown by immunostaining and cellular fractionation assays. It binds to CaM, undergoes autophosphorylation, and phosphorylates an exogenous substrate, the myosin light chain, in a Ca(2+)/CaM-dependent manner. The truncated protein, deleted of the CaM-regulatory domain, was converted into a constitutively active kinase. Ectopically expressed DRP-1 induced apoptosis in various types of cells. Cell killing by DRP-1 was dependent on two features: the status of the catalytic activity, and the presence of the C-terminal 40 amino acids shown to be required for self-dimerization of the kinase. Interestingly, further deletion of the CaM-regulatory region could override the indispensable role of the C-terminal tail in apoptosis and generated a "superkiller" mutant. A dominant negative fragment of DAP kinase encompassing the death domain was found to block apoptosis induced by DRP-1. Conversely, a catalytically inactive mutant of DRP-1, which functioned in a dominant negative manner, was significantly less effective in blocking cell death induced by DAP kinase. Possible functional connections between DAP kinase and DRP-1 are discussed.

Amino Acid Sequence↗
PubMed1996

Current misinterpretations of the linear no-threshold hypothesis.

Contrary to the "linear no-threshold hypothesis," which implies that "any amount, however small" of radiation energy is a serious cancer threat, it is shown here that only relatively quite large amounts of such energy can pose such a threat to a person or population. Key to doing this is to make a sharp distinction between the actual amount of the radiation agent imparted energy, epsilon, which must be expressed in units of joules, and the average concentration or density of energy, epsilon/m (i.e., absorbed dose), which is expressed in units of Gy. With any cellular system, e.g., in tissue culture, one can easily adjust the numbers of cells used at each dose point so that a clearly significant number of radiation-induced quantal responses (e.g., mutations, chromosome aberrations, malignant transformations, cell death), in the absorbed dose range of about 0.7 to 3 or more Gy, can be observed. However, if the number of cells is held constant as the absorbed dose is progressively reduced, a point is reached at which no significant excess is observable. This situation is frequently "remedied" by including more cells at that point, which, of course, can increase the number of malignant transformations sufficiently to render the excess statistically valid. However, because both axes are expressed in relative terms, the data point, despite having gained statistical significance, remains at the same location on the graph. This gives the false impression that no more of the agent energy was added or needed to achieve significance. However, if both coordinates are put in absolute terms, i.e., the actual number of quantal responses vs. imparted energy, and the same exercise of "improving the statistics" at low exposures is attempted, it then becomes evident that any point thus rendered significant must be relocated at a substantially higher energy point on the graph. This demonstrates unequivocally the fallacy in the proof of the "linear hypothesis" which is based on agent concentration response curves and not agent amount. It shows that the smaller the agent concentration (absorbed dose; epsilon/m), the larger the amount of radiation energy that must be added to the system in order to demonstrate a radiation-induced response. This suggests a minimum average energy requirement for production of a radiation-attributable cancer. It Ls concluded that the "linear hypothesis" should be abandoned as the cornerstone of radiation protection and practice.

Dose-Response Relationship, Radiation↗
PubMed1996

The biological effects of Auger electrons compared to alpha-particles and Li ions.

The present study reports the results of V-79 Chinese hamster cell survival studies in which Auger electron emission was stimulated in gadolinium (Gd) after thermal neutron capture. When a porphyrin that had previously been labeled with boron (10BOPP) was also labeled with Gd (Gd-10BOPP), the cells were incubated with Gd-10BOPP to assess the compound's ability to physiologically transport the Gd into the cell, and localize the Gd atoms in or near the cell's critical target, presumably the DNA. It was anticipated that Auger electron emission, stimulated during the 157Gd (n, gamma)158Gd interaction, would impart additional high LET damage to that observed from the alpha-particle and Li ion during the 10B(n, alpha) 7Li reaction. Following irradiation with thermal neutrons from the Brookhaven Medical Research Reactor, the effectiveness of the Auger electrons was determined by comparing the response of cells incubated with 10BOPP, where damage was imparted by the boron neutron capture (BNC) products, to that from Gd-10BOPP, with equal concentration of 10B in both solutions. An Auger effectiveness factor of approximately 2 was found for the Gd-10BOPP cells. The Auger effectiveness observed with Gd strongly suggested that the 10BOPP molecule physiologically transported the Gd3+ ion intracellularly where it probably bound to DNA. Others have reported that Gd3+ does, in fact, complex with DNA. While depositing less energy per interaction than the high LET BNC reaction by-products, Auger electron ionization was more effective.

Animals↗
PubMed1989

The chicken homeo box genes CHox1 and CHox3: cloning, sequencing and expression during embryogenesis.

Several Drosophila genes involved in the control of segmentation and segment identity share a 183-bp conserved sequence termed homeo box. Homeo box sequences have been detected and cloned from the genomes of insects like Drosophila to vertebrates such as mouse and man. Two chicken homeo box genes CHox1 and CHox3, are described. Cloning of the CHox1 and CHox3 homeo boxes was performed using Drosophila and murine homeo box sequences as probes under low-stringency conditions. Analysis of both chicken homeo box sequences revealed them to be homeo boxes that have diverged from the Antennapedia class with homologies to homeo boxes of other organisms in the range of 75-42% at the nucleotide level and 69-41% at the protein level. Analysis of CHox3 expression during early embryo development showed that the gene codes for five transcripts 1.3, 1.9, 2.6, 5.6 and 7.9 kb in size. Three of the transcripts (1.3, 1.9 and 5.6 kb) are also recognized by a flanking non-homeo box containing probe. The levels of the different transcripts changed during the first five days of development. The most abundant transcripts (1.3 and 1.9 kb) are already present at the time the egg is laid. Their transcription peaks at day 1 of incubation and then decreases. The CHox1 transcripts are present at very low levels between days 2.5 and 4 of development. These two chicken genes represent bona fide Hox genes in a branch of vertebrates that evolved parallel to mammals.

Amino Acid Sequence↗