Involvement of mu- and m-calpains and protein kinase C isoforms in L8 myoblast differentiation

The objectives were to investigate the roles of different calpains and protein kinase C (PKC) isoforms in muscle differentiation. Concentrations of mu- and m-calpain increased significantly whereas PKCalpha and delta declined significantly during L8 myoblast differentiation. Both mu-calpain and m-calpain antisense oligonucleotides inhibited myotube formation and creatine kinase activity during L8 myoblast differentiation. These results implied that both mu- and m-calpain were involved in L8 myoblast differentiation. To investigate the involvement of calpain in regulation of PKC concentrations, mu-calpain antisense oligonucleotides were added to L8 myoblasts. PKCalpha remained unchanged and PKCdelta declined. By adding m-calpain antisense oligonucleotides instead, PKCalpha level remained unchanged and PKCdelta concentrations increased significantly during differentiation. These results suggest that PKCalpha, but not PKCdelta, is the substrate for mu-calpain and PKCalpha and delta are the substrates for the m-calpain. In addition, more phosphorylated myogenin was found in day 2 antisense oligonucleotides treated L8 cells. It is concluded that the decline of PKCalpha mediated by m- and mu-calpain is essential for L8 myoblast differentiation. The decline of PKC during myoblast differentiation may cause hypo-phosphorylation of myogenin, which in turn activates muscle-specific genes during myogenesis.     

The role of autolysis in activity of the Ca2+-dependent proteinases (mu-calpain and m-calpain)

A recent hypothesis suggests that proteolytic activity of the micromolar and millimolar Ca2+-requiring forms of the Ca2+-dependent proteinases (mu- and m-calpain, respectively) is regulated in vivo by their association with a phosphatidylinositol-containing site on the plasma membrane followed by autolysis of the proteinases. Phosphatidylinositol association lowers the Ca2+ concentration needed for autolysis, and autolysis, in turn, lowers the Ca2+ concentration needed for proteolytic activity. To test this hypothesis, we have compared the Ca2+ concentrations needed for autolysis and for proteolytic activity of the calpains both in the presence and the absence of phosphatidylinositol. Bovine skeletal muscle mu-calpain required 40-50 microM Ca2+ for half-maximal rate of proteolysis of a casein substrate, 140-150 microM Ca2+ for half-maximal autolysis in the presence of 80 microM phosphatidylinositol, and 190-210 microM Ca2+ for half-maximal autolysis in the absence of phosphatidylinositol. Consequently, mu-calpain is an active proteinase and does not require autolysis for activation. Bovine skeletal muscle m-calpain required 700-740 microM Ca2+ for half-maximal rate of proteolysis of a casein substrate, 370-400 microM Ca2+ for half-maximal autolysis in the presence of 80 microM phosphatidylinositol, and 740-780 microM Ca2+ for half-maximal autolysis in the absence of phosphatidylinositol. These results are consistent with the idea that m-calpain functions in its autolyzed form, but the results do not demonstrate that unautolyzed m-calpain is inactive. 80 microM phosphatidylinositol had no effect on the Ca2+ requirement of the autolyzed forms of either mu- or m-calpain but lowered the specific activity of mu-calpain to 20% of its activity in the absence of phosphatidylinositol. Of the four forms of the calpains, unautolyzed m-calpain, autolyzed m-calpain, and unautolyzed mu-calpain would not be proteolytically active at the free Ca2+ concentrations of 300-1200 nM present inside normal cells, and neither mu- nor m-calpain would undergo autolysis at these Ca2+ concentrations, even in the presence of phosphatidylinositol. Cells must contain a mechanism other than or in addition to membrane association and autolysis to activate the calpains.     

mu-Calpain and calpain-3 are not autolyzed with exhaustive exercise in humans

mu-Calpain and calpain-3 are Ca2+-dependent proteases found in skeletal muscle. Autolysis of calpains is observed using Western blot analysis as the cleaving of the full-length proteins to shorter products. Biochemical assays suggest that mu-calpain becomes proteolytically active in the presence of 2-200 microM Ca2+. Although calpain-3 is poorly understood, autolysis is thought to result in its activation, which is widely thought to occur at lower intracellular Ca2+ concentration levels ([Ca2+]i; approximately 1 microM) than the levels at which mu-calpain activation occurs. We have demonstrated the Ca2+-dependent autolysis of the calpains in human muscle samples and rat extensor digitorum longus (EDL) muscles homogenized in solutions mimicking the intracellular environment at various [Ca2+] levels (0, 2.5, 10, and 25 microM). Autolysis of calpain-3 was found to occur across a [Ca2+] range similar to that for mu-calpain, and both calpains displayed a seemingly higher Ca2+ sensitivity in human than in rat muscle homogenates, with approximately 15% autolysis observed after 1-min exposure to 2.5 microM Ca2+ in human muscle and almost none after 1- to 2-min exposure to the same [Ca2+]i level in rat muscle. During muscle activity, [Ca2+]i may transiently peak in the range found to autolyze mu-calpain and calpain-3, so we examined the effect of two types of exhaustive cycling exercise (30-s "all-out" cycling, n = 8; and 70% VO2 peak until fatigue, n = 3) on the amount of autolyzed mu-calpain or calpain-3 in human muscle. No significant autolysis of mu-calpain or calpain-3 occurred as a result of the exercise. These findings have shown that the time- and concentration-dependent changes in [Ca2+]i that occurred during concentric exercise fall near but below the level necessary to cause autolysis of calpains in vivo.     

Activation of protein kinase calpha in the lysophosphatidic acid-induced bovine sperm acrosome reaction and phospholipase D1 regulation

Protein kinase C (PKC) has been implicated in the sperm acrosome reaction. In the present study, we demonstrate induction of the acrosome reaction and activation of sperm PKCalpha by lysophosphatidic acid (LPA), which is known to induce signal transduction cascades in many cell types via binding to specific cell-surface receptors. Under conditions by which LPA activates PKCalpha, there is significant stimulation of the acrosome reaction, which is inhibited by PKC inhibitors. Protein kinase Calpha belongs to the Ca(2+)-dependent classical PKC family of isoforms, and indeed we show that its activation depends upon the presence of Ca(2+) in the incubation medium. Protein kinase Calpha is a known regulator of phospholipase D (PLD). We investigated the possible regulatory relationships between PKCalpha and PLD1. Using specific antibodies against PLD1, we demonstrate for the first time its presence in bovine sperm. Furthermore, PLD1 coimmunoprecipitates with PKCalpha and the PKCalpha-PLD1 complex decomposes after treatment of the cells with LPA or 12-O:-tetradecanoyl phorbol-13-acetate, resulting in the translocation of PKCalpha to the plasma membrane and translocation of PLD1 to the particulate fraction. A possible bilateral regulation of PKCalpha and PLD1 activation during the sperm acrosome reaction is suggested.     

Effect of Ca2+ on binding of the calpains to calpastatin

Autolyzed mu-calpain, unautolyzed mu-calpain, autolyzed m-calpain, and unautolyzed m-calpain (mu-calpain is the micromolar Ca2+-requiring proteinase, m-calpain is the millimolar Ca2+-requiring proteinase) were passed through a calpastatin-affinity column at different free Ca2+ concentrations, and binding of the calpains to calpastatin was compared with proteolytic activity of that calpain at each Ca2+ concentration. Unautolyzed m-calpain, autolyzed m-calpain, and autolyzed mu-calpain required less Ca2+ for half-maximal binding to calpastatin than for half-maximal activity. Unautolyzed mu-calpain, however, required slightly more Ca2+ for half-maximal binding to calpastatin than for half-maximal activity. Half-maximal binding of oxidatively inactivated mu- or m-calpain to calpastatin required approximately the same Ca2+ concentrations as half-maximal binding of unautolyzed mu- or m-calpain, respectively, to calpastatin. Binding of unautolyzed m-calpain and autolyzed mu-calpain to calpastatin occurred over a wide range of Ca2+ concentrations, and it seems likely that two or more Ca2+-binding sites with different Ca2+-binding constants are involved in binding of the calpains to calpastatin. Proteolytic activity occurs at different Ca2+ concentrations than calpastatin binding, suggesting a second set of Ca2+-binding sites associated with proteolytic activity. Third and fourth sets of Ca2+-binding sites may be involved in autolysis and in binding to phosphatidylinositol or cell membranes; these four Ca2+-dependent properties of the calpains may require the eight potential Ca2+-binding sites that amino acid sequences predict are present in the calpain molecules.     

Competitive binding of protein kinase Calpha to membranes and Rho GTPases

Previously, we have shown that protein kinase C (PKC) forms a direct high-affinity, isozyme-specific and membrane lipid-independent interaction with Rho GTPases [Slater, S. J., Seiz, J. L., Stagliano, B. A., and Stubbs, C. D. (2001) Biochemistry 40, 4437-4445]. Since the cellular activation of PKCalpha involves an initial translocation from cytosolic to membrane compartments, the present study investigates the interdependence between the direct protein-protein interaction of PKCalpha with the Rho GTPase, Cdc42, and the protein-lipid interactions of PKCalpha with membranes. It was hypothesized that the interaction of PKCalpha with membrane-bound Cdc42 would contribute to the overall membrane-binding affinity of the kinase by providing an additional anchor. However, it was found that the incorporation of isoprenylated Cdc42 into membranes resulted in an apparent decrease in the membrane-binding affinity of PKCalpha, whereas the association of PKCbetaI, PKCdelta, PKCepsilon, and PKCzeta was each unaffected. The presence of membrane-bound Cdc42 resulted in a rightward shift in both the PS- and Ca2+-concentration response curves for PKCalpha membrane association and for the ensuing activation, whereas the maximal levels of binding and activation attained at saturating PS and Ca2+ concentrations were in each case unaffected. Overall, these findings suggest that PKCalpha undergoes a isozyme-specific interaction with membrane-bound Cdc42 to form a PKCalpha-Cdc42 complex, which possesses a membrane-binding affinity that is reduced relative to that of the individual components due to competition between Cdc42 and PS/Ca2+ for binding to PKCalpha. Consistent with this, it was found that the interaction of PKCalpha with membrane-bound Cdc42 was accompanied by the physical dissociation of the PKCalpha-Cdc42 complex from membranes. Thus, the study provides a novel mechanism by which the membrane association and activation of PKCalpha and Cdc42 may be regulated by competing protein-protein and protein-lipid interactions.     

Phospholipase D1 in caveolae: regulation by protein kinase Calpha and caveolin-1

Caveolae are small plasma membrane invaginations that have been implicated in cell signaling, and caveolin is a principal structural component of the caveolar membrane. Previously we have demonstrated that protein kinase Calpha (PKCalpha) directly interacts with phospholipase D1 (PLD1), activating the enzymatic activity of PLD1 in the presence of phorbol 12-myristate 13-acetate (PMA) [Lee, T. G., et al. (1997) Biochim. Biophys. Acta 1347, 199-204]. In this study, using a detergent-free procedure for the purification of a caveolin-enriched membrane fraction (CEM) and immunoblot analysis, we show that PLD1 is enriched in the CEMs of 3Y1 rat fibroblasts. Purified PLD1 directly bound to a glutathione S-transferase-caveolin-1 fusion protein in in vitro binding assays. The association of PLD1 with caveolin-1 could be completely eliminated by preincubation of PLD1 with an oligopeptide corresponding to the scaffolding domain (amino acids 82-101) of caveolin-1, indicating that caveolin-1 interacts with PLD1 through the scaffolding domain. The peptide also inhibited PKCalpha-stimulated PLD1 activity and the interaction between PLD1 and PKCalpha with an IC50 of 0.5 microM. PMA elicits translocation of PKCalpha to the CEMs, inducing PLD activation through the interaction of PKCalpha with PLD1 in the CEMs. Caveolin-1 also coimmunoprecipitated with PLD1 in the absence of PMA, and the amounts of coimmunoprecipitated caveolin-1 decreased in response to treatment with PMA. Taken together, our results suggest a new mechanism for the regulation of the PKCalpha-dependent PLD activity through the molecular interaction between PLD1, PKCalpha, and caveolin-1 in caveolae.     

Agonist-induced association of tropomyosin with protein kinase Calpha in colonic smooth muscle

Smooth muscle contraction regulated by myosin light chain phosphorylation is also regulated at the thin-filament level. Tropomyosin, a thin-filament regulatory protein, regulates contraction by modulating actin-myosin interactions. Present investigation shows that acetylcholine induces PKC-mediated and calcium-dependent phosphorylation of tropomyosin in colonic smooth muscle cells. Our data also shows that acetylcholine induces a significant and sustained increase in PKC-mediated association of tropomyosin with PKCalpha in the particulate fraction of colonic smooth muscle cells. Immunoblotting studies revealed that in colonic smooth muscle cells, there is no significant change in the amount of tropomyosin or actin in particulate fraction in response to acetylcholine, indicating that the increased association of tropomyosin with PKCalpha in the particulate fraction may be due to acetylcholine-induced translocation of PKCalpha to the particulate fraction. To investigate whether the association of PKCalpha with tropomyosin was due to a direct interaction, we performed in vitro direct binding assay. Tropomyosin cDNA amplified from colonic smooth muscle mRNA was expressed as GST-tropomyosin fusion protein. In vitro binding experiments using GST-tropomyosin and recombinant PKCalpha indicated direct interaction of tropomyosin with PKCalpha. PKC-mediated phosphorylation of tropomyosin and direct interaction of PKCalpha with tropomyosin suggest that tropomyosin could be a substrate for PKC. Phosphorylation of tropomyosin may aid in holding the slided tropomyosin away from myosin binding sites on actin, resulting in actomyosin interaction and sustained contraction.     

1-Butanol interferes with phospholipase D1 and protein kinase Calpha association and inhibits phospholipase D1 basal activity

1-Butanol is commonly used as a substrate for phospholipase D (PLD) activity measurement. Surprisingly we found that, in the presence of 30 mM 1-butanol (standard PLD assay conditions), PLD1 activity in COS-7 cells was lost after incubation for 2 min. In contrast, in the presence of the protein kinase C (PKC) inhibitor staurosporine or dominant negative PKCalpha D481E, the activity was sustained for at least 30min. The binding between PLD1 and PKCalpha was also lost after 2 min incubation with 30 mM 1-butanol while staurosporine and D481E maintained the binding. 1-Butanol at 2 mM did not inhibit PLD1 basal activity or PLD1 binding to PKCalpha, and staurosporine and PKCalpha D481E produced a constant increase in PLD1 basal activity of 2-fold. These results indicate that 1-butanol is inhibitory to PLD1 activity by reducing its association with PKCalpha, and that the concentration of 1-butanol is an important consideration in assaying basal PLD1 activity.     

Association of diacylglycerol kinase zeta with protein kinase C alpha: spatial regulation of diacylglycerol signaling

Activation of PKC depends on the availability of DAG, a signaling lipid that is tightly and dynamically regulated. DAG kinase (DGK) terminates DAG signaling by converting it to phosphatidic acid. Here, we demonstrate that DGKzeta inhibits PKCalpha activity and that DGK activity is required for this inhibition. We also show that DGKzeta directly interacts with PKCalpha in a signaling complex and that the binding site in DGKzeta is located within the catalytic domain. Because PKCalpha can phosphorylate the myristoylated alanine-rich C-kinase substrate (MARCKS) motif of DGKzeta, we tested whether this modification could affect their interaction. Phosphorylation of this motif significantly attenuated coimmunoprecipitation of DGKzeta and PKCalpha and abolished their colocalization in cells, indicating that it negatively regulates binding. Expression of a phosphorylation-mimicking DGKzeta mutant that was unable to bind PKCalpha did not inhibit PKCalpha activity. Together, our results suggest that DGKzeta spatially regulates PKCalpha activity by attenuating local accumulation of signaling DAG. This regulation is impaired by PKCalpha-mediated DGKzeta phosphorylation.     

Proteolysis of acidic calponin by mu-calpain

Acidic calponin is an actin binding protein expressed in smooth muscle and brain. Although the role of smooth muscle calponin (basic calponin) has been well studied, few studies have been performed on acidic calponin. In the present study, we demonstrated that acidic calponin binds to filamentous actin, but not monomeric actin. A co-sedimentation assay indicated that acidic calponin binds to actin with an apparent binding constant of 4 x 10(5) M(-1). In the presence of an excess amount of calmodulin, the binding of acidic calponin to actin was inhibited. The binding of acidic calponin to calmodulin was Ca(2+)-dependent with K(d) of 31 microM. We next investigated whether or not acidic calponin could be a substrate for mu-calpain in vitro, since it has been shown that basic calponin is cleaved by mu-calpain. The results showed that acidic calponin was also cleaved by mu-calpain. Neither the proteolytic pattern nor velocity of acidic calponin was different in the absence or presence of calmodulin. When acidic calponin had bound to actin, however, the susceptibility of the acidic calponin to mu-calpain was significantly reduced, which was reversed by the addition of calmodulin. Our results suggest that acidic calponin might be involved in the mu-calpain-regulated actin cytoskeleton.     

Rapid in vitro conformational changes of the catalytic site of PKC alpha assessed by FIM-1 fluorescence

To study the activation process of protein kinase C (PKCalpha), we used a fluorescent probe, FIM-1, a bis-indolylmaleimide derivative, which binds to the ATP-binding site on the catalytic domain [Chen, C. S., and Poenie, M. (1993) J. Biol. Chem. 268, 15812]. This enabled us to directly observe the microenvironment of the ATP-binding site in vitro during the activation process. The FIM-1 binding affinity for PKCalpha (EC(50) between 6 and 10 nM) was affected neither by PKCalpha activating conditions nor by enzyme proteolysis. The fluorescence yield of the PKCalpha-FIM-1 complex depended on the PKCalpha activation state. This fluorescence yield was decreased upon proteolysis, which allowed us to study the rate of PKC proteolysis by mu-calpain and its modification by cofactors. Two binding sites were also observed for Ca2+ on the partially activated PKCalpha. After phorbol ester (TPA) application, PKC activation was characterized by biexponential kinetics, including a rapid phase completed within 5 min and a slow phase lasting at least 30 min, which reflected several activation steps. Two different binding sites for TPA were revealed on membrane-associated PKCalpha (EC(50) = 31 +/- 12 and 580 +/- 170 nM), and their modulation by phosphatidylserine and Ca2+ was characterized. The high-affinity TPA binding site was highly conserved, even on the soluble enzyme. Our study shows that binding of low concentrations of TPA triggers conformational changes in the soluble PKCalpha, which affect the microenvironment of its catalytic domain.     

Regulation of protein kinase B/Akt activity and Ser473 phosphorylation by protein kinase Calpha in endothelial cells

Protein kinase Balpha (PKBalpha/Akt-1) is a key mediator of multiple signaling pathways involved in angiogenesis, cell proliferation and apoptosis among others. The unphosphorylated form of Akt-1 is virtually inactive and its full activation requires two phosphatidylinositol-3,4,5-triphosphate-dependent phosphorylation events, Thr308 by 3-phosphoinositide-dependent kinase-1 (PDK1) and Ser473 by an undefined kinase that has been termed PDK2. Recent studies have suggested that the Ser473 kinase is a plasma membrane raft-associated kinase. In this study we show that protein kinase Calpha (PKCalpha) translocates to the membrane rafts in response to insulin growth factor-1 (IGF-1) stimulation. Overexpression of PKCalpha increases Ser473 phosphorylation and Akt-1 activity, while inhibition of its activity or expression decreases IGF-1-dependent activation of Akt-1. Furthermore, in vitro, in the presence of phospholipids and calcium, PKCalpha directly phosphorylates Akt-1 at the Ser473 site. We conclude, therefore, that PKCalpha regulates Akt-1 activity via Ser473 phosphorylation and may function as PDK2 in endothelial cells.     

Functional interaction of protein kinase Calpha with the tyrosine kinases Syk and Src in human platelets

There is a high degree of cross-talk between tyrosine phosphorylation and the serine/threonine phosphorylation signaling pathways. Here we show a physical and functional interaction between the classical protein kinase C isoform (cPKC), PKCalpha, and two major nonreceptor tyrosine kinases in platelets, Syk and Src. In the presence of the cPKC-selective inhibitor Go6976, platelet 5-hydroxytryptamine release was abolished in response to co-activation of glycoproteins VI and Ib-IX-V by the snake venom alboaggregin A, whereas platelet aggregation was substantially inhibited. Of the two platelet cPKCs, PKCalpha but not PKCbeta was activated, occurring in an Syk- and phospholipase C-dependent manner. Syk and PKCalpha associate in a stimulation-dependent manner, requiring Syk but not PKC activity. PKCalpha and Syk also co-translocate from the cytosol to the plasma membrane upon platelet activation, in a manner dependent upon the activities of both kinases. Although PKCalpha is phosphorylated on tyrosine downstream of Syk, we provide evidence against phosphorylation of Syk by PKCalpha, consistent with a lack of effect of PKCalpha inhibition on Syk activity. PKCalpha also associates with Src; although in contrast to interaction with Syk, PKCalpha activity is required for the association of these kinases but not the stimulation-induced translocation of Src to the cell membrane. Finally, the activity of Src is negatively regulated by PKC, as shown by potentiation of Src activity in the presence of the PKC inhibitors GF109203X or Go6976. Therefore, there is a complex interplay between PKCalpha, Syk, and Src involving physical interaction, phosphorylation, translocation within the cell, and functional activity regulation.     

Identification of calpastatin and mu-calpain and studies of their association in pulmonary smooth muscle mitochondria

Using calpastatin antibody we have identified a 145 kDa major band along with two relatively minor bands at 120 kDa and 110 kDa calpastatin molecules in bovine pulmonary artery smooth muscle mitochondria. To the best of our knowledge this is first report regarding the identification of calpastatin in mitochondria. We also demonstrated the presence of micro-calpain in the mitochondria by immunoblot and casein zymogram studies. Immunoblot studies identified two major bands corresponding to the 80 kDa large and the 28 kDa small subunit of mu-calpain. Additionally 76 kDa, 40 kDa and 18 kDa immunoreactive bands have also been detected. Purification and N-terminal amino acid sequence analysis of the identified proteins confirmed their identity as mu-calpain and calpastatins. Immunoprecipitation study revealed molecular association between mu-calpain and calpastatin in the mitochondria indicating that calpastatin could play an important role in preventing uncontrolled activity of mu-calpain which otherwise may facilitate pulmonary hypertension, smooth muscle proliferation and apoptosis.     

In skeletal muscle advanced glycation end products (AGEs) inhibit insulin action and induce the formation of multimolecular complexes including the receptor for AGEs

Chronic hyperglycemia promotes insulin resistance at least in part by increasing the formation of advanced glycation end products (AGEs). We have previously shown that in L6 myotubes human glycated albumin (HGA) induces insulin resistance by activating protein kinase Calpha (PKCalpha). Here we show that HGA-induced PKCalpha activation is mediated by Src. Coprecipitation experiments showed that Src interacts with both the receptor for AGE (RAGE) and PKCalpha in HGA-treated L6 cells. A direct interaction of PKCalpha with Src and insulin receptor substrate-1 (IRS-1) has also been detected. In addition, silencing of IRS-1 expression abolished HGA-induced RAGE-PKCalpha co-precipitation. AGEs were able to induce insulin resistance also in vivo, as insulin tolerance tests revealed a significant impairment of insulin sensitivity in C57/BL6 mice fed a high AGEs diet (HAD). In tibialis muscle of HAD-fed mice, insulin-induced glucose uptake and protein kinase B phosphorylation were reduced. This was paralleled by a 2.5-fold increase in PKCalpha activity. Similarly to in vitro observations, Src phosphorylation was increased in tibialis muscle of HAD-fed mice, and co-precipitation experiments showed that Src interacts with both RAGE and PKCalpha. These results indicate that AGEs impairment of insulin action in the muscle might be mediated by the formation of a multimolecular complex including RAGE/IRS-1/Src and PKCalpha.     

Protein kinase Calpha regulates insulin receptor signaling in skeletal muscle

Certain PKC isoforms are stimulated by insulin and interact with IR as well as with IRS, but it is still not clear if specific PKC isoforms regulate IR signaling directly or through IRS-1. PKCalpha may regulate IRS activity in response to insulin. We investigated the possibility that PKCalpha may be important in insulin signaling. Studies were conducted on skeletal muscle in adult mice and on L6 skeletal cells. PKCalpha is constitutively associated with IRS-1, and insulin stimulation of PKCalpha causes disassociation of the two proteins within 5 min. Blockade of PKCalpha inhibited insulin-induced disassociation of PKCalpha from IRS1. Selective inhibition of PKCalpha increased the ability of insulin to reduce blood glucose levels. Insulin stimulation activates PKB and increases the association of PKCalpha with PKB. Blockade of PKCalpha increased threonine phosphorylation of PKB. We suggest that PKCalpha regulates insulin signaling in skeletal muscle through its disassociation from IRS-1 and association with PKB.     

Identification of glycogen phosphorylase and creatine kinase as calpain substrates in skeletal muscle

The goal of this study was to identify calpain substrates in muscle cells. Our hypothesis was that the yeast two-hybrid method could be used to identify novel calpain substrates. To accomplish this, native mu- and m-calpains, as well as a variety of calpain DNA fragments, were expressed in yeast cells and used to screen for binding proteins in a human skeletal muscle cDNA library. Calpain constructs that were used in the screening process included native mu- and m-calpains, a dominant negative (DN) m-calpain (i.e. active site modified), N-terminal truncated DN m-calpain (i.e. autolyzed DN-m-calpain) and, finally, an N- and C-terminal truncated m-calpain (i.e. autolyzed DN-m-calpain lacking a calcium-binding domain). Yeast cells were transformed using yeast two-hybrid expression vectors containing the different calpain constructs as "baits". Beta-galactosidase activity was assayed as an index of interaction between calpain and its potential target proteins. From this analysis, four clones (Ca2+-ATPase, novel nebulin-related protein (N-RAP), creatine kinase and glycogen phosphorylase) were recovered. Two of these, creatine kinase and glycogen phosphorylase, were selected for further study. In in-vitro assays, calpain was able to partially digest both proteins, suggesting that both creatine kinase and glycogen phosphorylase are natural calpain substrates.