Phosphorylation of the glucose transporter in rat adipocytes. Identification of the intracellular domain at the carboxyl terminus as a target for phosphorylation in intact-cells and in vitro

Phosphorylation of the insulin-regulatable glucose transporter (IRGT) is increased by incubating rat adipocytes with isoproterenol or by incubating microsomal membranes with cAMP-dependent protein kinase. To attempt to locate the sites of phosphorylation, the IRGT (apparent Mr = 46,000) was immunoprecipitated from 32P-labeled adipocytes and cleaved with CNBr or trypsin. Essentially all of the 32P could be recovered in a single CNBr fragment, denoted CB-T (Mr = 8,000), which bound a polyclonal antibody (R820) against a peptide having the sequence of the last 12 amino acids in the COOH terminus of the IRGT. 32P-Labeling of the IRGT was also confined to CB-T when membranes were incubated with [gamma-32P]ATP and cAMP-dependent protein kinase. Isoproterenol increased phosphorylation of CB-T, but insulin was without effect. To resolve phosphorylation sites further, IRGT from 32P-labeled cells was subjected to exhaustive proteolysis with trypsin. Samples were applied to a C-18 column, and 32P-labeled fragments were resolved into three peak fractions by elution with an increasing gradient of acetonitrile. [32P]Phosphoserine was the only phosphoamino acid detected in any of the peaks. Peak III contained approximately 80% of the 32P and was increased by isoproterenol. Almost all of the 32P introduced by cAMP-dependent protein kinase in vitro eluted in Peak III. In all cases, the 32P-labeled species in Peak III were quantitatively immunoprecipitated by R820. Digesting the peptide(s) in Peak III with V8 protease generated a single peak of 32P which eluted at lower acetonitrile than Peak III and contained 32P-labeled species that did not interact with R820. Automated Edman degradation indicated that the serine residue in Peak III phosphorylated by cAMP-dependent protein kinase was the 3rd or 4th residue from the NH2 terminus of the peptide. These findings indicate that phosphorylation of the IRGT is restricted to the presumed intracellular domain at the COOH terminus and that Ser488 is a major site phosphorylated both by cAMP-dependent protein kinase in vitro and in response to isoproterenol in vivo.     

Glucagon-stimulated phosphorylation of rat liver glycogen synthase in isolated hepatocytes

Addition of glucagon (20 nM) to the isolated hepatocytes from 24-h starved male rats results in an inactivation of glycogen synthase. The A0.5 for glucose-6-P is increased 2-fold over the control but the S0.5 for UDP-glucose is not significantly affected. The glucagon-stimulated inactivation of glycogen synthase is also accompanied by a 60-120% increase in the phosphorylation of the synthase. Glycogen synthase labeled with 32P by incubation of the hepatocytes with [32P] PO4(3-) was recovered by immunoprecipitation and the resulting immunoprecipitate was subjected to tryptic digestion. Analysis of the 32P-labeled peptides reveals that the sites corresponding to those phosphorylated by cAMP-dependent protein kinase and glycogen synthase (casein) kinase-1 (Itarte, E., and Huang, K.-P. (1979) J. Biol. Chem. 254, 4052-4057) are rapidly phosphorylated in response to glucagon. These results demonstrate that glucagon not only triggers the activation of cAMP-dependent protein kinase through an increase in the intracellular level of cAMP but also, by an unknown mechanism, activates a Ca2+- and cAMP-independent protein kinase.     

Cyclic 3',5'-adenosine monophosphate-dependent kinase and phosphoproteins in human amnion

3',5'-Cyclic adenosine monophosphate (cAMP) modulates prostaglandin production in human amnion membranes. The major effects of cAMP are presumably mediated through the phosphorylation of specific regulatory phosphoproteins following cAMP activation of cAMP-dependent protein kinase. Cyclic AMP-dependent protein kinase and phosphoproteins have not previously been characterized in human amnion. Total homogenates, cytosol, and membrane fractions from human amnion were examined for [3H]cAMP binding activity and cAMP-dependent kinase activity. cAMP-dependent kinase activity was barely detectable in crude amnion fractions. Cytosol was therefore partially purified by DEAE column chromatography for further examination. Two peaks of coincident [3H]cAMP binding and cAMP-dependent kinase activity were demonstrated at 70 and 140 mM NaCl, characteristic of the Type I and Type II cAMP-dependent protein kinase isozymes. [3H]cAMP binding to the material from both peak fractions was saturable and reversible. Scatchard analysis of [3H]cAMP binding to the peak fractions was linear for peak I and curvilinear for peak II. Assuming a one-site model, [3H]cAMP binding to the Type I isozyme showed a KD = 4.17 x 10(-8) M and Bmax = 73 pmole/mg protein; using a two-site model, [3H]cAMP binding to the high-affinity site for the Type II isozyme had a KD = 3.94 x 10(-8) M and Bmax = 6.3 pmole/mg protein. Other cyclic nucleotides competed for these [3H]cAMP binding sites with a potency order of cAMP much greater than cGMP greater than (BU)2cAMP.cAMP caused a dose-dependent increase in cAMP-dependent kinase activity in the peak fractions; half-maximal activation was observed with 5.0 x 10(-8) M cAMP. The ability of cAMP to increase phosphorylation of endogenous proteins in both crude amnion cytosol and cytosol from cultures of amnion epithelial cells was assessed using [32P]ATP, SDS-polyacrylamide gel electrophoresis and autoradiography. cAMP stimulated 32P incorporation into three proteins having Mr = 80,000, 54,000, and 43,000 (P less than .01). Half-maximal 32P incorporation into these proteins occurred at 1.0 x 10(-7) M cAMP. cAMP-dependent kinase is present in human amnion; specific cAMP-enhanced phosphoproteins are also present. Hormones elevating cAMP levels in amnion may exert their effects by activating cAMP-dependent kinase and phosphorylating these phosphoproteins.     

CTP:phosphocholine cytidylyltransferase is a substrate for cAMP-dependent protein kinase in vitro

The role of phosphorylation/dephosphorylation in the regulation of CTP:phosphocholine cytidylyltransferase activity was investigated. Incubation of post mitochondrial supernatant with cAMP-dependent protein kinase (50 units) led to an increased (28%) recovery of the cytidylyltransferase in the cytosolic fraction, while incubation with an intestinal alkaline phosphatase (20 units) led to an increased (61%) recovery in the microsomal fraction. When pure cytidylyltransferase was incubated with washed microsomes in the presence of cAMP-dependent protein kinase (133 units), the enzyme associated with the supernatant fraction increased (3.12 +/- 0.02 to 3.77 +/- 0.03 nmol/min/ml) while that of the microsomal fraction decreased (1.36 +/- 0.01 to 0.56 +/- 0.05 nmol/min/ml) by 2.5-fold. The increase in the cytidylyltransferase activity in the supernatant corresponded to an increase in 32P incorporation into the cytidylyltransferase. Treatment with alkaline phosphatase (40 units) decreased the cytidylyltransferase activity in the supernatant (3.61 +/- 0.08 to 2.88 +/- 0.07 nmol/min/ml) while the activity in the microsomal fraction increased (0.56 +/- 0.08 to 1.16 +/- 0.06 nmol/min/ml) by 2-fold. The decrease in the cytidylyltransferase activity in the supernatant corresponded to a decrease in 32P incorporation into the cytidylyltransferase. Incubation of cytidylyltransferase with phosphatidylcholine vesicles in the presence of cAMP-dependent protein kinase (110 units) decreased the cytidylyltransferase activity by 30%. The decrease in cytidylyltransferase activity corresponded to an increase in 32P incorporation into the cytidylyltransferase. Treatment with alkaline phosphatase (20 units) resulted in a 41% increase in the cytidylyltransferase activity. The increase in cytidylyltransferase activity corresponded to a decrease in 32P incorporation into the cytidylyltransferase. Incubation of the cytidylyltransferase with [gamma-32P] ATP and cAMP-dependent protein kinase led to incorporation of 32P into the serine residues of cytidylyltransferase. If the cytidylyltransferase were preincubated with alkaline phosphatase prior to incubation with cAMP-dependent protein kinase, 2-fold more 32P (0.2 mol P/mol cytidylyltransferase) was incorporated into the cytidylyltransferase. Collectively, this data is in agreement with a role for reversible phosphorylation in the regulation of cytidylyltransferase.     

Self-phosphorylation of cyclic guanosine 3':5'-monophosphate-dependent protein kinase from bovine lung. Effect of cyclic adenosine 3':5'-monophosphate, cyclic guanosine 3':5'-monophosphate and histone.

Incubation of purified cyclic guanosine 3':5'-monophospate-dependent protein kinase with [gamma-32P]ATP and Mg2+ led to formation of one 32P-labeled protein, Mr = 75,000, which corresponded to the single protein band detected after polyacrylamide gel electrophoresis in sodium dodecyl sulfate. When electrophoresis was performed without detergent, the labeled protein coincided with the position of cGMP-dependent protein kinase activity. Phosphorylation was enhanced severalfold by either histone or cAMP and was inhibited by the addition of cGMP. Low concentrations of cGMP blocked the stimulatory effects of cAMP or histone (or both). Since neither cAMP-dependent protein kinase nor cGMP-dependent phosphoprotein phosphatase activities were detected in the purified enzyme, we concluded that the cGMP-dependent protein kinase is a substrate for its own phosphotransferase activity and that other protein substrates (histone) and cyclic nucleotides modulate the process of self-phosphorylation.     

Inhibition by insulin of glucagon-dependent phospholipid methyltransferase phosphorylation in rat hepatocytes

Addition of insulin to isolated rat hepatocytes prelabeled with [32P]phosphate inhibited glucagon-dependent phospholipid methyltransferase phosphorylation and activation. Insulin alone had no effect on either the phosphorylation of the enzyme or on its activity. The effect of insulin on glucagon-dependent phospholipid methyltransferase phosphorylation was dose-dependent and occurred at physiological doses of the hormone (10(-11)-10(-10) M). Analysis of 32P-labeled peptides after digestion with trypsin revealed only one site of phosphorylation regulated by glucagon (10(-8) M) in isolated rat hepatocytes. This site, as analyzed by HPLC and thin-layer chromatography, coincided with that phosphorylated by the cAMP-dependent protein kinase using purified rat liver phospholipid methyltransferase.     

Characterization of endogenous protein phosphorylation in isolated rat liver lysosomes

Membranes prepared from highly purified rat liver lysosomes contain endogenous protein-phosphorylation activities. The transfer of phosphate to membrane fractions from [gamma-32P]ATP was analyzed by gel electrophoresis under acidic denaturing conditions. Two phosphopeptides were detected, with molecular weights of 3,000 and 14,000. Phosphorylation of these proteins was unaffected by the addition of cAMP, cGMP, or the heat-stable inhibitor of cAMP-dependent protein kinase. No additional phosphorylation was observed when cAMP-dependent protein kinase was included in the reaction or when exogenous protein kinase substrates were added. The 14,000-dalton 32P-labeled product was formed rapidly in the presence of low concentrations (250 microM) of either Ca2+ or Mg2+. This product was labile under both acidic and alkaline conditions, suggesting that this protein contains an acyl phosphate, present presumably as a catalytic intermediate in a phosphotransferase reaction. The lower molecular weight species required a high concentration (5 mM) of Mg2+ for phosphorylation, and micromolar concentrations of Ca2+ stimulated the Mg2+-dependent activity. The addition of Ca2+ and calmodulin stimulated the phosphorylation reaction to a greater extent than with Ca2+ alone. This activity was strongly inhibited by 0.2 mM LaCl3 and to a lesser extent by 50 microM chlorpromazine or trifluoperazine. These results suggest that the 3000-dalton peptide may be phosphorylated by a Ca2+, calmodulin-dependent kinase associated with the lysosomal membrane.     

Phenylalanine positively modulates the cAMP-dependent phosphorylation and negatively modulates the vasopressin-induced and okadaic-acid-induced phosphorylation of phenylalanine 4-monooxygenase in intact rat hepatocytes.

The state of phosphorylation of phenylalanine hydroxylase was determined in isolated intact rat hepatocytes. 32P-labeled phenylalanine hydroxylase was immunoisolated from cells loaded with 32Pi or from cell extracts 'back-phosphorylated' with [gamma-32P]ATP by cAMP-dependent protein kinase. The rate of phenylalanine hydroxylase phosphorylation in cells with elevated cAMP was similar to that observed for the isolated enzyme phosphorylated by homogeneous cAMP-dependent protein kinase. The phosphorylation rate in cAMP-stimulated cells was increased up to four times (reaching 0.018 s-1) by the presence of phenylalanine, the phosphate content (mol/mol hydroxylase) increasing to 0.5 from the basal level (0.17) in 50 s. The half maximal effect of phenylalanine was obtained at a physiologically relevant concentration (110 microM). The synthetic phenylalanine hydroxylase cofactor dimethyltetrahydropterin also enhanced the cAMP-stimulated phosphorylation of phenylalanine hydroxylase, presumably by displacing the endogenous cofactor, tetrahydrobiopterin. Phenylalanine was a negative modulator of the phosphorylation of phenylalanine hydroxylase induced by incubating cells with vasopressin or with the phosphatase inhibitor okadaic acid. The same site on the phenylalanine hydroxylase was phosphorylated in response to these two agents as in response to elevated cAMP. The available evidence suggested that not only vasopressin, but also okadaic acid, acted by stimulating the multifunctional Ca2+/calmodulin-dependent protein kinase II or a kinase with closely resembling properties.     

Identification, characterization, and quantitative measurement of cyclic AMP receptor proteins in cytosol of various tissues using a photoaffinity ligand.

Two protein bands, present in cytosol fractions from each of seven rat tissues examined, specifically incorporated 32P-labeled 8-azidoadenosine 3':5'-monophosphate (8-N3-[32P]cAMP), a photoaffinity label for cAMP-binding sites. These proteins had apparent molecular weights of 47,000 and 54,000 on a sodium dodecyl sulfate-polyacrylamide gel electrophoresis system. These two proteins were characterized in three of the tissues, namely, heart, uterus, and liver, by the total amount of 8-N3-[32P]cAMP incorporation, by the dissociation constant (Kd) for 8-N3-[32P]cAMP, and by the nucleotide specific inhibition of 8-N3-[32P]cAMP incorporation. Several lines of evidence were obtained that the protein with an apparent molecular weight of 47,000 represents the regulatory subunit of a type I cAMP-dependent protein kinase, while the protein with an apparent molecular weight of 54,000 represents the regulatory subunit of a type II cAMP-dependent protein kinase. Almost all of the cAMP receptor protein found in the cytosol of these tissues, as measured by 8-N3-[32P]cAMP incorporation, was associated with these two protein kinases, in agreement with the idea that most effects of cAMP are mediated through protein kinases. The photoaffinity labeling with 8-N3-[32P]cAMP can be used to estimate quantitatively the amounts of regulatory subunit of type I and type II cAMP-dependent protein kinases in various tissues.     

Adenosine 3',5'-monophosphate-receptor protein and protein kinase in Coprinus macrorhizus

A single cAMP-receptor protein could be detected in mycelial extracts of Coprinun macrorhizus by using the photoaffinity cAMP-analogue, 8-N3-cAMP. The protein which specifically bound 32P-labeled 8-N3-cAMP had an apparent molecular weight of 46,000 as determined by an SDS-polyacrylamide gel electrophoresis system. The 46,000-dalton protein was characterized by the dissociation constant for [32P]-8-N3-cAMP, and by the nucleotide specific inhibition of [32P]-8-N3-cAMP binding. The 46,000-dalton protein was co-chromatographed on a DEAE-cellulose column with cAMP-dependent protein kinase. The levels of [32P]-8-N3-cAMP-binding and protein kinase activities in mycelial extracts of strains used was always in parallel. The result indicated that the 46,000-dalton protein may be a regulatory subunit of protein kinase with the capacity to bind cAMP. cAMP-dependent protein kinase of this fungus was immunologically different from those of higher animals.     

Demonstration of the phosphorylation of dihydropyridine-sensitive calcium channels in chick skeletal muscle and the resultant activation of the channels after reconstitution.

We have examined the effects of cAMP elevating agents on the phosphorylation of dihydropyridine-sensitive Ca2+ channels in intact newborn chick skeletal muscle. In situ treatment with the beta-adrenergic receptor agonist isoproterenol resulted in the phosphorylation of the 170-kDa alpha 1 subunit in the intact cells, as evidenced by a marked decrease in the ability of the alpha 1 peptide to serve as a substrate in in vitro back phosphorylation reactions with [gamma-32P]ATP and the purified catalytic subunit of cAMP-dependent protein kinase. The phosphorylation of the 52-kDa beta subunit was not affected. The effects of isoproterenol were time- and concentration-dependent and were mimicked by other cAMP elevating agents but not by the Ca2+ ionophore A23187 or a protein kinase C activator. To test for functional effects of the observed phosphorylation, purified channels were reconstituted into liposomes containing entrapped fluo-3, and depolarization-sensitive and dihydropyridine-sensitive Ca2+ influx was measured. Channels from isoproterenol-treated muscle exhibited an increased rate and extent of Ca2+ influx compared to control preparations. The effects of isoproterenol pretreatment could be mimicked by phosphorylating the channels with cAMP-dependent protein kinase in vitro. These results demonstrate that the alpha 1 subunit of the dihydropyridine-sensitive Ca2(+)-channels is the primary target of cAMP-dependent phosphorylation in intact muscle and that the phosphorylation of this protein leads to activation of channel activity.     

Phosphorylation of Endogenous Proteins by Adenosine 3':5'-Monophosphate-Dependent Protein Kinase in Mouse Neuroblastoma Cells

Abstract: Increased intracellular adenosine 3':5'-monophosphate (cAMP) levels and activation of cAMP-dependent protein kinases (ATP:protein phosphotransferase, EC 2.7.1.37) in vivo were correlated in mouse neuroblastoma cells grown in the presence of 1 mM-⁶ N.O ²-dibutyryl 3':5'-monophosphate (Bt₂cAMP). The time course for activation showed that cAMP-dependent protein kinases were activated by 30 min. A heat-stable inhibitor protein inhibited a majority of activated cAMP-dependent protein kinase. Activation of cAMP—dependent protein kinase caused additional phosphorylation of proteins when compared with untreated control cells, as demonstrated by endogenous phosphorylation of proteins in vitro using [γ-³²P]ATP and analysis by two—dimensional polyacrylamide gel electrophoresis. The phosphorylation data show selective phosphorylation of specific proteins by cAMP-independent and cAMP-dependent protein kinase. Among the proteins in the postmitochondrial supernatant fraction phosphorylated by cAMP-dependent protein kinases, two proteins with a molecular weight of 43,000 were heavily phosphorylated. It is suggested that phosphorylation of cellular proteins by cAMP-dependent protein kinases might be involved in the cAMP-modulated biochemical changes in neuroblastoma cells.     

cAMP analogues inhibit phosphatidylcholine biosynthesis in cultured rat hepatocytes

The effect of cAMP analogues on phosphatidylcholine formation via the CDP-choline pathway was investigated in cultured monolayers of rat hepatocytes. Treatment with chlorophenylthio-cAMP or the cAMP phosphodiesterase inhibitor, aminophylline, reduced the total uptake of [methyl-3H]choline by 32 and 26% (p less than 0.01), respectively. Chlorophenylthio-cAMP inhibited the incorporation of [methyl-3H]choline into phosphatidylcholine by 2.5-fold (p less than 0.001) and reduced the rate of phosphatidylcholine biosynthesis by approximately 40%. Aminophylline, 8-bromoadenosine 3':5'-monophosphate and N6,O2'-dibutyryladenosine 3':5'-monophosphate also inhibited [methyl-3H]choline incorporation into phosphatidylcholine. Although choline kinase and phosphocholinetransferase activities were stimulated by chlorophenylthio-cAMP treatment, CTP: phosphocholine cytidylyltransferase activity was reduced 46% (p less than 0.01). The results indicate that cytidylyltransferase may be phosphorylated and inhibited by cAMP-dependent protein kinases.     

Posttranslational modification of hepatic cytochrome P-450. Phosphorylation of phenobarbital-inducible P-450 forms PB-4 (IIB1) and PB-5 (IIB2) in isolated rat hepatocytes and in vivo

Phosphorylation of hepatic cytochrome P-450 was studied in isolated hepatocytes incubated in the presence of agents known to stimulate protein kinase activity. Incubation of hepatocytes isolated from phenobarbital-induced adult male rats with [32P]orthophosphate in the presence of N6,O2'-dibutyryl-cAMP (diBtcAMP) or glucagon resulted in the phosphorylation of microsomal proteins that are immunoprecipitable by polyclonal antibodies raised to the phenobarbital-inducible P-450 form PB-4 (P-450 gene IIB1). Little or no phosphorylation of these proteins was observed in the absence of diBtcAMP or glucagon or in the presence of activators of Ca2+-dependent protein kinases. Two-dimensional gel electrophoresis revealed that these 32P-labeled microsomal proteins consist of a mixture of P-450 PB-4 and the closely related P-450 PB-5 (gene IIB2), both of which exhibited heterogeneity in the isoelectric focusing dimension. Phosphorylation of both P-450 forms was markedly enhanced by diBtcAMP at concentrations as low as 5 microM. In contrast, little or no phosphorylation of P-450 forms reactive with antibodies to P-450 PB-1 (gene IIC6), P-450 2c (gene IIC11), or P-450 PB-2a (gene IIIA1) was detected in the isolated hepatocytes under these incubation conditions. Phosphoamino acid analysis of the 32P-labeled P-450 PB-4 + PB-5 immunoprecipitate revealed that these P-450s are phosphorylated on serine in the isolated hepatocytes. Peptide mapping indicated that the site of phosphorylation in hepatocytes is indistinguishable from the site utilized by cAMP-dependent protein kinase in vitro, which was previously identified as serine-128 for the related rabbit protein P-450 LM2.(ABSTRACT TRUNCATED AT 250 WORDS)     

In vivo phosphorylation of distinct domains of the 70-kilodalton neurofilament subunit involves different protein kinases

A combination of in vivo and in vitro approaches were used to characterize phosphorylation sites on the 70,000-kilodalton (kDa) subunit of neurofilaments (NF-L) and to identify the protein kinases that are likely to mediate these modifications in vivo. Neurofilament proteins in a single class of neurons, the retinal ganglion cells, were pulse-labeled in vivo by injecting mice intravitreously with [32P]orthophosphate. Radiolabeled neurofilaments were isolated after they had advanced along optic axons, and the individual subunits were separated on sodium dodecyl sulfate-polyacrylamide gels. Two-dimensional alpha-chymotryptic phosphopeptide map analysis of NF-L revealed three phosphorylation sites: an intensely labeled peptide (L-1) and two less intensely labeled peptides (L-2 and L-3). The alpha-chymotryptic peptide L-1 was identified as the 11-kDa segment containing the C terminus of NF-L. The ability of these peptides to serve as substrates for specific protein kinases were examined by incubating neurofilament preparations with [gamma-32P]ATP in the presence of purified cAMP-dependent protein kinase or appropriate activators and/or inhibitors of endogenous cytoskeleton-associated protein kinases. The heparin-sensitive, calcium- and cyclic nucleotide-independent kinase associated with the cytoskeleton selectively phosphorylated L-1 and L-3 but had little, if any, activity toward L-2. When this kinase was inhibited with heparin, cAMP addition to the neurofilament preparation stimulated the phosphorylation of L-2, and addition of the purified catalytic subunit of cAMP-dependent protein kinase induced intense labeling of L-2. At higher labeling efficiencies, the exogenous kinase also phosphorylated L-3 and several sites at which labeling was not detected in vivo; however, L-1 was not a substrate. Calcium and calmodulin added to neurofilament preparations in the presence of heparin modestly stimulated the phosphorylation of L-1 and L-3, but not L-2, and the stimulation was reversed by trifluoperazine. The selective phosphorylation of different polypeptide domains on NF-L by second messenger-dependent and -independent kinases suggests multiple functions for phosphate groups on this protein.     

Dephosphorylation of cardiac myofibril C-protein by protein phosphatase 1 and protein phosphatase 2A

C-protein purified from chicken cardiac myofibrils was phosphorylated with the catalytic subunit of cAMP-dependent protein kinase to nearly 3 mol [32P]phosphate/mol C protein. Digestion of 32P-labeled C-protein with trypsin revealed that the radioactivity was nearly equally distributed in three tryptic peptides which were separated by reversed-phase HPLC. Fragmentation of 32P-labeled C-protein with CNBr showed that the isotope was incorporated at different ratios in three CNBr fragments which were separated on polyacrylamide gels in the presence of sodium dodecyl sulfate. Phosphorylation was present in both serine and threonine residues. Incubation of 32P-labeled C-protein with the catalytic subunit of protein phosphatase 1 or 2A rapidly removed 30-40% of the [32P]phosphate. The major site(s) dephosphorylated by either one of the phosphatases was a phosphothreonine residue(s) apparently located on the same tryptic peptide and on the same CNBr fragment. CNBr fragmentation also revealed a minor phosphorylation site which was dephosphorylated by either of the phosphatases. Increasing the incubation period or the phosphatase concentration did not result in any further dephosphorylation of C-protein by phosphatase 1, but phosphatase 2A at high concentrations could completely dephosphorylate C-protein. These results demonstrate that C-protein phosphorylated with cAMP-dependent protein kinase can be dephosphorylated by protein phosphatases 1 and 2A. It is suggested that the enzyme responsible for dephosphorylation of C-protein in vivo is phosphatase 2A.     

Structural organization of the lens fiber cell plasma membrane protein MP18

The 18,000-dalton bovine lens fiber cell intrinsic membrane protein MP18 was phosphorylated on a serine residue by both cAMP-dependent protein kinase and protein kinase C. In addition, this protein bound calmodulin and was recognized by a monoclonal antibody (2D10). These different regions were localized using enzymatic and chemical fragmentation of electrophoretically purified MP18 that had been phosphorylated with either cAMP-dependent protein kinase or protein kinase C. Partial digestion of 32P-labeled MP18 with protease V8 resulted in a Mr = 17,000 peptide that bound calmodulin, but neither contained 32P or was recognized by the monoclonal antibody 2D10. Furthermore, the 17-kDa peptide had the same N-terminal amino acid sequence as MP18. Thus, the monoclonal antibody 2D10 recognition site and the protein kinase phosphorylation site(s) are close together and confined to a small region in the C terminus of MP18. This conclusion was confirmed in experiments where MP18 was fragmented with trypsin, endoproteinase Lys-C, or CNBr. The location of the phosphorylation site was confirmed by sequencing the small 32P-labeled, C-terminal peptide that resulted from protease V8 digestion of 32P-labeled MP18. This peptide contained a consensus sequence for cAMP-dependent protein kinase.     

The insulin-directed phosphorylation site on ATP-citrate lyase is identical with the site phosphorylated by the cAMP-dependent protein kinase in vitro

32P-labeled ATP-citrate lyase isolated from 32P-labeled hepatocytes treated with insulin contained 1.6-1.8-fold greater 32P-radioactivity per mg protein than control enzyme. Both enzyme preparations were digested in parallel with trypsin until 94% of all 32P-radioactivity was rendered acid soluble. Quantitative high performance liquid chromatographic peptide mapping of the tryptic digests revealed a principal 32P-peptide which accounted for at least 80% of the insulin induced increment in 32P-radioactivity of native lyase. This peptide was purified, sequenced, and the site of 32P-phosphorylation assigned by two methods: electrophoresis (pH 6.5) of residual peptide after each step of Edman degradation and solid phase sequencing. The site of insulin-directed phosphorylation of ATP-citrate lyase (Thr-Ala-Ser(32P)-Phe-Ser-Glu-Ser-Arg) is the same as that directed by glucagon, and, in turn, identical with that phosphorylated by the cAMP-dependent protein kinase in vitro.     

Activation of cyclic AMP-dependent protein kinase isoenzymes: studies using specific antisera

A novel method for rapidly determining the amount and degree of association-dissociation of the Type I and Type II cAMP-dependent protein kinases has been developed and validated. Antibodies directed against the regulatory subunits of Type I and Type II cAMP-dependent protein kinases were used. The antibodies formed complexes with holoenzymes and regulatory subunits which were precipitated by goat anti-rabbit IgG (immunoglobulin G). These complexes bound [3H]cAMP with an apparent Kb of 20 nM for protein kinase I and 80 nM for protein kinase II. Immunoprecipitated protein kinases I and II were catalytically active when incubated with cAMP, [gamma-32P]ATP, and histone H2B. When mixtures of the two kinase isoenzymes or cytosol were incubated with various amounts of [3H]cAMP and the isoenzymes were separated by precipitation with antisera specific for each isoenzyme, the amount of [3H]cAMP associated with immunoprecipitates was proportional to the concentration of [3H]cAMP. In contrast, the catalytic activity that was immunoprecipitated varied inversely with the concentration of [3H]cAMP, showing that the activation of protein kinase could be assessed by the disappearance of catalytic activity from the immunoprecipitates. In the absence of MgATP protein kinase I was activated by a 10-fold lower concentration of cAMP than protein kinase II. However, when MgATP was added to the incubation, there was no significant difference in the binding of [3H]cAMP or dissociation of catalytic subunits of the two isoenzymes. The anti-R antibodies were also used to rapidly quantitate the concentration of regulatory subunits and the relative ratio of protein kinases I and II in tissue cytosols.     

Threonine phosphorylation of rat liver glycogen synthase

32P-labeled glycogen synthase specifically immunoprecipitated from 32P-phosphate incubated rat hepatocytes contains, in addition to [32P] phosphoserine, significant levels of [32P] phosphothreonine (7% of the total [32P] phosphoaminoacids). When the 32P-immunoprecipitate was cleaved with CNBr, the [32P] phosphothreonine was recovered in the large CNBr fragment (CB-2, Mapp 28 Kd). Homogeneous rat liver glycogen synthase was phosphorylated by all the protein kinases able to phosphorylate CB-2 "in vitro" (casein kinases I and II, cAMP-dependent protein kinase and glycogen synthase kinase-3). After analysis of the immunoprecipitated enzyme for phosphoaminoacids, it was observed that only casein kinase II was able to phosphorylate on threonine and 32P-phosphate was only found in CB-2. These results demonstrate that rat liver glycogen synthase is phosphorylated at threonine site(s) contained in CB-2 and strongly indicate that casein kinase II may play a role in the "in vivo" phosphorylation of liver glycogen synthase. This is the first protein kinase reported to phosphorylate threonine residues in liver glycogen synthase.