Hypoxia-induced cell death of HepG2 cells involves a necrotic cell death mediated by calpain

To elucidate mechanism of cell death in response to hypoxia, we attempted to compare hypoxia-induced cell death of HepG2 cells with cisplatin-induced cell death, which has been well characterized as a typical apoptosis. Cell death induced by hypoxia turned out to be different from cisplatin-mediated apoptosis in cell viability and cleavage patterns of caspases. Hypoxia-induced cell death was not associated with the activation of p53 while cisplatin-induced apoptosis is p53 dependent. In order to explain these differences, we tested involvement of μ-calpain and m-calpain in hypoxia-induced cell death. Calpains, especially μ-calpain, were initially cleaved by hypoxia, but not by cisplatin. Interestingly, the treatment of a calpain inhibitor restored PARP cleavage that was absent during hypoxia, indicating the recovery of activated caspase-3. The inhibition of calpains prevented proteolysis induced by hypoxia. In addition, hypoxia resulted in a necrosis-like morphology while cisplatin induced an apoptotic morphology. The calpain inhibitor prevented necrotic morphology induced by hypoxia and converted partially to apoptotic morphology with nuclear segmentation. Our result suggests that calpains are involved in hypoxia-induced cell death that is likely to be necrotic in nature and the inhibition of calpain switches hypoxia-induced cell death to apoptotic cell death without affecting cell viability.     

Calpain inhibitor 1 activates p53-dependent apoptosis in tumor cell lines.

Reports suggest a role of calpains in degradation of wild-type p53, which may regulate p53 induction of apoptosis. A calpain inhibitor, n-acetyl-leu-leu-norleucinal (calpain inhibitor 1), was assessed for ability to enhance p53-dependent apoptosis in human tumor cell lines with endogenous wild-type p53 and in altered p53 cell lines with the replacement of wild-type p53 by a recombinant adenovirus (rAd-p53). Calpain inhibitor 1 treatment resulted in increased levels of activated p53, increased p21 protein, and activation of caspases. Cell lines with wild-type, but not mutated or null, p53 status arrested in G0/G1 and were sensitive to calpain inhibitor-induced apoptosis. Regardless of endogenous p53 status, calpain inhibitor treatment combined with rAd-p53, but not empty vector virus, enhanced apoptosis in tumor cell lines. These results demonstrate p53-dependent apoptosis induced by a calpain inhibitor and further suggest a role for calpains in the regulation of p53 activity and induction of apoptotic pathways.     

Conditional disruption of ubiquitous calpains in the mouse

Ubiquitous mu- and m-calpain proteases are implicated in development and apoptosis. They are heterodimers consisting of 80-kDa catalytic subunits encoded by capn1 and capn2, respectively, and a common 28-kDa regulatory subunit encoded by capn4. The regulatory subunit is required to maintain stability and activity of mu- and m-calpains; thus, genetic disruption of capn4 was predicted to eliminate both calpain activities. Germline disruption of capn4 caused embryonic lethality, hampering the use of those mouse models to explore physiological calpain functions. Here we describe a loxP/cre conditional capn4 targeted mouse model that enables tissue-specific and temporal deletion of calpain activity. Disruption of the floxed capn4 gene using a ubiquitous cytomegalovirus promoter driven Cre recombinase transgene led to midgestation embryonic lethality. Fibroblasts from these embryos lacked detectable regulatory subunit expression, had reduced levels of the mu- and m-calpain catalytic subunits, and had no detectable mu- and m-calpain activities. These defects were corrected with a capn4-encoding lentivirus.     

Mutations in calpain 3 associated with limb girdle muscular dystrophy: analysis by molecular modeling and by mutation in m-calpain

Limb-girdle muscular dystrophy type 2A (LGMD2A) is an autosomal recessive disorder characterized by selective atrophy of the proximal limb muscles. Its occurrence is correlated, in a large number of patients, with defects in the human CAPN3 gene, a gene that encodes the skeletal muscle-specific member of the calpain family, calpain 3 (or p94). Because calpain 3 is difficult to study due to its rapid autolysis, we have developed a molecular model of calpain 3 based on the recently reported crystal structures of m-calpain and on the high-sequence homology between p94 and m-calpain (47% sequence identity). On the basis of this model, it was possible to explain many LGMD2A point mutations in terms of calpain 3 inactivation, supporting the idea that loss of calpain 3 activity is responsible for the disease. The majority of the LGMD2A mutations appear to affect domain/domain interaction, which may be critical in the assembly and the activation of the multi-domain calpain 3. In particular, we suggest that the flexibility of protease domain I in calpain 3 may play a critical role in the functionality of calpain 3. In support of the model, some clinically observed calpain 3 mutations were generated and analyzed in recombinant m-calpain. Mutations of residues forming intramolecular domain contacts caused the expected loss of activity, but mutations of some surface residues had no effect on activity, implying that these residues in calpain 3 may interact in vivo with other target molecules. These results contribute to an understanding of structure-function relationships and of pathogenesis in calpain 3.     

Tobacco-specific nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone induces phosphorylation of mu- and m-calpain in association with increased secretion, cell migration, and invasion

Mounting evidence indicates that cigarette smoking not only promotes tumorigenesis but also may increase the spread of cancer cells in the body. However, the intracellular mechanism(s) by which cigarette smoking promotes metastasis of human lung cancer remains enigmatic. Nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) is an important component in cigarette smoke and is formed by nitrosation of nicotine. mu- and m-calpain (calpain I and calpain II) are major members of the calpain family, which are ubiquitously expressed in both small cell lung cancer and non-small cell lung cancer cells. Our findings indicated that NNK potently induces phosphorylation of both mu- and m-calpain in association with their activation and increased migration as well as invasion of lung cancer cells. Treatment of cells with PD98059 blocked phosphorylation of m- and mu-calpain and resulted in suppression of NNK-induced cell migration and invasion. p44 MAPK/extracellular signal-regulated kinase 1 (ERK1) and p42 MAPK/ERK2 were activated by NNK, co-localized with mu- and m-calpain in cytoplasm, and directly phosphorylated mu- and m-calpain in vitro. These findings suggest a role for the ERK1/2 kinases as NNK-activated physiological calpain kinases. Specific knock-down of mu- and/or m-calpain expression by RNA interference blocked NNK-stimulated migration and invasion, suggesting that mu- and m-calpain may act as required targets in a NNK-induced metastatic signaling pathway. Furthermore, NNK promotes secretion of active mu- and m-calpain from lung cancer cells through vesicles, which may have the potential to cleave substrates in the extracellular matrix. Thus, NNK-induced cell migration and invasion may occur, at least in part, through a novel mechanism involving phosphorylation of calpains that leads to their activation and secretion, which may contribute to metastasis and/or progression of lung cancer.     

Proteolytic cleavage of human p53 by calpain: a potential regulator of protein stability.

The p53 tumor suppressor protein is activated in cells in response to DNA damage and prevents the replication of cells sustaining genetic damage by inducing a cell cycle arrest or apoptosis. Activation of p53 is accompanied by stabilization of the protein, resulting in accumulation to high levels within the cell. p53 is normally degraded through the proteasome following ubiquitination, although the mechanisms which regulate this proteolysis in normal cells and how the p53 protein becomes stabilized following DNA damage are not well understood. We show here that p53 can also be a substrate for cleavage by the calcium-activated neutral protease, calpain, and that a preferential site for calpain cleavage exists within the N terminus of the p53 protein. Treatment of cells expressing wild-type p53 with an inhibitor of calpain resulted in the stabilization of the p53 protein. By contrast, in vitro or in vivo degradation mediated by human papillomavirus E6 protein was unaffected by the calpain inhibitor, indicating that the stabilization did not result from inhibition of the proteasome. These results suggest that calpain cleavage plays a role in regulating p53 stability.     

Knockdown of m-calpain increases survival of primary hippocampal neurons following NMDA excitotoxicity

The calpain family of cysteine proteases has a well-established causal role in neuronal cell death following acute brain injury. However, the relative contribution of calpain isoforms to the various forms of injury has not been determined as available calpain inhibitors are not isoform-specific. In this study, we evaluated the relative role of m-calpain and μ-calpain in a primary hippocampal neuron model of NMDA-mediated excitotoxicity. Baseline mRNA expression for the catalytic subunit of m-calpain ( capn2 ) was found to be 50-fold higher than for the μ-calpain catalytic subunit ( capn1 ) based on quantitative real-time PCR. Adeno-associated viral vectors designed to deliver short hairpin RNAs targeting capn1 or capn2 resulted in 60% and 90% knockdown of message respectively. Knockdown of capn2 but not capn1 increased neuronal survival after NMDA exposure at 21 days in vitro . Nuclear translocation of calpain substrates apoptosis inducing factor, p35/p25 and collapsin response mediator protein (CRMP) 2–4 was not detected after NMDA exposure in this model. However, nuclear translocation of CRMP-1 was observed and was prevented by capn2 knockdown. These findings provide insight into potential mechanisms of calpain-mediated neurodegeneration and have important implications for the development of isoform-specific calpain inhibitor therapy.     

Susceptibility to p53 dependent apoptosis correlates with increased levels of Gas2 and Gas3 proteins

p53 dependent apoptosis is a critical regulator of tumorigenesis. In this paper we demonstrate that BALB/c cells transformed with a LT mutant perturbing pRb but not p53 functions (LT-2809) show unrestrained cell division under low serum condition which is actively counterbalanced by apoptosis. BALB/c cells transformed with a LT mutant perturbing p53 but not pRb functions (LT-K1), show similar unrestrained cell division but no evident signs of apoptosis when grown in low serum. Such apoptotic response of LT-2809 cells is characterised by increased expression of Gas2 which becomes proteolytically processed. Similarly Gas3 expression is markedly increased in LT-2809 cells with respect to LT-K1. Since both Gas2 and Gas3 have been previously associated with the apoptotic response at growth arrest, our observations suggest that they could also contribute to the regulation of cellular susceptibility to p53 dependent apoptosis.     

Involvement of calpains in growth factor-mediated migration

Previous research in our laboratory has already shown the importance of the role played by ubiquitous calpains during myoblast migration. The aim of this study was to investigate calpain expression during myoblast migration and, to enhance this phenomenon via calpain stimulation. Ubiquitous calpains are members of a large family of calcium-dependent cysteine proteases. They play an important role in numerous biological and pathological phenomena, such as signal transduction, apoptosis, cell-cycle regulation, cell spreading, adhesion, invasion, myogenesis, and motility. Myoblast migration is a crucial step in myogenesis, as it is necessary for myoblast alignment and fusion to form myotubes. This study started by examining changes in calpain expression during migration, then investigated the possibility of activating myoblast migration via the stimulation of calpain expression and/or activity. The migration rate of myoblasts overexpressing mu- or milli-calpain was quantified. The results showed that calpain overexpression dramatically inhibited myoblast migration. Growth-factor treatments were then used to enhance myoblast migration. The results showed that treatment with IGF-1, TGF-β1, or insulin induced a major increase in migration and caused a significant increase in m-calpain expression and activity. The increase in migration was totally inhibited by adding calpeptin, a calpain-specific inhibitor. These findings suggest that milli-calpain is involved in growth factor-mediated migration.     

Expression of the gene for large subunit of m-calpain is elevated in skeletal muscle from Duchenne muscular dystrophy patients

Calpain is an intracellular nonlysosomal protease involved in essential regulatory or processing functions of the cell, mediated by physiological concentrations of Ca²⁺. However, in an environment of abnormal intracellular calcium, such as that seen in Duchenne muscular dystrophy (DMD), calpain is suggested to cause degeneration of muscle owing to enhanced activity. To test whether the reported increase in calpain activity in DMD results fromde novo synthesis of the protease, we have assessed the quantitative changes in mRNA specific for m-calpain. mRNA isolated from DMD and control muscle was analysed by dot blot hybridization using a cDNA probe for the large subunit of m-calpain. Compared to control a four-fold increase in specific mRNA was observed in dystrophic muscle. This enhanced expression of the m-calpain gene in dystrophic condition suggests that the reported increase in m-calpain activity results fromde novo synthesis of protease and underlines the important role of m-calpain in DMD.     

Ubiquitous calpains promote caspase-12 and JNK activation during endoplasmic reticulum stress-induced apoptosis

Ubiquitously expressed mu- and m-calpain proteases are implicated in development and apoptosis. They consist of 80-kDa catalytic subunits encoded by the capn1 and capn2 genes, respectively, and a common 28-kDa regulatory subunit encoded by the capn4 gene. The regulatory subunit is required to maintain the stability and activity of mu- and m-calpains. Accordingly, genetic disruption of capn4 in the mouse eliminated both ubiquitous calpain activities. In embryonic fibroblasts derived from these mice, calpain deficiency correlated with resistance to endoplasmic reticulum (ER) stress-induced apoptosis, and this was directly related to a calpain requirement for activation of both caspase-12 and the ASK1-JNK cascade. This study provides compelling genetic evidence for calpain's role in caspase-12 activation at the ER, and reveals a novel role for the ubiquitous calpains in ER-stress induced apoptosis and JNK activation.     

Dysregulation of the calpain-calpastatin system plays a role in the development of cerulein-induced acute pancreatitis in the rat

Calpain, a calcium-dependent cytosolic cysteine protease, is implicated in a multitude of cellular functions but also plays a role in cell death. Recently, we have shown that two ubiquitous isoforms, termed micro-calpain and m-calpain, are expressed in rat pancreatic acinar cells and that calcium ionophore-induced calpain activation leads to acinar cell injury. On the basis of these observations, we have now investigated the role of both calpain forms and the endogenous calpain inhibitor calpastatin in acute pancreatitis. After treatment of rats either without or with calpain inhibitor Z-Val-Phe methyl ester (ZVP; 60 mg/kg i.p.), pancreatitis was induced by cerulein injections (10 microg/kg i.p.; 5 times at hourly intervals). Calpain activation and calpastatin expression in the pancreatic tissue were studied by Western blot analysis. Pancreatic injury was assessed by plasma amylase activity, pancreatic wet/dry weight ratio (edema), histological and electron-microscopic analyses, as well as fluorescence labeling of actin filaments. Cerulein caused an activation of both micro-calpain and m-calpain, accompanied by degradation of calpastatin. Prophylactic administration of ZVP reduced the cerulein-induced calpain activation but had no effect on calpastatin alterations. In correlation to the diminished calpain activity, the severity of pancreatitis decreased as indicated by a decline in amylase activity (P < 0.01), pancreatic edema formation (P < 0.05), histological score for eight parameters (P < 0.01), and actin filament alterations. Our findings support the hypothesis that dysregulation of the calpain-calpastatin system may play a role in the onset of acute pancreatitis.     

m-Calpain colocalizes with the calcium-sensing receptor (CaR) in caveolae in parathyroid cells and participates in degradation of the CaR

The calcium-sensing receptor (CaR) is a G protein-coupled, seven-transmembrane receptor and resides within caveolin-rich membrane domains in bovine parathyroid cells. The proenzyme of calpain 2 (m-calpain) is a heterodimeric calcium-dependent cysteine protease consisting of catalytic and regulatory subunits. The effects of calcium on the enzyme include activation, autolysis, and subunit dissociation. Here, we examine the potential role of caveolin-1 and caveolae in regulating the cellular distribution and function of m-calpain in parathyroid cells. We show that the inactive heterodimeric forms of m-calpain are concentrated in caveolin-rich membrane fractions prepared from parathyroid cells incubated with low extracellular calcium (Ca2+(o)). In contrast, in cells incubated with 3 mm Ca2+(o), which activates the CaR and increases intracellular calcium, there is a reduction in m-calpain in association with an increase in CaR protein and phosphorylated protein kinase C alpha and beta in caveolin-rich fractions. To assess the impact of activation of calpain on CaR protein in caveolar fractions, we analyzed the effects of m-calpain on the CaR. Activation of the CaR with high Ca2+(o) induced the release of lower molecular weight fragments of the receptor into the cell culture medium, and calpain inhibitors blocked this effect. Moreover, the fragments of the CaR as well as caveolin-1, m-calpain, and alkaline phosphatase were localized in membrane vesicles shed by parathyroid cells, supporting the association of these proteins in living cells. Treatment of CaR proteins in vitro with m-calpain also resulted in the appearance of lower molecular weight fragments of the CaR. Our data suggest that localization of m-calpain within caveolae may contribute to maintenance of the enzyme in an inactive state and that m-calpain may also contribute to the regulation of CaR levels.     

Group B Streptococcus induces macrophage apoptosis by calpain activation

Group B Streptococcus (GBS) has developed several strategies to evade immune defenses. We show that GBS induces macrophage (Mphi) membrane permeability defects and apoptosis, prevented by inhibition of calcium influx but not caspases. We analyze the molecular mechanisms of GBS-induced murine Mphi apoptosis. GBS causes a massive intracellular calcium increase, strictly correlated to membrane permeability defects and apoptosis onset. Calcium increase was associated with activation of calcium-dependent protease calpain, demonstrated by casein zymography, alpha-spectrin cleavage to a calpain-specific fragment, fluorogenic calpain-substrate cleavage, and inhibition of these proteolyses by calpain inhibitors targeting the calcium-binding, 3-(4-Iodophenyl)-2-mercapto-(Z)-2-propenoic acid, or active site (four different inhibitors), by calpain small-interfering-RNA (siRNA) and EGTA. GBS-induced Mphi apoptosis was inhibited by all micro- and m-calpain inhibitors used and m-calpain siRNA, but not 3-(5-Fluoro-3-indolyl)-2-mercapto-(Z)-2-propenoic acid (micro-calpain inhibitor) and micro-calpain siRNA indicating that m-calpain plays a central role in apoptosis. Calpain activation is followed by Bax and Bid cleavage, cytochrome c, apoptosis-inducing factor, and endonuclease G release from mitochondria. In GBS-induced apoptosis, cytochrome c did not induce caspase-3 and -7 activation because they and APAF-1 were degraded by calpains. Therefore, apoptosis-inducing factor and endonuclease G seem the main mediators of the calpain-dependent but caspase-independent pathway of GBS-induced apoptosis. Proapoptotic mediator degradations do not occur with nonhemolytic GBS, not inducing Mphi apoptosis. Apoptosis was reduced by Bax siRNA and Bid siRNA suggesting Bax and Bid degradation is apoptosis correlated. This signaling pathway, different from that of most pathogens, could represent a GBS strategy to evade immune defenses.     

Effect of mu-calpain on m-calpain

The free Ca(2+) concentrations required for half-maximal proteolytic activity of m-calpain are in the range of 400-800 microM and are much higher than the 50-500 nM free Ca(2+) concentrations that exist in living cells. Consequently, a number of studies have attempted to find mechanisms that would lower the Ca(2+) concentration required for proteolytic activity of m-calpain. Although autolysis lowers the Ca(2+) concentration required for proteolytic activity of m-calpain, 90-400 microM Ca(2+) is required for a half-maximal rate of autolysis of m-calpain, even in the presence of phospholipid. It has been suggested that mu-calpain, which has a lower Ca(2+) requirement than m-calpain, might proteolyze m-calpain and reduce its Ca(2+) requirement to a level that would allow it to be active at physiological Ca(2+) concentrations. We have incubated m-calpain with mu-calpain for 60 min at a ratio of 1:50 mu-calpain:m-calpain, in the presence of 50 microM free Ca(2+); this Ca(2+) concentration is high enough for more than half-maximal activity of mu-calpain, but does not activate m-calpain. Under these conditions, mu-calpain caused no detectable proteolytic degradation of the m-calpain polypeptide and did not change the Ca(2+) concentration required for proteolytic activity of m-calpain. mu-Calpain also did not degrade the m-calpain polypeptide at 1000 microM Ca(2+), which is a Ca(2+) concentration high enough to completely activate m-calpain. It seems unlikely that mu-calpain could act as an "activator" of m-calpain in living cells. Because m-calpain rapidly degrades itself (autolyzes) at 1000 microM Ca(2+) and because the subsite specificities of mu- and m-calpain are very similar if not identical, failure of mu-calpain to rapidly degrade m-calpain at 1000 microM Ca(2+) suggests a unique role of autolysis in calpain function.     

Mechanical stimulation of C2C12 cells increases m-calpain expression, focal adhesion plaque protein degradation

Myogenesis is a complex sequence of events, including the irreversible transition from the proliferation-competent myoblast stage into fused, multinucleated myotubes. During embryonic development, myogenic differentiation is regulated by positive and negative signals from surrounding tissues. Stimulation due to stretch- or load-induced signaling is now beginning to be understood as a factor which affects gene sequences, protein synthesis and an increase in Ca2+ influx in myocytes. Evidence of the involvement of Ca2+ -dependent activity in myoblast fusion, cell membrane and cytoskeleton component reorganization due to the activity of the ubiquitous proteolytic enzymes, calpains, has been reported. Whether there is a link between stretch- or load-induced signaling and calpain expression and activation is not known. Using a magnetic bead stimulation assay and C2C12 mouse myoblasts cell population, we have demonstrated that mechanical stimulation via laminin receptors leads to an increase in m-calpain expression, but no increase in the expression of other calpain isoforms. Our study revealed that after a short period of stimulation, m-calpain relocates into focal adhesion complexes and is followed by a breakdown of specific focal adhesion proteins previously identified as substrates for this enzyme. We show that stimulation also leads to an increase in calpain activity in these cells. These data support the pivotal role for m-calpain in the control of muscle precursor cell differentiation and thus strengthen the idea of its implication during the initial events of muscle development.     

Domain II of m-calpain is a Ca(2+)-dependent cysteine protease

Calpain, a Ca(2+)-dependent cytosolic cysteine protease, proteolytically modulates specific substrates involved in Ca(2+)-mediated intracellular events, such as signal transduction, cell cycle, differentiation, and apoptosis. The 3D structure of m-calpain, in the absence of Ca(2+), revealed that the two subdomains (domains IIa and IIb) of the protease domain (II) have an 'open' conformation, probably due to interactions with other domains. Although the presence of an EF-hand structure was once predicted in the protease domain, no explicit Ca(2+)-binding structure was identified in the 3D structure. Therefore, it is predicted that if the protease domain is excised from the calpain molecule, it will have a Ca(2+)-independent protease activity. In this study, we have characterized a truncated human m-calpain that consists of only the protease domain. Unexpectedly, the proteolytic activity was Ca(2+)-dependent, very weak, and not effectively inhibited by calpastatin, a calpain inhibitor. Ca(2+)-dependent modification of the protease domain by the cysteine protease inhibitor, E-64c, was clearly observed as a SDS-PAGE migration change, indicating that the conformational changes of this domain are a result of Ca(2+) binding. These results suggest that the Ca(2+) binding to domain II, as well as to domains III, IV, and VI, is critical in the process of complete activation of calpain.     

Inhibition of mutant p53 phosphorylation at serine 15 or serine 315 partially restores the function of wild-type p53.

The tumor suppressor protein p53 is a phosphoprotein and has growth and transformation suppression functions. Phosphorylation of wild-type p53 is known to modulate its function. To investigate the role of phosphorylation in modulating the functions of mutant p53, we constructed a series of phosphorylation site mutants based on mutant p53 Ala143 (p53-143) and p53 His175 (p53-175). When transfected into p53-negative Saos-2 cells, parental mutant p53-143 and p53-175 abolished both growth suppression and induction of apoptosis. However, DNA-activated protein kinase (DNA-PK) or cyclin-dependent kinase (cdks) phosphorylation site double mutants partially restored the growth suppression and induction of apoptosis and recovered the p53-specific DNA binding activity. We also observed a difference in sensitivity to calpain from parental mutants p53-175 and p53-175/15 or p53-175/315. These results suggest that the lack of phosphorylation at either the DNA-PK or cdks site in p53 mutants partially restores the wild-type functions by altering their conformation.     

Activation of m-calpain (calpain II) by epidermal growth factor is limited by protein kinase A phosphorylation of m-calpain

We have shown previously that the ELR-negative CXC chemokines interferon-inducible protein 10, monokine induced by gamma interferon, and platelet factor 4 inhibit epidermal growth factor (EGF)-induced m-calpain activation and thereby EGF-induced fibroblast cell motility (H. Shiraha, A. Glading, K. Gupta, and A. Wells, J. Cell Biol. 146:243-253, 1999). However, how this cross attenuation could be accomplished remained unknown since the molecular basis of physiological m-calpain regulation is unknown. As the initial operative attenuation signal from the CXCR3 receptor was cyclic AMP (cAMP), we verified that this second messenger blocked EGF-induced motility of fibroblasts (55% +/- 4.5% inhibition) by preventing rear release during active locomotion. EGF-induced calpain activation was inhibited by cAMP activation of protein kinase A (PKA), as the PKA inhibitors H-89 and Rp-8Br-cAMPS abrogated cAMP inhibition of both motility and calpain activation. We hypothesized that PKA might negatively modulate m-calpain in an unexpected manner by directly phosphorylating m-calpain. A mutant human large subunit of m-calpain was genetically engineered to negate a putative PKA consensus sequence in the regulatory domain III (ST369/370AA) and was expressed in NR6WT mouse fibroblasts to represent about 30% of total m-calpain in these cells. This construct was not phosphorylated by PKA in vitro while a wild-type construct was, providing proof of the principle that m-calpain can be directly phosphorylated by PKA at this site. cAMP suppressed EGF-induced calpain activity of cells overexpressing a control wild-type human m-calpain (83% +/- 3.7% inhibition) but only marginally suppressed that of cells expressing the PKA-resistant mutant human m-calpain (25% +/- 5.5% inhibition). The EGF-induced motility of the cells expressing the PKA-resistant mutant also was not inhibited by cAMP. Structural modeling revealed that new constraints resulting from phosphorylation at serine 369 would restrict domain movement and help "freeze" m-calpain in an inactive state. These data point to a novel mechanism of negative control of calpain activation, direct phosphorylation by PKA.