Cyclic-AMP-dependent phosphorylation of voltage-sensitive sodium channels in primary cultures of rat brain neurons

We have studied cAMP-dependent phosphorylation of sodium channels in rat brain neurons maintained in primary culture. In back phosphorylation studies, cells were treated with drugs to increase intracellular cAMP and sodium channels were solubilized and isolated by immunoprecipitation. Surface and intracellular pools of sodium channels were isolated separately. Purified channels were then phosphorylated with [gamma-32P]ATP by the catalytic subunit of cAMP-dependent protein kinase to incorporate 32P into available cAMP-dependent phosphorylation sites. The amount of 32P incorporated in vitro is inversely proportional to the extent of endogenous phosphorylation. Incubation of cells with forskolin (0.1-100 microM), 8-Br-cAMP (0.1-10 mM), or isobutylmethylxanthine (0.01-1.0 mM) inhibited subsequent incorporation of 32P into isolated sodium channels by 70-80%, indicating that treatment of cells with these drugs had increased endogenous phosphorylation to nearly maximum levels. The phosphopeptides phosphorylated in vivo and in vitro were identical. To examine the magnitude of basal phosphorylation and the extent of stimulated phosphorylation, the amount of 32P incorporated into sodium channels from control and stimulated cells was compared to that from matched samples which had been dephosphorylated with calcineurin. Sodium channels from control cells incorporated approximately 2-fold more 32P after dephosphorylation, indicating that cAMP-dependent sites on the channel are at least 47% phosphorylated in the basal state. Sodium channels from forskolin-treated cells incorporated 7-8-fold more 32P after dephosphorylation, indicating that cAMP-dependent phosphorylation sites are 80-90% phosphorylated after stimulation. Cell surface and intracellular pools of sodium channels were phosphorylated similarly. In cells metabolically labeled with 32P, cell surface sodium channels incorporated 2.7 mol of phosphate/mol of channel. Forskolin stimulated 32P incorporation into sodium channels 1.3-fold, consistent with the results obtained by back phosphorylation. We conclude that the rat brain sodium channel is substantially phosphorylated in both the cell surface and intracellular pools in vivo in unstimulated rat brain neurons, and the extent of phosphorylation is increased to 80-90% of maximum phosphorylation by agents that elevate intracellular cAMP.     

Two apparent molecular weight forms of human and monkey phenylalanine hydroxylase are due to phosphorylation

Two-dimensional polyacrylamide gel analyses of purified human and monkey liver phenylalanine hydroxylase reveal that the enzyme consists of two different apparent molecular weight forms of polypeptide, designated H (Mr = 50,000) and L (Mr = 49,000), each containing three isoelectric forms. The two apparent molecular weight forms, H and L, represent the phosphorylated and dephosphorylated forms of phenylalanine hydroxylase, respectively. After incubation of purified human and monkey liver enzyme with purified cAMP-dependent protein kinase and [gamma-32P]ATP, only the H forms contained 32P. Treatment with alkaline phosphatase converted the phenylalanine hydroxylase H forms to the L forms. The L forms but not the H forms could be phosphorylated on nitrocellulose paper after electrophoretic transfer from two-dimensional gels. Phosphorylation and dephosphorylation of human liver phenylalanine hydroxylase is not accompanied by significant changes in tetrahydrobiopterin-dependent enzyme activity. Peptide mapping and acid hydrolysis confirm that the apparent molecular weight heterogeneity (and charge shift to a more acidic pI) in human and monkey liver enzyme results from phosphorylation of a single serine residue. However, phosphorylation by the catalytic subunit of cAMP-dependent protein kinase does not account for the multiple charge heterogeneity of human and monkey liver phenylalanine hydroxylase.     

Cyclic AMP-dependent phosphorylation of two size forms of alpha 1 subunits of L-type calcium channels in rat skeletal muscle cells

Skeletal muscle dihydropyridine-sensitive calcium channels are in vitro substrates for cAMP-dependent protein kinase. In the present work, alpha 1 subunits were isolated from cultured skeletal muscle cells by immunoprecipitation with a specific monoclonal antibody under conditions where proteolysis and dephosphorylation were prevented. Two forms of alpha 1 subunit, 200 and 160 kDa, were identified by back phosphorylation in vitro with cAMP-dependent protein kinase, specific immunoprecipitation, and phosphopeptide mapping. Treatment of cells with forskolin, isoproterenol, calcitonin gene-related peptide, or 8-bromo-cAMP to increase intracellular cAMP reduced 32P incorporation into all phosphopeptides in vitro by 60-80% indicating that increases in cAMP caused endogenous phosphorylation of all sites on both alpha 1(200) and alpha 1(160) to nearly maximal levels. The extents of basal and stimulated phosphorylation in vivo were estimated by back phosphorylation methods to be 35-40% and 83-86%, respectively. In muscle cells metabolically labeled with 32P, 3 mol of phosphate were incorporated into alpha 1 subunits. Forskolin stimulated 32P incorporation into alpha 1 subunits 1.6-fold. Taken together, our results show that skeletal muscle cells contain two forms of the alpha 1 subunit which both are basally phosphorylated on cAMP-dependent phosphorylation sites and are further phosphorylated in response to agents that increase intracellular cAMP.     

Ca2+-dependent and cAMP-dependent control of nicotinic acetylcholine receptor phosphorylation in muscle cells

Mouse BC3H1 myocytes were incubated with 32Pi before acetylcholine receptors were solubilized, immunoprecipitated, and subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis. More than 90% of the 32P found in the receptor was bound to the delta subunit. Two phosphorylation sites in this subunit were resolved by reverse phase high performance liquid chromatography after exhaustive proteolysis of the protein with trypsin. Sites 1 and 2 were phosphorylated to approximately the same level in control cells. The divalent cation ionophore, A23187, increased 32P in site 1 by 40%, but did not affect the 32P content of site 2. In contrast, isoproterenol increased 32P in site 2 by more than 60%, while increasing 32P in site 1 by only 20%. When dephosphorylated receptor was incubated with [gamma-32P]ATP and the catalytic subunit of cAMP-dependent protein kinase, the delta subunit was phosphorylated to a maximal level of 1.6 phosphates/subunit. Approximately half of the phosphate went into site 2, with the remainder going into a site not phosphorylated in cells. The alpha subunit was phosphorylated more slowly, but phosphorylation of both alpha and delta subunits was blocked by the heat-stable protein inhibitor of cAMP-dependent protein kinase. Phosphorylation of the receptor was also observed with preparations of phosphorylase kinase. In this case phosphorylation occurred in the beta subunit and site 1 of the delta subunit, neither of which were phosphorylated by cAMP-dependent protein kinase. The rate of receptor phosphorylation by phosphorylase kinase was slow relative to that catalyzed by cAMP-dependent protein kinase. Therefore, it can not yet be concluded that phosphorylase kinase phosphorylates the beta subunit and the delta subunit site 1 in cells. However, the results strongly support the hypothesis that phosphorylation by cAMP-dependent protein kinase accounts for phosphorylation of the alpha subunit and the delta subunit site 2 in response to elevations in cAMP.     

In vitro phosphorylation of phosphatidylethanolamine N-methyltransferase by cAMP-dependent protein kinase: lack of in vivo phosphorylation in response to N6-2'-O-dibutryladenosine 3',5'-cyclic monophosphate

Phosphorylation of rat liver phosphatidylethanolamine (PE) N-methyltransferase by cAMP-dependent protein kinase was investigated. The 18 kDa methyltransferase was found to be phosphorylated in vitro by cAMP-dependent protein kinase on a serine residue. The stoichiometry of phosphate incorporation reached a maximum of 0.25 mol phosphate/mol methyltransferase at 30 min. Resolution of the phosphorylated methyltransferase by two-dimensional gel electrophoresis showed that two isoproteins were substrates. Phosphorylation of the purified PE N-methyltransferase for up to 1 h had no effect on the methylation of PE, PMME or PDME. To test for in vivo phosphorylation, isolated rate hepatocytes were exposed to 0.5 mM N6-2'-O-dibutryladenosine 3':5'-cyclic monophosphate (DiB-cAMP) and the phosphorylation state of microsomal proteins evaluated by two-dimensional gel electrophoresis, nitrocellulose blotting and autoradiography. The same nitrocellulose blots were probed with a rabbit anti-PE N-methyltransferase antibody, immunochemically stained and aligned with the autoradiogram. No phosphorylated proteins co-migrated with the methyltransferase under non-phosphorylating conditions, or when hepatocytes were exposed to the cAMP analogue for up to 2 h. Oddly, DiB-cAMP increased both PE- and PMME-dependent activity in isolated microsomes, but decreased PE to PC conversion measured in intact hepatocytes. The results indicated that PE N-methyltransferase is poorly phosphorylated by cAMP-dependent protein kinase in vitro, and is not phosphorylated in intact hepatocytes treated with a cAMP analogue.     

Phosphorylation of myocardial fructose-6-phosphate,2-kinase: fructose-2,6-bisphosphatase by cAMP-dependent protein kinase and protein kinase C. Activation by phosphorylation and amino acid sequences of the phosphorylation sites

Phosphorylation of pure fructose-6-phosphate,2-kinase:fructose-2,6-bisphosphatase from bovine heart by cAMP-dependent protein kinase and protein kinase C was investigated. The major enzyme form (subunit Mr of 58,000) was rapidly phosphorylated by both cAMP-dependent protein kinase and protein kinase C, incorporating 0.8 and 1.0 mol/mol of subunit, respectively. The rate of phosphorylation of the heart enzyme by cAMP-dependent protein kinase was 10 times faster than that of the rat liver enzyme. The minor enzyme (subunit Mr of 54,000), however, was phosphorylated only by protein kinase C and was phosphorylated much more slowly with a phosphate incorporation of less than 0.1 mol/mol of subunit. Phosphorylation by either cAMP-dependent protein kinase or protein kinase C activated the enzyme, but each phosphorylation affected different kinetic parameters. Phosphorylation by cAMP-dependent protein kinase lowered the Km value for fructose 6-phosphate from 87 to 42 microM without affecting the Vmax, whereas the phosphorylation by protein kinase C increased the Vmax value from 55 to 85 milliunits/mg without altering the Km value. The phosphorylated peptides were isolated, and their amino acid sequences were determined. The phosphorylation sites for both cAMP-dependent protein kinase and protein kinase C were located in a single peptide whose sequence was Arg-Arg-Asn-Ser-(P)-Phe-Thr-Pro-Leu-Ser-Ser-Ser-Asn-Thr(P)-Ile-Arg-Arg-Pro. The seryl residue nearest the N terminus was the residue specifically phosphorylated by cAMP-dependent protein kinase, whereas the threonine residue nearest the C terminus was phosphorylated by protein kinase C.     

Selective phosphorylation of the alpha subunit of the sodium channel by cAMP-dependent protein kinase

The alpha subunit of the sodium channel purified from rat brain is rapidly and selectively phosphorylated by the catalytic subunit of cAMP-dependent protein kinase to a level of 3 to 4 mol of 32P/mol of saxitoxin-binding activity. The rate of phosphorylation is comparable to that of the synthetic peptide analog of the phosphorylation site of pyruvate kinase, one of the best substrates for cAMP-dependent protein kinase. An endogenous cAMP-dependent protein kinase that is present in the partially purified sodium channel preparations also selectively phosphorylates the alpha subunit. The specificity and rapidity of the phosphorylation reaction are consistent with the hypothesis that the alpha subunit is phosphorylated by cAMP-dependent protein kinase in vivo.     

Phosphorylation of a single subunit of the epithelial Na+ channel protein following vasopressin treatment of A6 cells

Arginine vasopressin (antidiuretic hormone, ADH) stimulation of sodium transport in high electrical resistance epithelia is accompanied by adenylate cyclase stimulation and cAMP accumulation. The hypothesis of direct phosphorylation of the purified amiloride-blockable epithelial Na+ channel protein by cAMP-dependent protein kinase A after ADH treatment of cultured cells was investigated in this study. Phosphate-depleted A6 cells (a cell line derived from toad kidney) were exposed to 32PO4(3-) in the absence or presence of basolateral ADH (100 milliunits/ml). After 20 min (the time needed for ADH to increase maximally Na+ transport), the Na+ channels were extracted from the cells and purified. At every stage of purification, only one subunit of the Na+ channel, namely, the 315-kDa subunit, was specifically phosphorylated as assessed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography or scintillation counting. In addition, a polyclonal antibody raised against purified epithelial Na+ channel protein was able to immunoprecipitate the phosphorylated channel protein from a detergent-solubilized fraction of vasopressin-treated A6 cells. This same subunit was also specifically phosphorylated in vitro when the purified Na+ channel protein was incubated with gamma-[32P]ATP and the purified catalytic subunit of the cAMP-dependent protein kinase. Thus, only a single component, the 315-kDa subunit, of the Na+ channel protein complex (which is composed of six subunits) can be phosphorylated both in vivo and in vitro. This subunit is selectively phosphorylated by the catalytic subunit of cAMP-dependent protein kinase to a level of 2-3 mol of 32P/mol of protein.     

Different effects on activity caused by phosphorylation of tyrosine hydroxylase at serine 40 by three multifunctional protein kinases.

Tyrosine hydroxylase was maximally phosphorylated by protein kinase C, with a stoichiometry of 0.43 mol of phosphate/mol of tyrosine hydroxylase subunit at Ser40, and by calmodulin-dependent protein kinase II, with stoichiometries of 0.43 mol/mol at Ser40 and 0.76 mol/mol at Ser19, respectively, without undergoing any significant direct activation. In contrast, the enzyme was maximally phosphorylated with a stoichiometry of 0.78 mol of phosphate/mol of subunit at Ser40 by cAMP-dependent protein kinase, which resulted in a large activation of the enzyme (about 3-fold activation under the assay conditions). Incubation of the enzyme, which had previously been maximally phosphorylated by calmodulin-dependent protein kinase II, with protein kinase C under phosphorylating conditions resulted in no additional incorporation of phosphate into the enzyme, suggesting that both protein kinases phosphorylated Ser40 of the same subunits of the enzyme. Since tyrosine hydroxylase is thought to be composed of four identical subunits, the results may indicate that calmodulin-dependent protein kinase II or protein kinase C phosphorylates only two of the four subunits of the enzyme at Ser40 without affecting the enzyme activity and that cAMP-dependent protein kinase phosphorylates Ser40 of all four subunits of the enzyme molecule, causing a marked activation. Based on a linear relationship between phosphorylation and the resulting activation of the enzyme by cAMP-dependent protein kinase, possible mechanisms for the activation of the enzyme by the protein kinase are discussed.     

The multifunctional Ca2+/calmodulin-dependent protein kinase mediates Ca2+-dependent phosphorylation of tyrosine hydroxylase

Stimulation of rat pheochromocytoma PC12 cells with ionophore A23187, carbachol, or high K+ medium, agents which increase intracellular Ca2+, results in the phosphorylation and activation of tyrosine hydroxylase (Nose, P., Griffith, L. C., and Schulman, H. (1985) J. Cell Biol. 101, 1182-1190). We have identified three major protein kinases in PC12 cells and investigated their roles in the Ca2+-dependent phosphorylation of tyrosine hydroxylase and other cytosolic proteins. A set of PC12 proteins were phosphorylated in response to both elevation of intracellular Ca2+ and to protein kinase C (Ca2+/phospholipid-dependent protein kinase) activators. In addition, distinct sets of proteins responded to either one or the other stimulus. The three major regulatory kinases, the multifunctional Ca2+/calmodulin-dependent protein kinase, the cAMP-dependent protein kinase, and protein kinase C all phosphorylate tyrosine hydroxylase in vitro. Neither the agents which increase Ca2+ nor the agents which directly activate kinase C (12-O-tetradecanoylphorbol-13-acetate or 1-oleyl-2-acetylglycerol) increase cAMP or activate the cAMP-dependent protein kinase, thereby excluding this pathway as a mediator of these stimuli. The role of protein kinase C was assessed by long term treatment of PC12 cells with 12-O-tetradecanoylphorbol-13-acetate, which causes its "desensitization." In cells pretreated in this manner, agents which increase Ca2+ influx continue to stimulate tyrosine hydroxylase phosphorylation maximally, while protein kinase C activators are completely ineffective. Comparison of tryptic peptide maps of tyrosine hydroxylase phosphorylated by the three protein kinases in vitro with phosphopeptide maps generated from tyrosine hydroxylase phosphorylated in vivo indicates that phosphorylation by the Ca2+/calmodulin-dependent kinase most closely mirrors the in vivo phosphorylation pattern. These results indicate that the multifunctional Ca2+/calmodulin-dependent protein kinase mediates phosphorylation of tyrosine hydroxylase by hormonal and electrical stimuli which elevate intracellular Ca2+ in PC12 cells.     

Modulation of keratin intermediate filament distribution in vivo by induced changes in cyclic AMP-dependent phosphorylation.

Treatment of PtK1 cells with 5 mM acrylamide for 4 hr induces reversible dephosphorylation of keratin in concert with reversible aggregation of intermediate filaments (Eckert and Yeagle, Cell Motil. Cytoskeleton 11:24-30, 1988). We have examined this phenomenon by 1) in vitro phosphorylation of isolated PtK1 keratin filaments and 2) combined treatments of PtK1 cells with both acrylamide and agents which elevate intracellular cAMP levels. PtK1 keratins were incubated in gamma-32P-ATP in the presence or absence of cAMP-dependent kinase (A-kinase) and cAMP. Levels of phosphorylation were analyzed by electrophoresis and autoradiography. Phosphorylation of keratin polypeptides (56 kD, 53 kD, 45 kD, 40 kD) occurred without added kinase, suggesting the presence of an endogenous kinase which remains with intermediate filaments in residues of Triton X-100 extracted cells. Phosphorylation levels were increased by A-kinase but not by cAMP alone, indicating the presence of cAMP-dependent phosphorylation sites in addition to sites phosphorylated by the endogenous kinase. To study the possible role of cAMP-dependent phosphorylation in acrylamide-induced aggregation of keratin filaments, we treated cells with acrylamide in the presence of 8-bromo-cAMP (brcAMP), pertussis toxin (PT), isobutylmethylxanthine (IBMX), or forskolin, which increase intracellular cAMP levels. The distribution and phosphorylation levels of keratin filaments, as well as intracellular cAMP levels, were determined for each of these treatments. In addition to aggregation and dephosphorylation of keratin filaments reported previously, treatment of cells with acrylamide alone also results in reduced levels of intracellular cAMP. 8-bromo-cAMP, IBMX, and forskolin prevent acrylamide-induced aggregation of keratin filaments and result in both normal levels of keratin phosphorylation and normal intracellular cAMP levels. PT was apparently ineffective. These observations suggest that 1) PtK1 keratins are phosphorylated by cAMP-dependent kinase and an endogenous, cAMP-independent kinase and 2) alteration of levels of cAMP-dependent phosphorylation may be involved in aggregation of keratin filaments in response to acrylamide.     

In vitro phosphorylation of type I collagen by cyclic AMP-dependent protein kinase

Both the triple-helical and denatured forms of nonfibrillar bovine dermal type I collagen were tested as substrates for the catalytic subunit of cAMP-dependent protein kinase in an in vitro reaction. Native, triple-helical collagen was not phosphorylated, but collagen that had been thermally denatured into individual alpha chains was a substrate for the protein kinase. Catalytic subunit of cAMP-dependent protein kinase phosphorylated denatured collagen to between 3 to 4 mol of phosphate/mol of (alpha 1(I)2 alpha 2(I). Pepsin-solubilized and intact collagens were phosphorylated similarly, as long as each was in a nonhelical conformation. The first 2 mol of phosphate incorporated into type I collagen by the protein kinase were present in the alpha 2(I) chain. The alpha 1(I) chain was only phosphorylated during long incubations in which the stoichiometry exceeded 2 mol of phosphate/mol of (alpha 1(I)2 alpha 2(I). Phosphoserine was the only phosphoamino acid identified in collagen that had been phosphorylated to any degree by the protein kinase. The 2 mol of phosphate incorporated into the alpha 2(I) chain were localized to the alpha 2(I)CB4 cyanogen bromide fragment. The catalytic subunit of cAMP-dependent protein kinase phosphorylated denatured pepsin-solubilized collagen with a Km of 8 microM and a Vmax of approximately 0.1 mumol/min/mg of enzyme. Denatured, but not triple-helical, type I collagen was also phosphorylated by cGMP-dependent protein kinase, although it was a poorer substrate for this enzyme than for the cAMP-dependent protein kinase. Collagen was not a substrate for phospholipid-sensitive Ca2+-dependent protein kinase. These results suggest the potential for nascent alpha chains of type I collagen to be susceptible to phosphorylation by cAMP-dependent protein kinase in vivo prior to triple-helix formation. Such a phosphorylation of collagen could be relevant to the action of cAMP to increase the intracellular degradation of newly synthesized collagen.     

Phosphorylation of DARPP-32, a dopamine- and cAMP-regulated phosphoprotein, by casein kinase II

DARPP-32 (dopamine- and cAMP-regulated phosphorprotein, Mr = 32,000 as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis) is an inhibitor of protein phosphatase-1 and is enriched in dopaminoceptive neurons possessing the D1 dopamine receptor. Purified bovine DARPP-32 was phosphorylated in vitro by casein kinase II to a stoichiometry greater than 2 mol of phosphate/mol of protein whereas two structurally and functionally related proteins, protein phosphatase inhibitor-1 and G-substrate, were poor substrates for this enzyme. Sequencing of chymotryptic and thermolytic phosphopeptides from bovine DARPP-32 phosphorylated by casein kinase II suggested that the main phosphorylated residues were Ser45 and Ser102. In the case of rat DARPP-32, the identification of these phosphorylation sites was confirmed by manual Edman degradation. The phosphorylated residues are located NH2-terminal to acidic amino acid residues, a characteristic of casein kinase II phosphorylation sites. Casein kinase II phosphorylated DARPP-32 with an apparent Km value of 3.4 microM and a kcat value of 0.32 s-1. The kcat value for phosphorylation of Ser102 was 5-6 times greater than that for Ser45. Studies employing synthetic peptides encompassing each phosphorylation site confirmed this difference between the kcat values for phosphorylation of the two sites. In slices of rat caudate-putamen prelabeled with [32P]phosphate, DARPP-32 was phosphorylated on seryl residues under basal conditions. Comparison of thermolytic phosphopeptide maps and determination of the phosphorylated residue by manual Edman degradation identified the main phosphorylation site in intact cells as Ser102. In vitro, DARPP-32 phosphorylated by casein kinase II was dephosphorylated by protein phosphatases-1 and -2A. Phosphorylation by casein kinase II did not affect the potency of DARPP-32 as an inhibitor of protein phosphatase-1, which depended only on phosphorylation of Thr34 by cAMP-dependent protein kinase. However, phosphorylation of DARPP-32 by casein kinase II facilitated phosphorylation of Thr34 by cAMP-dependent protein kinase with a 2.2-fold increase in the Vmax and a 1.4-fold increase in the apparent Km. Phosphorylation of DARPP-32 by casein kinase II in intact cells may therefore modulate its phosphorylation in response to increased levels of cAMP.     

Phosphorylation of L-type pyruvate kinase by a Ca2+/calmodulin-dependent protein kinase

Rat liver L-type pyruvate kinase was phosphorylated in vitro by a Ca2+/calmodulin-dependent protein kinase purified from rabbit liver. The calmodulin (CaM)-dependent kinase catalyzed incorporation of up to 1.7 mol of 32P/mol of pyruvate kinase subunit; maximum phosphorylation was associated with a 3.0-fold increase in the K0.5 for P-enolpyruvate. This compares to incorporation of 0.7 to 1.0 mol of 32P/mol catalyzed by the cAMP-dependent protein kinase with a 2-fold increase in K0.5 for P-enolpyruvate. When [32P]pyruvate kinase, phosphorylated by the CaM-dependent protein kinase, was subsequently incubated with 5 mM ADP and cAMP-dependent protein kinase (kinase reversal conditions), 50-60% of the 32PO4 was removed from pyruvate kinase, but the K0.5 for P-enolpyruvate decreased only 20-30%. Identification of 32P-amino acids after partial acid hydrolysis showed that the CaM-dependent protein kinase phosphorylated both threonyl and seryl residues (ratio of 1:2, respectively) whereas the cAMP-dependent protein kinase phosphorylated only seryl groups. The two phosphorylation sites were present in the same 3-4-kDa CNBr fragment located near the amino terminus of the enzyme subunit. These results indicate that the CaM-dependent protein kinase catalyzed phosphorylation of L-type pyruvate kinase at two discrete sites. One site is apparently the same serine which is phosphorylated by the cAMP-dependent protein kinase. The second site is a unique threonine residue whose phosphorylation also inactivates pyruvate kinase by elevating the K0.5 for P-enolpyruvate. These results may account for the Ca2+-dependent phosphorylation of pyruvate kinase observed in isolated hepatocytes.     

cAMP-dependent phosphorylation and hexamethylene-bis-acetamide induced dephosphorylation of p19 in murine erythroleukemia cells

The objective of this study was to investigate cyclic-adenosinemonophosphate (cAMP)-dependent phosphorylation in murine erythroleukemia (MEL) cells and to identify either direct substrates of cAMP-dependent kinase or downstream effectors of cAMP dependent phosphorylation with a potential function in growth and differentiation. MEL-cells rendered deficient in cAMP-dependent protein kinase (A-kinase) activity by stable transfection with DNA encoding for either a mutant regulatory subunit or a specific peptide inhibitor of A-Kinase (PKI) are unable to differentiate normally in response to chemical inducers. We have identified by 2-D Western blotting 2 phosphorylated forms of p19, a highly conserved 18-19 kDa cytosolic protein that is frequently upregulated in transformed cells and undergoes phosphorylation in mammalian cells upon activation of several signal transduction pathways. The phosphorylation of the more acidic phosphorylated form is increased in a cAMP-dependent fashion and impaired in cells deficient in cAMP-dependent kinase (A-kinase). Treatment of MEL-cells with the chemical inducer of differentiation hexamethylene-bisacetamide (HMBA) led to dephosphoryation of this phosphoform. Our data are compatible with previous observations which imply that phosphorylation of Ser 38 in p19 by p34cdc2-kinase leads to a more basic phosphoform and simultaneous phosphorylation by mitogen-activated kinase of Ser 25 in response to protein kinase C and the cAMP- dependent kinase creates the more acidic species.     

Evidence for the activation of the multifunctional Ca2+/calmodulin-dependent protein kinase in response to hormones that increase intracellular Ca2+

The phosphorylation state of six cytoplasmic proteins is increased following treatment of isolated rat hepatocytes with hormones that elevate free intracellular Ca2+ levels (Garrison, J. C. and Wagner, J. D. (1982) J. Biol. Chem. 257, 13135-13143). Tryptic 32P-phosphopeptide maps of two of the substrates, pyruvate kinase and a 49,000-dalton protein, the major 32P-labeled protein in hepatocytes, were prepared following stimulation of cells with vasopressin, a Ca2+-linked hormone. Peptide maps of the 49,000-dalton protein phosphorylated in vitro with the recently identified multifunctional Ca2+/calmodulin-dependent protein kinase contained phosphopeptides identical to those observed in the intact cell, suggesting that this kinase is activated in response to Ca2+-mobilizing hormones. Similar in vitro phosphorylation experiments with pyruvate kinase suggested that the Ca2+/calmodulin-dependent protein kinase can phosphorylate not only the serine residues observed following vasopressin stimulation of the intact cell but also additional threonine residues. Both pyruvate kinase and the 49,000-dalton protein are also phosphorylated in the hepatocyte in response to glucagon and in vitro by the cAMP-dependent protein kinase. Both vasopressin and glucagon appear to stimulate the phosphorylation of identical serine residues in pyruvate kinase but only vasopressin enhances the phosphorylation of certain sites in the 49,000-dalton protein. Comparison of the tryptic phosphopeptide maps of these substrates phosphorylated in vitro with either the Ca2+/calmodulin-dependent protein kinase or the cAMP-dependent protein kinase suggests that the Ca2+-dependent kinase can phosphorylate unique sites in both substrates. It appears to share specificity at other sites with the cAMP-dependent protein kinase. Overall, the results suggest that the multifunctional Ca2+/calmodulin-dependent protein kinase plays an important role in the response of the hepatocyte to a Ca2+ signal.     

cAMP-dependent phosphorylation in the pig brain

Protein phosphorylation in the cytosol, membranes and nuclei of pig brain cells was investigated. Endogenous cAMP-dependent phosphorylation was observed in all fractions studied. The degree of activation of this process by cAMP was different, depending on the fraction. The molecular weights of the endogenous protein substrates were found to be equal to 60 000, 45 000 and 28 000 for the nuclei, 80 000, 71 000, 31 000, 25 000, 16 000 and 11 000 for the membranes and 280 000, 80 000, 67 000, 53 000, 32 000, 25 000 and 22 000 for cytosol. It was shown that the majority of these proteins can be phosphorylated by the exogenous cAMP-dependent pig brain protein kinase.     

The effect of cyclic nucleotides and cholera toxin on in vivo and in vitro phosphorylation of small intestinal brush border membranes

The effect of cyclic nucleotides and cholera toxin on the phosphorylation of the brush border membrane proteins of the rat jejunum was studied. Phosphorylation was analyzed by autoradiography of brush border membrane proteins separated by SDS-polyacrylamide gel electrophoresis. Phosphorylation was performed either in vivo by perfusion of the jejunum with [³²P]orthophosphate followed by an analysis of the isolated membranes or in vitro by phosphorylation of isolated brush border membranes by [γ-³²P]ATP in the presence of saponin. The addition of cholera toxin (10 μg/ml) or dibutyryl-cAMP (5 mmol/l) to the perfusate was unable to produce significant changes in the phosphoprotein pattern. On the other hand, cAMP (at 5 μmol/l) induced an increase of the phosphorylation of a 86 kDa protein when freshly isolated brush border membranes were phosphorylated by [γ-³²P]ATP. However, the same effect could also be induced by low concentrations of cGMP (0.1 μmol/l). It is concluded that brush border membranes from rat jejunum do not contain cAMP-dependent protein kinase activity and that cAMP-dependent protein phosphorylation of this membrane does probably not represent the final event of cholera toxin-induced secretion.     

Regulation of pigment organelle translocation. II. Participation of a cAMP-dependent protein kinase

In intact goldfish xanthophores, the phosphorylation of a pigment organelle (carotenoid droplet) protein, p57, appears to play an important role in adrenocorticotropin (ACTH)- or cAMP-induced pigment organelle dispersion while the dephosphorylation of this protein upon withdrawal of ACTH or cAMP is implicated in pigment aggregation. In this paper, we report the cAMP-dependent phosphorylation of this protein in cell-free extracts of xanthophores as determined by the incorporation of 32P from [gamma-32P]ATP. As is the case in intact cells, p57 is the predominant protein phosphorylated in the presence of cAMP. The cAMP-dependent protein kinase which phosphorylates p57 is not bound to the isolated organelles but is found in the soluble portion of the cell extracts. Hence, the phosphorylation of p57 requires the carotenoid droplets bearing the substrate, soluble extract containing the kinase, cAMP (half-maximal activation at 0.5 microM), and Mg2+ (optimal at 5 mM or higher). The presence of protein phosphatase(s) in these extracts was shown indirectly by the stimulation of phosphorylation by fluoride. The phosphorylation of p57 does not appear to require a cell-specific kinase as soluble extracts of goldfish dermal nonpigment cells also phosphorylate p57 associated with isolated carotenoid droplets. Furthermore, using a constant amount of carotenoid droplets, a linear relationship was demonstrated between the rate of p57 phosphorylation and the amount of extract present in the assays. These results suggest that p57 is phosphorylated directly by a cAMP-dependent protein kinase and that the activity of this enzyme is important in regulating the intracellular movement of the pigment organelles of the xanthophore.     

In vitro phosphorylation of bovine adrenal tyrosine hydroxylase by adenosine 3':5'-monophosphate-dependent protein kinase.

We have studied the effects of adenosine 3':5'-monophosphate (cAMP)-dependent protein kinase on the phosphorylative and functional modification of bovine adrenal tyrosine hydroxylase. Incubation of partially purified tyrosine hydroxylase with cAMP-dependent protein kinase in the presence of [gamma32P]ATP and 5 micron cAMP led to a 3- to 5-fold activation of tyrosine hydroxylase and to incorporation of [32P]phosphate into protein. When tyrosine hydroxylase preparations activated by exposure to enzymatic phosphorylating conditions were analyzed by sucrose density gradient centrifugation, polyacrylamide gel electrophoresis, and gel electrofocusing, the radioactivity of 32P was coincident with the activity of tyrosine hydroxylase, suggesting incorporation of 32P from [gamma-32P]ATP into tyrosine hydroxylase. Polyacrylamide gel electrophoresis of the phosphorylated tyrosine hydroxylase preparation in the presence of 0.1% sodium dodecyl sulfate revealed that the 60,000-dalton polypeptide subunit of tyrosine hydroxylase served as the phosphate acceptor.