Search

To relate the alterations occurring in neurodegenerations with Ca2+ homeostasis dysregulation, we analyzed the functional properties of the Ca2+-dependent calpain/calpastatin system in neuronal cells of transgenic mice overexpressing human mutated -G93A(+) SOD1. Motor cortex and spinal cord lower segments from transgenic mice show a very large increase in free Ca2+ ions and evident calpain activation, identified onthe basis of its consumption and substrates digestion. Changes in calpastatin intracellular localization and calpain conformation state further support these observations. Moreover, calpastatin is significantly expressed, counteracting its calpain-mediated degradation. Thus, the calpain /calpastatin system may be a target for new therapeutic approaches to neurodegenerative diseases.

We have recently demonstrated that in resting conditions calpain 1, but not calpain 2, is specifically associated to the N-Methyl-D-Aspartate receptor (NMDAR) multiprotein complex. We are here reporting that in SKNBE neuroblastoma cells or in freshly isolated nerve terminals from adult rat hippocampus, the proteolytic activity of calpain 1 resident at the NMDAR is very low under basal conditions and greatly increases following NMDAR stimulation. Since the protease resides at the NMDAR in saturating amounts, variations in Ca2+ influx promote an increase in calpain 1 activity without affecting the amount of the protease originally associated to NMDAR. In all the conditions examined, resident calpain 1 specifically cleaves NR2B at the C-terminal region, leading to its internalization together with NR1 subunit. While in basal conditions intracellular membranes include small amounts of NMDAR containing the calpain-digested NR2B, upon NMDAR stimulation nearly all the receptor molecules are internalized. We here propose that resident calpain 1 is involved in NMDAR turnover, and following an increase in Ca2+ influx, the activated protease, by promoting the removal of NMDAR from the plasma membranes, can decrease Ca2+ entrance through this channel. Due to the absence of calpastatin in such cluster, the activity of resident calpain 1 may be under the control of HSP90, whose levels are directly related to the activation of this protease. Observations of different HSP90/calpain 1 ratios in different ultrasynaptic compartments support this conclusion.

Here we demonstrate that heat shock protein 90 (HSP90) interacts with calpain-1, but not with calpain-2, and forms a discrete complex in which the protease maintains its catalytic activity, although with a lower affinity for Ca2+. Equilibrium gel distribution experiments show that this complex is composed by an equal number of molecules of each protein partner. Moreover, in resting cells, cytosolic calpain-1 is completely associated with HSP90. Since calpain-1, in association with HSP90, retains its proteolytic activity, and the chaperone is displaced by calpastatin also in the absence of Ca2+, the catalytic cleft of the protease is not involved in this association. Thus, calpain-1 can form two distinct complexes depending on the availability of calpastatin in the cytosol. The occurrence of a complex between HSP90 and calpain-1, in which the protease is still activable, can prevent the complete inhibition of the protease even in the presence of high calpastatin levels. We also demonstrate that in basal cell conditions HSP90 and calpain-1, but not calpain-2, are inserted in the multi-protein N-Methyl-D-Aspartate receptor (NMDAR) complex. The amount of calpain-1 at the NMDAR cluster is not modified in conditions of increased [Ca2+]i, and this resident protease is involved in the processing of NMDAR components. Finally, the amount of calpain-1 associated with NMDAR cluster is independent from Ca2+-mediated translocation. Our findings show that HSP90 plays an important role in maintaining a given and proper amount of calpain-1 at the functional sites.

A basal calpain activity promotes the limited proteolysis of wild type (WT) cystic fibrosis conductance regulator (CFTR), inducing the internalization of the split channel. This process contributes to the regulation in the level of the active CFTR at the plasma membranes. In peripheral blood mononuclear cells (PBMC) from 16 healthy donors, the inhibition of calpain activity induces a 3-fold increase in the amount of active WT CFTR at the plasma membranes. Instead, in PBMC from cystic fibrosis (CF) patients, calpain activity is expressed at aberrant levels causing the massive removal of F(508)del-CFTR from the cell surface. In these patients, the inhibition of such abnormal proteolysis rescues physiological amounts of active mutated CFTR in 90% of the patients (25 over 28). The recovery of functional F(508)del-CFTR at the physiological location, in cells treated with a synthetic calpain inhibitor, indicates that F(508)del-CFTR folding, maturation, and trafficking operate in CF-PBMC at significant rate. Thus, an increase in the basal calpain activity seems primarily involved in the CFTR defect observed in various CF cells. Furthermore, in CF-PBMC the recovery of the scaffolding protein Na(+)/H(+) exchanger regulatory factor 1 (NHERF-1), occurring following inhibition of the aberrant calpain activity, can contribute to rescue CFTR-functional clusters.

Background: Extracellular high mobility group box 1 (HMGB1) protein can operate in a synergistic fashion with different signal molecules promoting an increase of cell Ca(2+) influx. However, the mechanisms responsible for this effect of HMGB1 are still unknown., Principal Findings: Here we demonstrate that, at concentrations of agonist per se ineffective, HMGB1 potentiates the activation of the ionotropic glutamate N-methyl-D-aspartate receptor (NMDAR) in isolated hippocampal nerve terminals and in a neuroblastoma cell line. This effect was abolished by the NMDA channel blocker MK-801. The HMGB1-facilitated NMDAR opening was followed by activation of the Ca(2+)-dependent enzymes calpain and nitric oxide synthase in neuroblastoma cells, resulting in an increased production of NO, a consequent enhanced cell motility, and onset of morphological differentiation. We have also identified NMDAR as the mediator of HMGB1-stimulated murine erythroleukemia cell differentiation, induced by hexamethylenebisacetamide. The potentiation of NMDAR activation involved a peptide of HMGB1 located in the B box at the amino acids 130-139. This HMGB1 fragment did not overlap with binding sites for other cell surface receptors of HMGB1, such as the advanced glycation end products or the Toll-like receptor 4. Moreover, in a competition assay, the HMGB1((130-139)) peptide displaced the NMDAR/HMGB1 interaction, suggesting that it comprised the molecular and functional site of HMGB1 regulating the NMDA receptor complex., Conclusion: We propose that the multifunctional cytokine-like molecule HMGB1 released by activated, stressed, and damaged or necrotic cells can facilitate NMDAR-mediated cell responses, both in the central nervous system and in peripheral tissues, independently of other known cell surface receptors for HMGB1.

Persistent dysregulation in Ca(2+) homeostasis is a pervasive pathogenic mechanism in most neurodegenerative diseases, and accordingly, calpain activation has been implicated in neuronal cells dysfunction and death. In this study we examined the intracellular functional state of the calpain-calpastatin system in -G93A(+) SOD1 transgenic mice to establish if and how uncontrolled activation of calpain can be prevented in vivo during the course of prolonged [Ca(2+)](i) elevation. The presented data indicate that 1) calpain activation is more extensive in motor cortex, in lumbar, and sacral spinal cord segments compared with the lower or almost undetectable activation of the protease in other brain areas, 2) direct measurements of the variations of Ca(2+) levels established that the degree of the protease activation is correlated to the extent of elevation of [Ca(2+)](i), 3) intracellular activation of calpain is always associated with diffusion of calpastatin from perinuclear aggregated forms into the cytosol and the formation of a calpain-calpastatin complex, and 4) a conservative fragmentation of calpastatin is accompanied by its increased expression and inhibitory capacity in conditions of prolonged increase in [Ca(2+)](i). Thus, calpastatin diffusion and formation of the calpain-calpastatin complex together with an increased synthesis of the inhibitor protein represent a cellular defense response to conditions of prolonged dysregulation in intracellular Ca(2+) homeostasis. Altogether these findings provide a new understanding of the in vivo molecular mechanisms governing calpain activation that can be extended to many neurodegenerative diseases, potentially useful for the development of new therapeutic approaches.

Although several reports have indicated that eNOS is a highly sensitive calpain substrate, the occurrence of a concomitant Ca(2+)-dependent activation of the synthase and of the protease has never been analyzed in specific direct experiments. In this study, we have explored in vivo how eNOS can undergo Ca(2+)-dependent translocation and activation, protected against degradation by activated calpain. Here we demonstrate that following a brief exposure to Ca(2+)-loading, the cytosolic eNOS-HSP90 complex recruits calpain in a form in which the chaperone and the synthase are almost completely resistant to digestion by the protease. Furthermore, in the presence of the HSP90 inhibitor geldanamycin, a significant decrease in NO production and an extensive degradation of eNOS protein occurs, indicating that dissociation from membranes and association with the chaperone is correlated to the protection of the synthase. Experiments with isolated membrane preparations confirm the primary role of HSP90 in dissociation of eNOS from caveolae. Prolonged exposure of cells to Ca(2+)-loading resulted in an extensive degradation of both eNOS and HSP90, accompanied by a large suppression of NO production. We propose that the protective effect exerted by HSP90 on eNOS degradation mediated by calpain represents a novel and critical mechanism that assures the reversibility of the intracellular trafficking and activation of the synthase.

We have shown previously that isolated heat shock protein 90 (HSP90) and nitric oxide synthase (NOS), once associated in a heterocomplex, become completely resistant to calpain digestion. In this study, it is shown that, in vivo, under conditions of calpain activation, the protection of NOS degradation occurs. In addition, the extent of NOS degradation is a function of the level of HSP90 expression. Thus, in rat brain, which contains a large excess of HSP90, almost all neuronal NOS is associated with the chaperone protein. In this condition, neuronal NOS retains its full catalytic activity, although limited proteolytic conversion to still active low-molecular-mass (130 kDa) products takes place. In contrast, in aorta, which contains much smaller amounts of HSP90, endothelial NOS is not completely associated with the chaperone, and undergoes extensive degradation with a loss of protein and catalytic activity. On the basis of these findings, we propose a novel role of the HSP90-NOS heterocomplex in protecting in vivo NOS from proteolytic degradation by calpain. The efficiency of this effect is directly related to the level of intracellular HSP90 expression, generating a high HSP90 to NOS ratio, which favours both the formation and stabilization of the HSP90-NOS heterocomplex. This condition seems to occur in rat brain, but not in aorta, thus explaining the higher vulnerability to proteolytic degradation of endothelial NOS relative to neuronal NOS.

Ca2+ loading of Jurkat and bovine aorta endothelium cells induces the degradation of the neuronal and endothelial nitric oxide synthases that are selectively expressed in these cell lines. For neuronal nitric oxide synthase, this process involves a conservative limited proteolysis without appreciable loss of catalytic activity. By contrast, endothelial nitic oxide synthase digestion proceeds through a parallel loss of protein and catalytic activity. The chaperone heat shock protein 90 (HSP90) is present in a large amount in Jurkat cells and at significantly lower levels in bovine aorta endothelium cells. The differing ratios of HSP90/nitric oxide synthase (NOS) occurring in the two cell types are responsible for the conservative or nonconservative digestion of NOS isozymes. Consistently, we demonstrate that, in the absence of Ca2+, HSP90 forms binary complexes with NOS isozymes or with calpain. When Ca2+ is present, a ternary complex containing the three proteins is produced. In this associated state, HSP90 and NOS forms are almost completely resistant to calpain digestion, probably due to a structural hindrance and a reduction in the catalytic efficiency of the protease. Thus, the recruitment of calpain in the HSP90-NOS complexes reduces the extent of the proteolysis of these two proteins. We have also observed that calpastatin competes with HSP90 for the binding of calpain in reconstructed systems. Digestion of the proteins present in the complexes can occur only when free active calpain is present in the system. This process can be visualized as a novel mechanism involving the association of NOS with HSP90 and the concomitant recruitment of active calpain in ternary complexes in which the proteolysis of both NOS isozymes and HSP90 is significantly reduced.

Activation of calpain occurs as an early event in correlation with an increase in [Ca2+]i induced in rat brain upon treatment with a high salt diet for a prolonged period of time. The resulting sequential events have been monitored in the brain of normal and hypertensive rats of the Milan strain, diverging for a constitutive alteration in the level of [Ca2+]i found to be present in nerve cells of hypertensive animals. After 2 weeks of treatment, the levels of the plasma membrane Ca2+-ATPase and of native calpastatin are profoundly decreased. These degradative processes, more pronounced in the brain of hypertensive rats, are progressively and efficiently compensated in the brain of both rat strains by different incoming mechanisms. Along with calpastatin degradation, 15-kDa still-active inhibitory fragments are accumulated, capable of efficiently replacing the loss of native inhibitor molecules. A partial return to a more efficient control of Ca2+ homeostasis occurs in parallel, assured by an early increase in the expression of Ca2+-ATPase and of calpastatin, both producing, after 12 weeks of a high salt (sodium) diet, the restoration of almost original levels of the Ca2+ pump and of significant amounts of native inhibitor molecules. Thus, conservative calpastatin fragmentation, associated with an increased expression of Ca2+-ATPase and of the calpain natural inhibitor, has been demonstrated to occur in vivo in rat brain. This represents a sequential adaptive response capable of overcoming the effects of calpain activation induced by a moderate long term elevation of [Ca2+]i.

It is generally accepted that the Ca(2+)-dependent interaction of calpain with calpastatin is the most relevant mechanism involved in the regulation of Ca(2+)-induced proteolysis. We now report that a calpain-calpastatin association can occur also in the absence of Ca(2+) or at very low Ca(2+) concentrations, reflecting the physiological conditions under which calpain retains its inactive conformational state. The calpastatin binding region is localized in the non-inhibitory L-domain containing the amino acid sequences encoded by exons 4-7. This calpastatin region recognizes a calpain sequence located near the end of the DII-domain. Interaction of calpain with calpastatins lacking these sequences becomes strictly Ca(2+)-dependent because, under these conditions, the transition to an active state of the protease is an obligatory requirement. The occurrence of the molecular association between Ca(2+)-free calpain and various recombinant calpastatin forms has been demonstrated by the following experimental results. Addition of calpastatin protected calpain from trypsin digestion. Calpain was coprecipitated when calpastatin was immunoprecipitated. The calpastatin molecular size increased following exposure to calpain. The two proteins comigrated in zymogram analysis. Furthermore, calpain-calpastatin interaction was perturbed by protein kinase C phosphorylation occurring at sites located at the exons involved in the association. At a functional level, calpain-calpastatin interaction at a physiological concentration of Ca(2+) represents a novel mechanism for the control of the amount of the active form of the protease potentially generated in response to an intracellular Ca(2+) influx.

Conformational changes in the calpain molecule following interaction with natural ligands can be monitored by the binding of a specific monoclonal antibody directed against the catalytic domain of the protease. None of these conformational states showed catalytic activity and probably represent intermediate forms preceding the active enzyme state. In its native inactive conformation, calpain shows very low affinity for this monoclonal antibody, whereas, on binding to the ligands Ca(2+), substrate or calpastatin, the affinity increases up to 10-fold, with calpastatin being the most effective. This methodology was also used to show that calpain undergoes similar conformational changes in intact cells exposed to stimuli that induce either a rise in intracellular [Ca(2+)] or extensive diffusion of calpastatin into the cytosol without affecting Ca(2+) homeostasis. The fact that the changes in the calpain state are also observed under the latter conditions indicates that calpastatin availability in the cytosol is the triggering event for calpain-calpastatin interaction, which is presumably involved in the control of the extent of calpain activation through translocation to specific sites of action.

HMGB1 (high-mobility group box 1) protein, a pleiotropic cytokine released by several cell types under physiological and pathological conditions, has been identified as a signal molecule active on A431 cells. Although extracellular HMGB1 itself does not trigger any detectable signalling effect on these cells, it induces an increased susceptibility to EGF (epidermal growth factor) stimulation. Specifically, at concentrations of EGF which promote undetectable or limited cell responses, the addition of sub-nanomolar concentrations of HMGB1 potentiates the effect of EGF by specifically activating a downstream pathway that leads to enhanced cell motility through an increase in Ca2+ influx, activation of extracellular-signal-regulated kinase 1/2 and remodelling of the actin cytoskeleton. These results, which identify extracellular HMGB1 as an activator of human tumour cell migration operating in concert with EGF, have important implications in the search for novel strategies to control tumour progression and metastatic invasion.

The amount of calpastatin directly available in cytosol is under the control of [Ca2+] and [cyclic AMP]. Prolonged calpain activation also promotes degradation of calpastatin. The fluctuation of calpastatin concentration in cell soluble fraction is accompanied by an initial decrease in calpastatin gene expression, followed by a fivefold increase in its expression when the inhibitor protein is degraded. This process can be conceptualized as a mechanism to regulate calpastatin availability in the cell. This conclusion is supported by the fact that calpain, the other component of this proteolytic system, undergoes changes in its levels of expression in a much more limited manner. Furthermore, this process can be observed both in cells exposed to different natural stimuli, or in other cell lines. Modification of calpastatin gene expression might represent a new tool for the in vivo control of the regulatory machinery required for the modulation of Ca(2+)-dependent proteolysis.

Multiple protein kinase C (PKC) theta species, identified in an erythroleukaemia cell line, have been characterised in terms of their molecular properties and intracellular distribution. PKCthetas localised in the detergent-soluble cell fraction have an Mr of 76 kDa (theta-76) and contain Thr538 or pThr538 in the kinase activation loop. In contrast, PKCthetas localised in the Golgi complex have an Mr of 85 kDa (theta-85) and, although unphosphorylated at Thr538, are catalytically active. Strikingly, only theta-76 species which are unphosphorylated at Thr538 can undergo autocatalytic conversion to theta-85. Moreover, a Thr538-->Ala PKCtheta mutant is constitutively localised in the Golgi complex, confirming that changes in the phosphorylation state of this residue play a pivotal role in the overall control of catalytic properties and localisation of this kinase.

Human circulating PBMC (peripheral blood mononuclear cells) contain three calpain isoforms distinguishable on the basis of their chromatographic properties. Two of these proteases belong to the ubiquitous calpain subfamily, corresponding to the classical mu- and m-calpain forms. The third, which shows peculiar activating and regulatory properties, is an alternatively spliced calpain 3 (p94) form. This new calpain differs from calpain 3 in that it has lost IS1 insertion and exon 15, a lysine-rich sequence regarded as a nuclear translocation signal. PBMC p94-calpain undergoes activation and inactivation without the accumulation of a low-Ca2+-requiring form that is typical of the classical activation processes of mu- and m-calpain. Furthermore, it differs from the ubiquitous forms in that it displays a lower sensitivity to calpastatin. On the basis of these selective properties, it can be postulated that PBMC p94-calpain can be activated in response to specific stimuli that are not effective on the other calpain isoenzymes. The enzyme is preferentially expressed in B- and T-lymphocytes, whereas it is poorly expressed in natural killer cells and almost undetectable in polymorphonuclear cells. This distribution might reflect the specific function of this protease, which is preferentially present in cells devoted to the production of the humoral, rather than to the cellular, immune response.

In several cell types the binding of extracellular high-mobility group-box protein 1 (HMGB1) with the receptor for advanced glycation end-products (RAGE) induces cytoskeletal reorganization and cell motility. To establish whether RAGE is also involved in murine erythroleukaemia (MEL) cell differentiation stimulated by HMGB1, we have demonstrated that these cells express a 51 kDa protein identified as RAGE, and then we have produced stable transfectants overexpressing wild-type (wt) RAGE or a dominant negative (dn) RAGE mutant lacking the cytoplasmic domain to analyse the differentiation process in these cells. Several experimental findings indicated that RAGE was not involved in the MEL cell differentiation programme. This was also supported by the identical stimulatory effect exerted by HMGB1 on both wt- or dn-RAGE transfectants. We have also observed that HMGB1 binds a 65 kDa protein on the surface of MEL cells, supporting the hypothesis that alternative targets of HMGB1 are expressed on the MEL cell membrane and may be involved as mediators of its signalling.

Hypertensive rats from the Milan strain show a significant decrease in calpastatin activity as compared with normotensive control animals. Calpastatin deficiency is age-related and highly relevant in kidney, heart, and erythrocytes and of minor entity in brain tissue. In normotensives the changes during aging in the levels of calpastatin activity and mRNA are consistent with an increase of calpastatin protein. In hypertensive rats such a relationship during aging is not observed, because a progressive accumulation of mRNA is accompanied by a lower amount of calpastatin protein as compared with control rats. Together with the low level of calpastatin in kidney of hypertensive rats, a progressive accumulation of an active 15-kDa calpastatin fragment, previously shown to represent a typical product of calpain-mediated calpastatin degradation, is also observed. Evidence for such intracellular proteolysis by Ca(2+)-activated calpain is provided by the normalization of the calpastatin level, up to that of control animals, in hypertensive rats treated with drugs known to reduce both blood pressure and intracellular Ca(2+) influx. Further evidence is provided by the disappearance, in these conditions, of the 15-kDa calpastatin fragment. These data allow the conclusion that calpastatin degradation is a relevant part of the overall mechanism for regulating calpain activity.

The release of amphoterin by murine erythroleukaemia cells exposed to the chemical inducer hexamethylenebisacetamide represents an essential step for the process of their terminal differentiation. Once exported in the culture medium, amphoterin undergoes limited proteolysis, catalysed by a serine proteinase also secreted by stimulated cells. The isolated proteinase is responsible for degradation of amphoterin, with the production of a 10-amino-acid-residue fragment, specifically retaining the cell-differentiation-stimulating activity of the native protein molecule. This peptide does not express other properties of amphoterin, such as protein kinase C-stimulating activity or systemic toxicity. These findings define a selective mechanism accounting for extracellular amphoterin functional maturation.

We have previously reported that, in neuroblastoma LAN-5 cells, calpastatin is in an aggregated state, close to the cell nucleus [de Tullio, Passalacqua, Averna, Salamino, Melloni and Pontremoli (1999) Biochem. J. 343, 467-472]. In the present paper, we demonstrate that aggregated calpastatin is predominantly in a phosphorylated state. An increase in intracellular free [Ca2+] induces both dephosphorylation of calpastatin, through the action of a phosphoprotein phosphatase, and its redistribution as a soluble inhibitor species. cAMP, but not PMA-induced phosphorylation, reverses calpastatin distribution favouring its aggregation. This intracellular reversible mechanism, regulating the level of cytosolic calpastatin, could be considered a strategy through which calpain can escape calpastatin inhibition, especially during earlier steps of its activation process.

In neuroblastoma LAN-5 cells during calpain activation, in addition to the two expressed 70 kDa and 30 kDa calpastatin forms, other inhibitory species are produced, having molecular masses of 50 kDa and 15 kDa. At longer times of incubation, both native and new calpastatin species disappear. The formation of these new calpastatins as well as the decrease in intracellular total calpastatin activity are mediated by calpain itself, as indicated by the effect of the synthetic calpain inhibitor I, which prevents both degradative processes. Analysis of the calcium concentrations required for the two processes indicates that the first conservative proteolytic event is mediated by micro-calpain, whereas the second one is preferentially carried out by m-calpain. The appearance of the 15 kDa form, containing only the calpastatin repetitive inhibitory domain and identified also in red cells of hypertensive rats as the major inhibitor form, can be considered a marker of intracellular calpain activation, and it can be used for the monitoring of the involvement of calpain in pathological situations.

Acyl-CoA-binding protein, a 20-kDa homodimer that exerts many physiological functions, promotes activation of the classic calpain forms, most markedly that of the m-isozyme. This protein factor was purified from rat skeletal muscle and was also expressed in Escherichia coli. Both native and recombinant acyl-CoA-binding proteins show the same molecular properties and an identical capacity to decrease the [Ca(2+)] required for m-calpain activity. The binding of long-chain acyl-CoAs to acyl-CoA-binding protein does not modify the activating effect on calpains. Acyl-CoA-binding protein seems to be involved in the m-calpain regulation process, whereas the previously identified UK114 activator is a specific modulator of micro-calpain. Acyl-CoA-binding protein is proposed as a new component of the Ca(2+)-dependent proteolytic system. A comparative analysis among levels of classic calpains and their activator proteins is also reported.

Localization of the two main components of the Ca(2+)-dependent proteolytic system has been investigated in human neuroblastoma LAN-5 cells. Using a monoclonal antibody which recognizes the N-terminal calpastatin domain, it has been shown that this inhibitory protein is almost completely confined in two granule-like structures not surrounded by membranes. Similar calpastatin distribution has been found in other human and in murine cell types, indicating that aggregation of calpastatin is a general property and not an exclusive characteristic of neuronal-like cells. The existence of such calpastatin aggregates is confirmed by the kinetics of calpastatin-activity release during rat liver homogenization, which does not correspond to the rate of appearance of cytosolic proteins or to the disruption of membrane-surrounded organelles. Calpastatin distribution is affected by the intracellular increase in free Ca(2+), which results in calpastatin progressively becoming a soluble protein. However, calpain is distributed in the soluble cell fraction and, in activating conditions, partially accumulates on the plasma membrane. Similar behaviour has been observed in calpastatin localization in LAN-5 cells induced with retinoic acid, suggesting that the proteolytic system is activated during the differentiation process of these cells. The involvement of calpastatin in controlling calpain activity, rather than its activation process, and the utilization of changes in calpastatin localization as a marker of activation of the system is discussed.

Protein kinase C-theta is a member of the n-protein kinase C subfamily that in mitotic cells translocates to centrosomes and kinetochores. Although this kinase is expressed in comparable amounts in murine erythroleukaemia cells during the interphase or metaphase, when localized in the mitotic structures, it selectively phosphorylates a 66 kDa protein, also associated to chromosomes. Moreover, protein kinase C-theta immunoprecipitated from cells at the metaphase results four times more active in the absence of lipid cofactors as compared with the kinase obtained from cells in the interphase. This activation is accomplished by interaction of protein kinase C-theta with a protein factor which also promotes an increased autophosphorylation of the kinase. These findings indicate that in the mitotic phase of the cell cycle, protein kinase C-theta recognizes a protein factor which operates as a positive modulator of the kinase activity in the absence lipids.

Calpastatin, the natural inhibitor of calpain, is present in rat brain in multiple forms, having different molecular masses, due to the presence of one (low Mr form) or four (high Mr form) repetitive inhibitory domains. Recombinant and native calpastatin forms are substrates of protein kinase C, which phosphorylates a single serine residue at their N-terminus. Furthermore, both low and high Mr calpastatins are phosphorylated by protein kinase C at the same site. These calpastatin forms are phosphorylated also by protein kinase A, although with a lower efficiency. The incorporation of a phosphate group determines an increase in the concentration of Ca2+ required to induce the formation of the calpain-calpastatin complex. This effect results in a large decrease of the inhibitory efficiency of calpastatins. We suggest that phosphorylation of calpastatin represents a mechanism capable to balance the actual amount of active calpastatin to the level of calpain to be activated.

In this study we provide evidence that the protein kinase C (PKC)-straight theta isoenzyme is recruited on to the mitotic spindle in dividing murine erythroleukaemia (MEL) cells and associates specifically with centrosome and kinetochore structures. None of the other PKC isoenzymes (-alpha, -delta, -epsilon, -mu and -zeta) expressed by MEL cells shows this localization on the mitotic spindle. An identical subcellular distribution of PKC-straight theta is also observed in dividing murine P3 myeloma cells and human LAN-5 neuroblastoma cells, indicating that this PKC isoenzyme interacts with the mitotic apparatus in mammalian cells. In phorbol-ester-treated non-growing MEL cells, a rapid change in the intracellular distribution of PKC-straight theta occurs. Under these conditions, PKC-straight theta is translocated from the nuclear to the cytosolic cell compartment, an event that is accompanied by phosphorylation of the PKC-straight theta molecule and is followed by its down-regulation. The recovery of cell growth capacity results in the concomitant reappearance of PKC-straight theta. Furthermore, when MEL cells acquire the differentiated non-growing phenotype, the level of PKC-straight theta is reduced to less than 5%, suggesting that this PKC isoenzyme is no longer required. We propose that, unlike other members of the PKC family, PKC-straight theta may play a role in cell proliferation.

Four recombinant calpastatin forms, deduced from rat brain mRNAs and differing in the number of inhibitory repetitive domains from zero to four, were expressed and characterized for their inhibitory efficiency on mu- and m-calpain. Although the most effective one is a truncated calpastatin form composed of the N-terminal region (domain L) and a single inhibitory domain, all inhibitors are more active against mu-calpain, but are preferentially degraded and inactivated by m-calpain. The protein form composed exclusively of a domain L is deprived of any inhibitory activity but prevents inhibition of calpain by the other calpastatin forms, indicating that this calpastatin region could be relevant in the recognition of the proteinase. A calpastatin form having molecular properties similar to those of the recombinant truncated calpastatin, has also been found in rat brain. It does not derive from proteolysis of a higher molecular mass precursor. The expression of multiple calpastatin forms may be relevant for the specific modulation of the different calpain isozymes normally present in a single cell type.