Inhibitory effect of calcium-binding protein regucalcin on protein kinase activity in the nuclei of regenerating rat liver

The effect of Ca²⁺-binding protein regucalcin on protein kinase activity in the nuclei of normal and regenerating rat livers was investigated. Protein kinase activity in the nuclei isolated from normal rat liver was significantly increased by addition of Ca²⁺ (500 μM) and calmodulin (10 μg/ml) in the enzyme reaction mixture. Nuclear protein kinase activity was significantly decreased in the presence of EGTA (1.0 mM), trifluoperazine (TFP; 20 μM), dibucaine (10⁻⁴ M), or staurosporine (10⁻⁷ M), indicating that Ca²⁺-dependent protein kinases are present in the nuclei. Protein kinase activity was significantly elevated in the liver nuclei obtained at 6 to 48 h after a partial hepatectomy. Hepatectomy-increased nuclear protein kinase activity was significantly decreased in the presence of EGTA (1.0 mM), TFP (20 μM), or staurosporine (10⁻⁷ M) in the enzyme reaction mixture. The presence of regucalcin (0.1–0.5 μM) caused a significant decrease in protein kinase activity in the nuclei obtained from normal and regenerating rat livers. Meanwhile, the nuclear protein kinase activity from normal and regenerating livers was significantly elevated in the presence of anti-regucalcin monoclonal antibody (50–200 ng/ml). The present study suggests that regucalcin plays a role in the regulation of protein kinase activity in the nuclei of proliferative liver cells. J. Cell. Biochem. 71:569–576, 1998. © 1998 Wiley-Liss, Inc.     

Inhibitory effect of calcium-binding protein regucalcin on Ca2+/calmodulin-dependent cyclic nucleotide phosphodiesterase activity in rat liver cytosol

The effect of regucalcin, a calcium-binding protein isolated from rat liver cytosol, on Ca²⁺/calmodulin-dependent cyclic nucleotide (AMP) phosphodiesterase activity in rat liver cytosol was investigated. The addition of Ca²⁺ (50 µM) and calmodulin 160 U/ml in the enzyme reaction mixture caused a significant increase in cyclic AMP phosphodiesterase activity. This increase was inhibited by the presence of regucalcin (0.5-3.0 µM); the inhibitory effect was complete at 1.0 µM. Regucalcin (1.0 µM) did not have an appreciable effect on basal activity without Ca²⁺ and calmodulin. The inhibitory effect of regucalcin was still evident even at several fold higher concentrations of calmodulin (160–480 U/ml). However, regucalcin (1.0 µM) did not inhibit Ca²⁺/calmodulin-dependent cyclic AMP phosphodiesterase activity in the presence of 100 and 200 µM Ca²⁺ added. Meanwhile, Cd² (25–100 µM)-induced decrease in Ca²⁺/calmodulin-dependent cyclic AMP phosphodiesterase activity was not reversed by the presence of regucalcin (1.0 µM). The present results suggest that regucalcin can regulate Ca²⁺/calmodulin-dependent cyclic AMP phosphodiesterase activity due to binding Ca²⁺ in liver cells.     

Inhibitory effect of regucalcin on Ca2+/calmodulin-dependent protein kinase activity in rat renal cortex cytosol

The effect of regucalcin on Ca²⁺/calmodulin-dependent protein kinase activity in the cytosol of rat renal cortex was investigated. Regucalcin is a calcium-binding protein which exists in rat liver and renal cortex. Protein kinase activity in renal cortex cytosol was markedly increased by the addition of CaCl₂ (0.5 mM) plus calmodulin (10 µg/ml) in the enzyme reaction mixture. This increase was completely prevented by the addition of trifluoperazine (25 µM), an antagonist of calmodulin. The cytosolic Ca²⁺/calmodulin- dependent protein kinase activity was clearly inhibited by the addition of regucalcin; an appreciable effect of regucalcin was seen at 0.01 µM. The cytosolic Ca²⁺/calmodulin-dependent protein kinase activity was fairly increased by increasing concentrations of added Ca²⁺ (100-1000 µM). This increase was markedly blocked by the presence of regucalcin (0.1 µM). The inhibitory effect of regucalcin on the protein kinase activity was also seen with varying concentrations of calmodulin (2-20 µg/ml). These results demonstrate that regucalcin can regulate Ca²⁺/calmodulin-dependent protein kinase activity in renal cortex cells.     

Inhibitory effect of calcium-binding protein regucalcin on Ca2(+)-activated DNA fragmentation in rat liver nuclei

Incubation of isolated rat liver nuclei with ATP, NAD+, and micromolar Ca2+ concentrations of various metal ions resulted in extensive DNA hydrolysis. Half-maximal activity occurred with 1.0 microM Ca2+ added, and saturation of the process was observed with 10 microM Ca2+. The Ca2+ (10 microM)-activated DNA fragmentation was inhibited by the presence of Ca2(+)-binding protein regucalcin isolated from rat liver cytosol. The inhibitory effect of regucalcin was complete at 0.5 microM. At 25 microM Ca2+ added, such an effect of regucalcin (1.0 microM) was not seen. Regucalcin also inhibited Ca2(+)-activated DNA fragmentation in the presence of calmodulin (10 and 20 micrograms). The results show that regucalcin can inhibit the Ca2(+)-activated DNA fragmentation due to binding the metal, suggesting a role in regulation of liver nuclear functions.     

Calcium-binding protein regucalcin increases calcium-independent proteolytic activity in rat liver cytosol

The effect of regucalcin, isolated from rat liver cytosol, on neutral proteolytic activity in the hepatic cytosol was investigated. The Ca²⁺-requiring proteinase required 5–10 µM Ca²⁺ for maximal activity in the presence of a protein substrate (globin). The proteinase activity was markedly elevated by the addition of regucalcin (0.25–2.0 µM) in the absence or presence of Ca²⁺ (5.0 µM) added. The effect of regucalcin, however, was the greater in the absence of Ca²⁺ than that in the presence. The pronounced effect of regucalcin on the proteinase activity was also seen in the presence of 1.0 mM EGTA with or without Ca²⁺ (5.0 µM). In the absence of Ca²⁺, the regucalcin-increased proteinase activity was clearly inhibited by the presence of anti-regucalcin antiserum (diluted to 240-fold), leupeptin (20 and 200 µg/ml), and heavy metals (25 µM cadmium or 25 µM zinc), although the inhibition was not complete at the concentration used. The present findings suggest that regucalcin increases proteolytic activity in rat liver cytosol, and that regucalcin may activate Ca²⁺-independent neutral cysteinyl-proteinase.     

Characterization of regucalcin effect on proteolytic activity in rat liver cytosol: relation to cysteinyl-proteases

The increasing effect of regucalcin, isolated from rat liver cytosol, on neutral proteolytic activity in the hepatic cytosol was characterized. The proteolytic activity was markedly elevated by the addition of regucalcin (0.1–0.5 M) in the absence of Ca²⁺. This increase was not significantly altered by the presence of diisopropylfluorophsophate (DPF;2.5 mM)—although DFP caused a significant decrease in the proteolytic activity. Regucalcin (0.25 M) additively enhanced the dithiothreitol (DTT; 1.0 mM)—increased proteolytic activity, while the regucalcin or DTT effect was completely abolished by NEM (5 mM), indicating that regucalcin may act on the SH group in proteases. Also, regucalcin (0.25 M) enhanced the effect of Ca²⁺ (10 M) increasing liver proteolytic activity, suggesting that regucalcin does not influence on the active sites for Ca²⁺ in proteases. Moreover, the proteolytic activity of regucalcin (0.25 M) was significantly decreased by the presence of calpastatin (24 g/ml), an inhibitor of Ca²⁺-activated neutral protease (calpain). Now, regucalcin (0.25 M) increased about 7-fold the activity ofm-calpain isolated from rabbit skeletal muscle. These observations demonstrate that regucalcin directly activates cysteinyl-proteases. Regucalcin may have a role as a potent proteolytic activator in the cytoplasm of liver cells.     

Inhibitory effect of regucalcin on Ca2+/calmodulin-dependent cyclic AMP phosphodiesterase activity in rat kidney cytosol

The effect of regucalcin, a novel Ca²⁺-binding protein, on Ca²⁺/ calmodulin-dependent cyclic adenosine monophosphate (AMP) phosphodiesterase activity in the cytosol of rat renal cortex was investigated. Regucalcin with physiologic concentration (10^-7 M) in rat kidney had no effect on cyclic AMP phosphodiesterase activity in the absence of CaCl₂ and calmodulin. However, the activatory effect of both CaCl₂ (10 µM) and calmodulin (20 U/ml) on cyclic AMP phosphodiesterase was markedly inhibited by the addition of regucalcin (10^-8 to 10^-6 M) in the enzyme reaction mixture. The inhibitory effect of regucalcin on the enzyme activity was also seen in the presence of CaCl₂ (5-50 µM) or calmodulin (5-50 U/ml) with increasing concentrations. The presence of trifluoperazine (10 µM), an antagonist of calmodulin, caused a partial inhibition of Ca²⁺ /calmodulin-dependent cyclic AMP phosphodiesterase activity. This inhibition was further enhanced by the addition of regucalcin (10^-7 M). The inhibitory effect of regucalcin (10^-7 M) was not seen in the presence of 20 µM trifluoperazine. Moreover, the activatory effect of calmodulin (20 U/ml) on cyclic AMP phosphodiesterase was not entirely seen, when calmodulin was added 10 min after incubation in the presence of CaCl₂ (10 µM) and regucalcin (10^-7 M). The present results demonstrates that regucalcin has an inhibitory effect on Ca²⁺ /calmodulin-dependent cyclic AMP phosphodiesterase activation in the cytosol of rat renal cortex.     

Inhibitory effect of regucalcin on protein phosphatase activity in the nuclei of rat kidney cortex

The role of regucalcin, which is a regulatory protein of calcium signaling, in the regulation of protein phosphatase activity in the nuclei of rat kidney cortex was investigated. Protein phosphatase activity towards phosphotyrosine, phosphoserine, and phosphothreonine was found in the nuclei. The enzyme activity towards three phosphoamino acids was significantly increased by the addition of calcium chloride (10-50 microM) in the enzyme reaction mixture. This increase was significantly inhibited by trifluoperazine (25 or 50 microM), an antagonist of calmodulin. The presence of regucalcin (50 or 100 nM) in the enzyme reaction mixture caused a significant decrease in protein phosphatase activity towards three phosphoamino acids. This effect was also seen in the presence of calcium (25 microM) and/or calmodulin (5 microg/ml). Protein phosphatase activity towards three phosphoamino acids was significantly increased in the presence of anti-regucalcin monoclonal antibody (25 or 50 ng/ml) in the enzyme reaction mixture. This effect was completely blocked by the addition of regucalcin (100 nM). The effect of antibody (25 ng/ml) in increasing protein phosphatase activity towards phosphotyrosine was significantly inhibited by vanadate (10(-4) M). Also, the antibody's effect towards phosphoserine and phosphothreonine was significantly inhibited by cyclosporin A (10(-5) M). Endogenous regucalcin was found in the nuclei of rat kidney cortex using Western blot analysis. Nuclear regucalcin level was significantly reduced by the administration of saline (0.9% NaCl) for seven days in rats. Protein phosphatase activity towards three phosphoamino acids was significantly decreased by saline administration. The effect of anti-regucalcin monoclonal antibody (25 ng/ml) in increasing protein phosphatase activity towards three phosphoamino acids was weakened in the renal cortex nuclei of saline-administrated rats. The present study demonstrates that endogenous regucalcin plays a suppressive role in the regulation of protein phosphatase activity in the nuclei of rat kidney cortex cells.     

Expression of calcium-binding protein regucalcin mRNA in fetal rat liver is stimulated by calcium administration

The expression of hepatic calcium-binding protein regucalcin mRNA in fetal rats was investigated. The alteration in regucalcin mRNA levels was analyzed by Northern blotting using liver regucalcin cDNA (0.9 kb with complete open reading frame). Hepatic regucalcin mRNA levels were progressively increased with fetal development; the mRNA was clearly expressed at 15 and 21 days of pregnancy but only slightly at the 8 days. Meanwhile, -actin mRNA levels in the fetal liver were remarkable at 8 and 15 days of pregnancy. The fetal liver regucalcin mRNA levels at 15 days of pregnancy were significantly decreased by overnight-fasting of maternal rats. The oral administration of calcium chloride (50 mg Ca/100 g body weight) to maternal rats at 15 days of pregnancy caused a remarkable elevation (about 2 fold) of regucalcin mRNA levels in the fetal liver; this increase was seen 60 and 180 min after the calcium administration. After birth, regucalcin mRNA was increasingly expressed in the livers of newborn and weanling rats, while hepatic -actin mRNA expression was not appreciably altered with increasing ages. These findings demonstrate that the expression of hepatic regucalcin mRNA is increased with fetal development, and that the gene expression may be stimulated by the ingestion of dietary calcium.     

Activatory effect of regucalcin on GTPase activity in rat liver plasma membranes

The effect of regucalcin, a regulatory protein of Ca²⁺ signaling, on guanosine-5-triphosphatase (GTPase) activity in isolated rat liver plasma membranes was investigated. GTPase activity was significantly increased by the addition of Ca²⁺ (25–100 M) in the enzyme reaction mixture. Such an increase was not seen by other metals (Mg, Co, Zn, Cu, Ni, and Mn) with 50 M. The activatory effect of calcium (50 M) was significantly decreased by calmodulin (2.5 and 5 g/ml), indicating that it does not depend on calmodulin. The presence of regucalcin (0.1–0.5 M) in the enzyme reaction mixture caused a significant increase in GTPase activity. This increase was not significantly enhanced by calcium (50 M). GTPase activity was significantly increased by dithiothreitol (DTT; 5 mM), a protecting reagent of thiol (SH)-groups, while it was decreased by N-ethylmaleimide (NEM; 5 mM), a modifying reagent of SH-groups. The effect of calcium or regucalcin in increasing GTPase activity was not seen in the presence of NEM. Also, the activatory effect of calcium or regucalcin on GTPase was not seen in the presence of vanadate, an inhibitor of protein phosphorylation, which could inhibit GTPase activity. Moreover, the effect of regucalcin was not seen in the presence of digitonin (0.01%), a solubilizing reagent of membranous lipids, while the effect of calcium was not inhibited by digitonin. The present study demonstrates that regucalcin has an activatory effect on GTPase activity independently of Ca²⁺ in rat liver plasma membranes.     

Effect of anti-regucalcin antibody on neutral phosphatase activity in rat liver cytosol: Involvement of endogenous regucalcin

The effect of anti-regucalcin monoclonal antibody on neutral phoshatase activity in rat liver cytosol was investigated. Phosphotyrosine, phosphoserine, and phosphothreonine were used as the substrate toward phosphatase asssy. Liver cytosolic phosphatase activity with three phosphoaminoacids was significantly increased in the presence of anti-regucalcin antibody (100 and 200 ng/ml) in the enzyme reaction mixture with calcium chloride (0.1 mM) or EGTA (1.0 mM). The effect of anti-regucalcin antibody was completely abolished in the presence of exogenous regucalcin (1.0 M), indicating the involvement of endogenous regucalcin. The anti-regucalcin anti body- increased phosphatase activity was not significantly altered in the presence of trifluoperazine (20 M), an antagonist of calmodulin, or akadaic acid (10 M), an inhibitor of protein phosphatase, although these inihibitors caused a slight decrease in liver cytosolic phosphatase activity. The effect of endogenous regucalcin might be not related to calmodulin, and it was insensitive to okadaic acid. The present findings suggest that endogenous regucalcin is involved in the regulation of protein phasphatase in rat liver cytoplasm.     

Calcium administration increases calcium-binding protein regucalcin concentration in the liver of rats

The alteration of regucalcin concentrations in the liver and serum of rats administered orally calcium is investigated. Rats received a single oral administration of calcium chloride solution (25, 50 and 75 mg Ca/100 g body weight). The administration of calcium (50 mg/100 g) produced a significant increase in liver regucalcin concentration between 30 and 180 min after the administration, while serum regucalcin concentration was not altered appreciably. The effect of calcium administration increasing liver regucalcin concentration was also seen with the dose of 25 mg/100 g. When liver cytosol prepared from normal rats was incubated for 6 h in the presence of 10 M Ca²⁺, the cytosolic regucalcin concentration at 3 and 6 h of incubation was decreased about 20% (p<0.05) as compared with the value at zero time point, indicating that the presence of Ca²⁺ does not inhibit the decomposition of liver cytosolic regucalcin. Moreover, serum regucalcin concentration was not significantly altered by the incubation for 6 h at 37°C, indicating a stability of regucalcin in rat serum. This suggests that the calcium administration-induced in liver regucalcin concentration is not based on the inhibition of regucalcin release from liver to serum. The present study demonstrates that regucalcin in the liver is clearly increased by calcium administration, presumably due to stimulating the protein synthesis.     

Stimulatory effect of regucalcin on proteolytic activity in rat renal cortex cytosol: Involvement of thiol proteases

The effect of regucalcin, a calcium-binding protein, on neutral proteolytic activity in the cytosol of rat kidney cortex was investigated. Proteolytic activity was significantly increased by the presence of regucalcin (0. 01-0. 25 M) in the enzyme reaction mixture. This increase was not significantly altered by the addition of CaCl2 (0.01 and 1.0 mM) or EGTA (1.0 mM), indicating that the effect of regucalcin was independent on Ca2+. The effect of regucalcin to increase proteolytic activity was completely prevented in the presence of N-ethylmaleimide (5 mM), a modifying reagent of thiol groups. Proteolytic activity was clearly elevated by dithiothreitol (5 mM). This elevation was further enhanced by regucalcin (0.1 M). Meanwhile, the stimulatory effect of regucalcin on proteolytic activity was not significantly altered in the presence of diisopropylfluorophosphate (2.5 mM), an inhibitor of serine proteases. Also, the regucalcin effect was not appreciably changed by the addition of EDTA (2.5 mM), a chelator of metal ions, indicating that it is not involved in metal-related proteases. These results demonstrate that regucalcin can increase proteolytic activity in the cytosol of rat kidney cortex. Regucalcin may activate thiol proteases independent on Ca2+.     

Expression of calcium-binding protein regucalcin mRNA in rat liver is stimulated by calcitonin: The hormonal effect is mediated through calcium

The involvement of a hypocalcemic hormone calcitonin (CT) in the expression of hepatic Ca²⁺-binding protein regucalcin mRNA was investigated. The change of regucalcin mRNA levels was analyzed by Northern blotting using liver regucalcin complementary DNA (0.9 kb). A single oral administration of calcium chloride (100 mg Ca/100 g body weight) to rats induced a remarkable increase in the serum calcium concentration and a corresponding elevation of the liver calcium content during 120 min after the administration. Thyroparathyroidectomy (TPTX) did not cause a significant increase in the liver calcium content after calcium administration. Hepatic regucalcin mRNA level was markedly elevated by calcium administration; the level was about 180% of controls at 60 min after the administration. This increase was completely abolished by TPTX. A single subcutaneous administration of CT (synthetic eel CT; 25–100 MRC mU/100 g) to TPTX rats received oral administration of calcium (100 mg/100 g) produced a remarkable increase in hepatic regucalcin mRNA levels; the level was about 280% of controls with the dose of 25 MRC mU CT/100 g. The present finding suggests that the expression of hepatic mRNA is stimulated by CT, and that the hormonal effect is mediated through Ca²⁺ in rat liver.     

Activatory effect of calcium-binding protein regucalcin on ATP-dependent calcium transport in the basolateral membranes of rat kidney cortex

The effect of regucalcin, a calcium-binding protein, on ATP-dependent Ca2+ transport in the basolateral membranes isolated from rat kidney cortex was investigated. The prepared membranes were in inside-out oriented and membrane vesicles. Ca2+-ATPase activity in the basolateral membranes was progressively elevated by increasing concentrations of regucalcin (10-8 to 10-6 M) in the reaction mixture. This increase was dependent on Ca2+ addition. The activatory effect of regucalcin on the enzyme is inhibited by the presence of digitonin (5 × 10-6%) which can solubilize the membranous lipids. Moreover, the regucalcin effect was clearly abolished by the presence of vanadate (0.1 mM) or N-ethylmaleimide (5.0 mM). However, the effect of calmodulin (6 × 10-7 M) to increase Ca2+-ATPase activity was not significantly inhibited by vanadate or N-ethylmaleimide, indicating that the action mode of regucalcin differs from that of calmodulin. Also, the activatory effect of regucalcin on Ca2+-ATPase was appreciably inhibited by addition of dibutyryl cAMP (10-5 and 10-3 M), while inositol 1,4,5-trisphosphate (10-7 and 10-5 M) had no effect. Dibutyryl cAMP itself did not have an effect on the enzyme activity. Furthermore, the 45Ca2+ uptake by the basolateral membranes was clearly increased by the presence of regucalcin (10-7 and 10-6 M). This increase was completely blocked by the presence of vanadate (0.1 mM), N-ethylmaleimide (5.0 mM) or dibutyryl cAMP (10-4 and 10-3 M) in the reaction mixture. These results clearly demonstrate that regucalcin, which is expressed in rat kidney cortex, can increase Ca2+-ATPase activity and Ca2+ uptake in the basolateral membranes. Regucalcin may play a cell physiologic role as an activator in the ATP-dependent Ca2+ pumps in the basolateral membranes from rat kidney cortex.     

Suppressive role of endogenous regucalcin in the regulation of protein phosphatase activity in rat renal cortex cytosol

The role of endogenous regucalcin, which is a regulatory protein of calcium signaling, in the regulation of protein phosphatase activity in the cytosol of rat renal cortex was investigated. Protein phosphatase activity toward phosphotyrosine, phosphoserine, and phosphothreonine was found in the cytosol of kidney cortex. The addition of regucalcin (50-250 nM) in the enzyme reaction mixture caused a significant decrease in protein phosphatase activity toward three phosphoamino acids. The effect of calcium (25 microM) and calmodulin (2.5 microg/ml) in increasing protein phosphatase activity toward three phosphoamino acids was significantly decreased by the addition of regucalcin (100 nM). Protein phosphatase activity toward three phosphoamino acids was significantly increased in the presence of anti-regucalcin monoclonal antibody (10-50 ng/ml) in the enzyme reaction mixture. The effect of antibody (25 ng/ml) in increasing the enzyme activity was significantly inhibited by cyclosporin A (10(-5) M) or vanadate (10(-5) M). Regucalcin in the kidney cortex cytosol was clearly decreased by the administration of saline (0.9% NaCl) for seven days in rats. Protein phosphatase activity toward three phosphoamino acids was significantly decreased by saline administration. The effect of anti-regucalcin antibody (25 ng/ml) in increasing protein phosphatase activity toward three phosphoamino acids was not seen in the renal cortex cytocol of saline-administered rats. The present study demonstrates that endogenous regucalcin plays a suppressive role in the regulation of protein phosphatase activity in the cytoplasm of rat kidney cortex.     

Effect of calcium-binding protein regucalcin on hepatic protein synthesis: Inhibition of aminoacyl-tRNA synthetase activity

The effect of regucalcin, a calcium-binding protein isolated from rat liver cytosol, onin vitro protein synthesis in the 5500g supernatant fraction of rat liver homogenate was investigated. Addition of Ca²⁺ up to 5.0 M in the reaction mixture caused a significant decrease in protein synthesis. This decrease was saturated at 10 M Ca²⁺. The Ca²⁺ effect was not reversed by the presence of regucalcin (2.0 M); the protein caused a remarkable decrease in hepatic protein synthesis, and it enhanced significantly the Ca^2– effect. Meanwhile, calmodulin (2.5-20 g/ml), a calcium-binding protein, did not have an appreciable effect on the Ca²⁺ (10 M)-induced decrease in hepatic protein synthesis. [³H]Leucyl-tRNA synthetase activity in the 105000g supernatant fraction (cytosol) of liver homogenate was markedly decreased by addition of Ca²⁺ (1.0–50 M). This decrease was not reversed by the presence of regucalcin (2.0 M); the protein (1.0–2.0 M) caused a remarkable decrease in the enzyme activity. The present results suggest that regucalcin can regulate protein synthesis in liver cells.     

Protease-activated protein kinase C in rat liver

In regenerating rat liver, an elevated protein kinase activity was detected which phosphorylated ribosomal protein S6 and histones. The properties of this enzyme were closely similar with those of protease-activated protein kinase C with Mr 45,000. During the study of the mechanism of proteolytic activation, type III protein kinase C (encoding alpha-sequence) was shown to be subjected to limited proteolysis by trypsin-like protease and converted to protein kinase M in ionic strength- and pH-dependent manner. This reaction was stimulated in the presence of Ca2+ and phospholipid under slightly higher ionic strength condition than physiological level (greater than 140 mM NaCl) and alkaline pH (7.5-8.0). These results suggest that activation of Na+/H+ exchanger in plasma membrane may trigger this type of proteolytic activation of protein kinase C. In addition to protein kinase M, another type of protease-activated kinase with Mr 80,000 was detected when limited proteolysis of protein kinase C was performed on inactive form of this enzyme (in the absence of either Ca2+ or phospholipid or both activators) under lower ionic strength condition. The molecular mass of this active enzyme was slightly smaller (approximately 200) than that of native protein kinase C. However, it is not clear at this time whether this small fragment was released from amino-terminal or carboxy-terminal domain to make protein kinase C partially active in the absence of Ca2+ and phospholipid. Although it has been proposed that proteolytic degradation of protein kinase C is involved in down regulation of this enzyme, the physiological significance of these two types of protease-activated forms of protein kinases in liver has remained obscure.     

Regulation of protein phosphatase activity by regucalcin localization in rat liver nuclei.

The regulatory role of regucalcin on protein phosphatase activity in isolated rat liver nuclei was investigated. Phosphatase activity toward phosphotyrosine and phosphoserine was significantly increased by the addition of CaCl(2) (10(-5) and 10(-4) M) in the enzyme reaction mixture. Trifluoperazine (25 and 50 microM), an antagonist of calmodulin, significantly inhibited protein phosphatase activity toward phosphoserine, while it had no effect on the enzyme activity toward phosphotysine and phosphothreonine. Cyclosporin A (10(-6)-10(-4) M), an inhibitor of Ca(2+)/calmodulin-dependent protein phosphatase activity toward phosphoserine, but not phosphotyrosine and phosphoserine. Thus, Ca(2+)/calmodulin-dependent phosphatases were present in liver nuclei. Regucalcin (0.25 and 0.5 microM) had an inhibitory effect on liver nuclear phosphatase activity toward phosphotyrosine, phosphoserine, and phosphothreonine. The presence of anti-regucalcin monoclonal antibody (25 and 50 ng/ml) in the enzyme reaction mixture caused a significant elevation of nuclear phosphatase activity toward three phosphoaminoacids. An analysis with sodium sulfate-polyacrylamide gel electrophoresis suggested a possibility of localization of regucalcin in liver nuclei. Moreover, regucalcin was determined in liver nuclei using enzyme-linked immunoadsorbent assay. The present study demonstrates that the endogenous regucalcin inhibits phosphatase activity in the liver nuclei.