A cyclic AMP-dependent pathway regulates the expression of acetylcholinesterase during myogenic differentiation of C2C12 cells

The expression of acetylcholinesterase (AChE) is markedly increased during myogenic differentiation of C2C12 myoblasts to myotubes; the expression is mediated by intrinsic factor(s) during muscle differentiation. In order to analyze the molecular mechanisms regulating AChE expression during myogenic differentiation, a approximately 2.2-kb human AChE promoter tagged with a luciferase reporter gene, namely pAChE-Luc, was stably transfected into C2C12 cells. The profile of promoter-driven luciferase activity during myogenic differentiation of C2C12 myotubes was found to be similar to that of endogenous expression of AChE catalytic subunit. The increase of AChE expression was reciprocally regulated by a cAMP-dependent signaling pathway. The level of intracellular cAMP, the activity of cAMP-dependent protein kinase, the phosphorylation of cAMP-responsive element binding protein and the activity of cAMP- responsive element (CRE) were down-regulated during the myotube formation. Mutating the CRE site of human AChE promoter altered the original myogenic profile of the promoter activity and its suppressive response to cAMP. In addition, the suppressive effect of the CRE site is dependent on its location on the promoter. Therefore, our results suggest that a cAMP-dependent signaling pathway serves as a suppressive element in regulating the expression of AChE during early myogenesis.     

Affinity purification of the C alpha and C beta isoforms of the catalytic subunit of cAMP-dependent protein kinase

A synthetic peptide of 18 amino acids corresponding to the inhibitory domain of the heat-stable protein kinase inhibitor was synthesized and shown to inhibit both the C alpha and C beta isoforms of the catalytic (C) subunit of cAMP-dependent protein kinase. Extracts from cells transfected with expression vectors coding for the C alpha or the C beta isoform of the C subunit required 200 nM protein kinase inhibitor peptide for half-maximal inhibition of kinase activity in extracts from these cells. An affinity column was constructed using this synthetic peptide, and the column was incubated with protein extracts from cells overexpressing C alpha or C beta. Elution of the affinity column with arginine allowed single step isolation of purified C alpha and C beta subunits. The C alpha and C beta proteins were enriched 200-400-fold from cellular extracts by this single step of affinity chromatography. No residual inhibitory peptide activity could be detected in the purified protein. The purified C subunit isoforms were used to demonstrate preferential antibody reactivity with the C alpha isoform by Western blot analysis. Furthermore, preliminary characterization showed both isoforms have similar apparent Km values for ATP (4 microM) and for Kemptide (5.6 microM). These results demonstrate that a combination of affinity chromatography employing peptides derived from the heat-stable protein kinase inhibitor protein and the use of cells overexpressing C subunit related proteins may be an effective means for purification and characterization of the C subunit isoforms. Furthermore, this method of purification may be applicable to other kinases which are known to be specifically inhibited by small peptides.     

Regulation of gene expression by transfected subunits of cAMP-dependent protein kinase

cAMP regulates the expression of several genes by activation of a promoter consensus sequence which functions as a cAMP-response element. Evidence indicated that this is accomplished via cAMP dissociation of cAMP-dependent protein kinase into its regulatory (R) and catalytic (C) subunits. Our investigations of the role of these two subunits in gene expression provide direct and quantitative evidence that the C subunit is required for cAMP stimulation of the cAMP-response element in the vasoactive-intestinal-peptide gene in rat pheochromocytoma cells. After cotransfection of a metallothionein-regulated C-subunit expression vector (pCEV) and a vasoactive-intestinal-peptide--chloramphenicol acetyltransferase construct containing a cAMP-response element, we could demonstrate expression of transfected C-alpha-subunit mRNA (truncated size 1.7 kb) by Northern blot and a concentration-dependent C subunit stimulation of chloramphenicol acetyltransferase activity. Basal activity was stimulated 12- and 50-fold by pCEV (30 micrograms), in the absence and presence, respectively, of Zn2+. Metallothionein-regulated expression of C was demonstrated by results that showed a 2-4-fold increase in chloramphenicol acetyltransferase activity in the presence versus the absence of 90 microM Zn2+. In contrast, overexpression of the R-II beta regulatory subunit did not stimulate chloramphenicol acetyltransferase activity, and R-II beta transfected together with C (ratio 2:1 and 4:1) inhibited the stimulation by the C subunit 70% and 90% respectively. Our results indicate that transfection of cAMP-dependent protein kinase subunits results in functional expression of both C-alpha and R-II beta subunits. Expression of the C subunit mediated cAMP-regulated gene expression but this expression could be inhibited by cotransfected R-II beta subunit, indicating intracellular reconstitution of the inactive holoenzyme of cAMP-dependent protein kinase.     

Calmodulin inhibits the phosphorylation of spectrin in vitro

In vitro phosphorylation of purified spectrin dimer was studied in the presence of Ca2+-calmodulin (CaM). CaM inhibited autophosphorylation of the beta subunit of spectrin. The inhibitory effect (65% at a 32-fold molar excess) appeared to be due to a weak interaction of CaM with spectrin. CaM was similarly effective in a phosphatase-stimulated autothiophosphorylation of the beta subunit with [gamma-35S]ATP. Hence, its inhibitory effect was not due to stimulation of a spectrin-associated phosphatase activity. Phosphorylation of spectrin by the catalytic subunit of a cAMP-dependent protein kinase occurred in both subunits (1984, FEBS Lett. 169, 323). CaM selectively inhibited a cAMP-dependent phosphorylation of the alpha subunit of spectrin to 30% at two CaM per spectrin. It was ineffective on the cAMP-dependent phosphorylation of the beta subunit up to a 32-fold molar excess. These results yield functional evidence for a CaM-spectrin interaction. They further suggest that CaM can regulate the extent of a cAMP-dependent phosphorylation of the alpha subunit of spectrin.     

Decreased catalytic subunit mRNA levels and altered catalytic subunit mRNA structure in a cAMP-resistant Chinese hamster ovary cell line.

The mechanisms responsible for decreased levels of cAMP-dependent protein kinase activity in a mutant Chinese hamster ovary cell line have been examined. The cAMP-resistant Chinese hamster ovary 10260 cell line was found to possess only 20% of the cAMP-dependent protein kinase activity found in wild-type cells. The presence of decreased concentrations of the catalytic subunit in these cells was confirmed through binding studies using a radiolabeled, heat-stable inhibitor of the kinase. Cloned Chinese hamster ovary catalytic subunit cDNAs were isolated, characterized, and used as hybridization probes to examine the relative concentrations of catalytic subunit mRNAs in the wild-type and 10260 cell lines. A 40-50% decrease in the concentration of the mRNA for the C alpha isozyme of the catalytic subunit was observed in 10260 cells, as compared with wild-type. This decrease in catalytic subunit mRNA concentration probably accounts for a portion of the decreased kinase activity in the mutant cells. Further analysis of C alpha mRNA by polymerase chain reaction confirmed the decreased expression of C alpha mRNA in 10260 cells and further demonstrated the presence of two different species of C alpha mRNA in the 10260 cells. One species of C alpha cDNAs was indistinguishable from the wild-type cDNA, but the other species was shorter. Nucleotide sequence analysis of the amplified cDNAs led to the identification of a 191-base pair deletion in the shorter cDNA. Gene transfer studies using wild-type and 10260 C alpha cDNAs demonstrated that the longer cDNA from the 10260 cells produced wild-type activity, but the shorter cDNA was inactive. These studies suggest that at least two alterations in gene expression are responsible for decreased cAMP-dependent protein kinase activity in the 10260 cell line. One alteration results in an approximately 2-fold decrease in the concentrations of C alpha mRNA in the cells. The other change produces two species of C alpha mRNA; one of the C alpha mRNAs does not encode an active kinase.     

Enhanced casein kinase II activity in COS-1 cells upon overexpression of either its catalytic or noncatalytic subunit.

Casein kinase II consists of catalytic (alpha) and regulatory (beta) subunits complexed into a heterotetrameric alpha 2 beta 2 structure. Full-length cDNAs encoding the alpha and beta subunits of human casein kinase II were subcloned into an expression vector containing the cytomegalovirus promotor, yielding the expression constructs pCMV-alpha and pCMV-beta. Northern analyses of total cellular RNA prepared from COS-1 fibroblasts 65 h after transfection with pCMV-alpha or pCMV-beta or with both expression constructs showed marked specific increases in corresponding alpha and beta subunit RNAs. Immunoblot analysis utilizing anti-casein kinase II antiserum of cytosolic extracts prepared from COS-1 cells co-transfected with pCMV-alpha and pCMV-beta showed 2- and 4-fold increases in immunoreactive alpha and beta subunit protein, respectively, relative to vector-transfected cells. These same cytosolic fractions exhibited an average 5-fold increase in casein kinase II catalytic activity. COS-1 cells transfected with pCMV-alpha alone exhibited a 3-fold increase in immunoreactive alpha subunit protein and a nearly 2-fold increase in cytosolic casein kinase II catalytic activity. Transfection with the cDNA coding for the noncatalytic beta subunit alone also caused a near doubling of cytosolic casein kinase II catalytic activity. No increase in immunoreactive alpha subunit protein was observed in pCMV-beta-transfected cells, and no increase in immunoreactive beta subunit protein was observed in pCMV-alpha-transfected cells. These results indicate that a portion of the endogenous cellular casein kinase II protein is not fully active and that raising the concentration of the alpha or beta subunit stimulates this latent activity.     

The S49 Kin- cell line transcribes and translates a functional mRNA coding for the catalytic subunit of cAMP-dependent protein kinase

The S49 mouse lymphoma mutant cell line Kin- is resistant to the cytotoxic effects of elevated cAMP levels, has no detectable cAMP-dependent protein kinase activity, and has depressed levels of cAMP-binding regulatory subunits. We demonstrate that although the Kin- cell line lacks detectable catalytic subunit protein, these cells express wild-type levels of mRNA for both C alpha and C beta catalytic subunit isoforms. Translation of C alpha mRNA appears to be normal in the Kin- cell, based on the observation that C alpha mRNA associates with large polyribosomes in both wild-type and Kin- cells. We cloned the C alpha cDNA from Kin- cells and show that its transient expression in another cell type leads to activation of a cAMP-sensitive luciferase reporter gene, suggesting that functional C alpha protein is made. In addition to having catalytic activity, the C alpha subunit from Kin- cells is inhibited in the presence of mouse RI alpha regulatory subunit, indicating that formation of the holoenzyme complex is normal. We suggest that the mutation responsible for the Kin- phenotype is in a cellular component that directly or indirectly causes Kin- catalytic subunit protein to be degraded rapidly.     

Subunit phosphorylation and activation of skeletal muscle phosphorylase kinase by the cAMP-dependent protein kinase. Divalent metal ion, ATP, and protein concentration dependence

This report provides a characterization of the effects of varying the concentrations of Mg2+, ATP, phosphorylase kinase, and the cAMP-dependent protein kinase on the activation and phosphorylation of phosphorylase kinase. The results show the following. (a) The Km for MgATP2- for the cAMP-dependent protein kinase-catalyzed phosphorylation is decreased by increasing Mg2+, probably as a consequence of decreasing the free ATP:MgATP2- ratio and increasing free Mg2+. (b) Whereas beta subunit phosphorylation of phosphorylase kinase plays a prominent role in determining its activity, alpha subunit phosphorylation can also modulate activity. (c) The phosphorylation of the alpha subunit, which occurs following the initial cAMP-dependent phosphorylation of the beta subunit, is catalyzed by the cAMP-dependent protein kinase and is not a consequence of EGTA-insensitive (or EGTA-sensitive) autophosphorylation occurring as a result of the enhanced phosphorylase kinase activity. (d) The relationship between subunit phosphorylation and phosphorylase kinase activation is complex and particularly dependent upon concentrations of cAMP-dependent protein kinase and phosphorylase kinase in the activation reaction. The data suggest the possibilities that the pathway of phospho-intermediates involved in the activation process probably varies with the activation conditions, that the efficacy of a specific site to be covalently modified is dependent upon the phosphorylation status of other sites, and that the effect of phosphorylation in regulating activity may also be dependent on the phosphorylation status of other sites. It is clear from the data that the activation process for phosphorylase kinase can be very complex, and it is possible that this complexity might have significant physiological ramifications.     

Phosphorylation of smooth muscle myosin light chain kinase by protein kinase C. Comparative study of the phosphorylated sites

Smooth muscle myosin light chain kinase is phosphorylated in vitro by protein kinase C purified from human platelets. When myosin light chain kinase which has calmodulin bound is phosphorylated by protein kinase C, 0.8-1.1 mol of phosphate is incorporated per mol of myosin light chain kinase with no effect on its enzyme activity. Phosphorylation of myosin light chain kinase with no calmodulin bound results in the incorporation of 2-2.4 mol of phosphate and significantly decreases the rate of myosin light chain kinase activity. The decrease in myosin light chain kinase activity is due to a 3.3-fold increase in the concentration of calmodulin necessary for the half-maximal activation of myosin light chain kinase. The sites phosphorylated by protein kinase C and the catalytic subunit of cAMP-dependent protein kinase were compared by two-dimensional peptide mapping following extensive tryptic digestion of phosphorylated myosin light chain kinase. The single site phosphorylated by protein kinase C when calmodulin is bound to myosin light chain kinase (site 3) is different from that phosphorylated by the catalytic subunit of cAMP-dependent protein kinase (site 1). The additional site that is phosphorylated by protein kinase C when calmodulin is not bound appears to be the same site phosphorylated by the catalytic subunit of cAMP-dependent protein kinase (site 2). These studies confirm the important role of site 2 in binding calmodulin to myosin light chain kinase. Sequential studies using both protein kinase C and the catalytic subunit of cAMP-dependent protein kinase suggest that the phosphorylation of site 1 also plays a part in decreasing the affinity of myosin light chain kinase for calmodulin.     

Cytochemical identification of the regulatory subunit of the cAMP-dependent protein kinase by use of fluorescently labeled catalytic subunit. Examination of protein kinase dissociation in hepatoma cells responding to 8-Br-cAMP stimulation

Homogeneous catalytic subunit from the cAMP-dependent protein kinase, when derivatized with a fluorophore, was used as a cytochemical probe to locate intracellular sites of the protein kinase regulatory subunit. After conjugation, the fluoresceinated catalytic subunit (F:C), derivatized to a stoichiometry of approximately 1 mol/mol, retained near full activity as judged by specific activity and by titration against either regulatory subunit or Inhibitor Protein of the protein kinase. With this molecular probe the dissociated regulatory subunit was localized by direct cytochemistry in Reuber H-35 hepatoma cells that had been exposed, while intact, for 0-120 min to 10(-4) M 8-Br-cAMP. After stimulation, cultures were fixed and washed and then incubated for 16 h with F:C. Following 8-Br-cAMP stimulation, extensive binding of the probe to both cytoplasmic and nucleolar sites was observed. This binding was diminished but not eliminated when 50 microM cAMP was present during the incubation of the fixed cells with F:C that was eliminated by a 40-fold molar excess of underivatized catalytic subunit but not by heat-denatured catalytic subunit, and was not reduced by a 20-fold molar excess of cGMP-dependent protein kinase, examined plus or minus cGMP. Collectively, the results allow the conclusion that the F:C probe binds free regulatory subunit. The time course of its change with 8-Br-cAMP (measured as the difference between binding in the presence or absence of cAMP during the postfixation treatment) mirrors that previously reported for changes in the catalytic subunit in these cells, also identified cytochemically (Byus, C. V., and Fletcher, W.H. (1982) J. Cell Biol. 93, 727-734). The binding of the F:C probe, detected when cAMP is present during postfixation treatment, may possibly represent binding to free Inhibitor Protein of the cAMP-dependent protein kinase. If so, it was at a level of approximately 20% of the maximal level of detectable regulatory subunit, and it also showed cytosolic and nucleolar localization.