Phosphorylation of keratin polypeptides

The phosphorylation of keratin polypeptides was examined in calf snout epidermis. When slices of epidermis were incubated in the medium containing ³²P_i, the radioactivity was incorporated into several proteins. The predominant phosphorylated proteins migrated in SDS-polyacrylamide gels with apparent molecular weight between 49000 and 69000 and coincided with keratin polypeptides. The extent of keratin phosphorylation was not altered in the presence of dibutyryl cyclic AMP or reagents which elevate intracellular cyclic AMP. When homogenates of epidermis were incubated with [γ-³²P]ATP, keratin polypeptides were the predominant species phosphorylated as was also observed in epidermal slices. The presence of cyclic AMP or heat-stable inhibitor of cyclic AMP-dependent protein kinase in the reaction mixture did not affect the phosphorylation of keratin polypeptides, although the phosphorylation of exogenously-added histone was stimulated and inhibited, respectively, by these additions. Keratin polypeptides extracted from calf snout epidermis by 8 M urea were phosphorylated by incubation with [γ-³²P]ATP and cyclic AMP-dependent protein kinase form calf snout epidermis or bovine heart. No proteins were phosphorylated without the addition of the enzymes. The presence of cyclic AMP in the reaction mixture stimulated the keratin phosphorylation, and further addition of heat-stable protein kinase inhibitor reduced this stimulation.     

Phosphorylation of different sites of acetyl CoA carboxylase by ATP-citrate lyase kinase and cyclic AMP-dependent protein kinase

Native acetyl CoA carboxylase was phosphorylated by catalytic subunit of cyclic AMP-dependent protein kinase and ATP-citrate lyase kinase to 1 and 0.5 mol/subunit respectively. Both protein kinases added together increased acetyl CoA carboxylase phosphorylation additively. Partial proteolysis of 32P-acetyl CoA carboxylase followed by electrophoretic analysis showed that the 32P-phosphopeptides generated from acetyl CoA carboxylase phosphorylated with lyase kinase were different from the peptides obtained from the enzyme phosphorylated by cyclic AMP-dependent protein kinase. Mapping of tryptic 32P-phosphopeptides by high performance liquid chromatography showed that the major phosphopeptides phosphorylated by ATP-citrate lyase kinase were different from the major phosphopeptides phosphorylated by cyclic AMP-dependent protein kinase. The results suggest that at least one different site on acetyl CoA carboxylase is preferentially phosphorylated by each protein kinase.     

Epinephrine-stimulated phosphorylation of pyruvate kinase in hepatocytes

Incubation of rat liver parenchymal cells with 10^?5m epinephrine or norepinephrine resulted in a rapid incorporation of ³²P into pyruvate kinase. Inclusion of α-adrenergic blocking agents (phenoxybenzamine or phentolamine) in the hepatocyte incubation medium prior to addition of epinephrine suppressed the subsequent phosphorylation of pyruvate kinase. On the other hand, inclusion of the β-adrenergic antagonist, propranolol, in the hepatocyte incubation medium prior to addition of epinephrine did not suppress the epinephrine-elicited phosphorylation of pyruvate kinase. Exogenous addition of either cyclic AMP or cyclic GMP to the hepatocyte incubation medium also resulted in increased phosphorylation of pyruvate kinase. To investigate whether the same amino acid residue(s) of liver pyruvate kinase was being phosphorylated in each instance, ³²P-labeled pyruvate kinase was isolated from hepatocytes after incubation in the presence or absence of either glucagon or epinephrine. In addition, purified liver pyruvate kinase was phosphorylated in vitro with a rat liver cyclic AMP-dependent protein kinase. Each ³²P-labeled pyruvate kinase was then subjected to tryptic digestion, two-dimensional thin-layer peptide mapping, and autoradiography. Each ³²P-labeled pyruvate kinase sample yielded 44 to 48 tryptic peptides upon staining with ninhydrin and 4 peptides that contain ³²P as detected by autoradiography. Furthermore, the same 4 peptides of pyruvate kinase were radiolabeled in each instance. Thus phosphorylation of pyruvate kinase in vitro with [γ-³²P]ATP or upon addition of either glucagon or epinephrine to hepatocytes incubated with ³²P_i resulted in phosphorylation of the same amino acid residues.     

Phosphorylation and dephosphorylation of the regulatory subunit of cyclic 3′,5′-monophosphate-dependent protein kinase (type II) in vivo and in vitro

A phosphorylated regulatory subunit of cyclic AMP-dependent protein kinase (type II) was purified to homogeneity from inorganic [³²P]phosphate-injected rats.A new method of measuring the phosphorylation reaction was developed. It was found that this regulatory subunit was phosphorylated in cells and comprised 60, 82 and 55% of the total regulatory subunit in brain, heart and liver cytosol fractions from rats, respectively.Dephosphorylation was stimulated by cyclic nucleotides. The K_a values for cyclic AMP and cyclic IMP were 0.30 and 1.0 μM, respectively. Purified phosphoprotein phosphatase could dephosphorylate the regulatory subunit and this reaction was also stimulated by cyclic nucleotides with similar K_a values. The inhibitors of phosphoprotein phosphatase, NaF and ZnCl₂, protected against dephosphorylation unless ADP or cyclic AMP were present.     

Protein kinase activities in rat pancreatic islets of Langerhans.

1. Protein kinase activities in homogenates of rat islets of Langerhans were studied. 2. On incubation of homogenates with [gamma-32P]ATP, incorporation of 32P into protein occurred: this phosphorylation was neither increased by cyclic AMP nor decreased by the cyclic AMP-dependent protein kinase inhibitor described by Ashby & Walsh [(1972) J. Biol. Chem. 247, 6637--6642]. 3. On incubation of homogenates with [gamma-32P]ATP and histone as exogenous substrate for phosphorylation, incorporation of 32P into protein was stimulated by cyclic AMP (approx. 2.5-fold) and was inhibited by the cyclic AMP-dependent protein kinase inhibitor. In contrast, when casein was used as exogenous substrate, incorporation of 32P into protein was not stimulated by cyclic AMP, nor was it inhibited by the cyclic AMP-dependent protein kinase inhibitor. 4. DEAE-cellulose ion-exchange chromatography resolved four peaks of protein kinase activity. One species was the free catalytic subunit of cyclic AMP-dependent protein kinase, two species corresponded to 'Type I' and 'Type II' cyclic AMP-dependent protein kinase holoenzymes [see Corbin, Keely & Park (1975) J. Biol. Chem. 250, 218--225], and the fourth species was a cyclic AMP-independent protein kinase. 5. Determination of physical and kinetic properties of the protein kinases showed that the properties of the cyclic AMP-dependent activities were similar to those described in other tissues and were clearly distinct from those of the cyclic AMP-independent protein kinase. 6. The cyclic AMP-independent protein kinase had an s20.w of 5.2S, phosphorylated a serine residue(s) in casein and was not inhibited by the cyclic AMP-dependent protein kinase inhibitor. 7. These studies demonstrate the existence in rat islets of Langerhans of multiple forms of cyclic AMP-dependent protein kinase and also the presence of a cyclic AMP-independent protein kinase distinct from the free catalytic subunit of cyclic AMP-dependent protein kinase. The presence of the cyclic AMP-independent protein kinase may account for the observed characteristics of 32P incorporation into endogenous protein in homogenates of rat islets.     

Basal phosphorylation of cyclic AMP-regulated phosphoproteins in intact S49 mouse lymphoma cells.

Protein phosphorylation in intact S49 mouse lymphoma cells was studied by using high-resolution two-dimensional gel electrophoresis of proteins labelled with [35S]methionine or [32P]Pi. In wild-type cells substrates for cyclic AMP-stimulatable phosphorylation exhibited high basal phosphorylation; in mutant cells deficient in activities of either cyclic AMP-dependent protein kinase or adenylate cyclase, basal phosphorylation of most of these substrates was negligible. Analysis of tryptic phosphopeptides from proteins labelled with [32P]Pi in wild-type cells suggested that identical sites were phosphorylated under conditions of both basal and hormonally elevated concentrations of cyclic AMP. These results argue that most basal phosphorylation is a consequence of partial activation of cyclic AMP-dependent protein kinase and that this activation is attributable to basal concentrations of cyclic AMP. For the intermediate filament protein vimentin, basal phosphorylation was largely at a site distinct from that stimulated by increased cyclic AMP, and basal phosphorylation was not markedly different in mutant and wild-type cells. Vimentin phosphorylated at both sites was not observed. Cyclic AMP treatment resulted in enhanced phosphorylation at the cyclic AMP-specific site and decreased phosphorylation at the cyclic AMP-independent site.     

Cyclic AMP-dependent protein kinase stimulates the formation of polyphosphoinositides in lymphocyte plasma membrane

Inside-out vesicles from lymphocyte plasma membrane were phosphorylated in the presence of [gamma -32P]ATP. The dissociated catalytic subunit of cyclic AMP-dependent protein kinase stimulated both membrane protein and membrane lipid phosphorylation, indicating the presence of a phosphorylation cascade. The phosphorylated membrane lipids were analyzed by thin-layer chromatography. Increase of 32P-labelling stimulated by the cyclic AMP-dependent protein kinase was found exclusively in polyphosphoinositides.     

The role of the cyclic AMP-dependent protein kinase in the glucagon-stimulated phosphorylation of ATP-citrate lyase

We have examined the mechanism whereby glucagon stimulates the phosphorylation of ATP-citrate lyase in intact rat hepatocytes. Purified ATP-citrate lyase is phosphorylated in vitro by the catalytic subunit of the cyclic AMP-dependent protein kinase, in a reaction wherein 2-3 mol phosphate/mol lyase are incorporated, at an initial rate that approaches that observed for mixed histone. This reaction is completely abolished by the protein kinase inhibitor protein. Limited tryptic digestion of ATP-citrate lyase phosphorylated in vitro by the cyclic AMP-dependent protein kinase yields a pattern of 32P-labeled peptides, indistinguishable from those observed in parallel digests of lyase isolated from 32P-labeled, glucagon-stimulated hepatocytes. Phosphorylase b kinase catalyzes the incorporation of 1 mol phosphate/mol lyase, albeit at less than 1/160 the rate observed for phosphorylase b. The phosphorylation of purified ATP-citrate lyase is also catalyzed by homogenates of hepatocytes. This reaction is stimulated by cyclic AMP. At 30 degrees C, in the presence of maximally stimulating concentrations of cyclic AMP, the addition of excess protein kinase inhibitor protein inhibits the phosphorylation of ATP-citrate lyase by 67%. Thus, hepatocytes contain both cyclic AMP-dependent and cyclic AMP-independent ATP-citrate lyase kinase activities. Pretreatment of hepatocytes with glucagon (10(-8) M for 2 min) prior to homogenization results in activation of an endogenous hepatocyte ATP-citrate lyase kinase, as well as histone kinase and phosphorylase b kinase; the glucagon-stimulated increment in lyase kinase (and histone kinase) is observed only when homogenates are assayed in the absence of added cyclic AMP, and is completely abolished by an excess of the protein kinase inhibitor protein. We conclude that the glucagon-stimulated phosphorylation of ATP-citrate lyase in intact hepatocytes is catalyzed directly by the cyclic AMP-dependent protein kinase.     

Evidence for selective regulation of the phosphorylation of myocyte proteins by isoproterenol and prostaglandin E1

Both isoproterenol and prostaglandin E₁ increased the activation state of cyclic AMP-dependent protein kinase in cultured myocytes; however, only isoproterenol enhanced phosphorylase activity and contractile state. Following the incubation of intact myocytes with ³²PO₄^3?, 32 phosphoproteins were resolved from total cellular proteins by electrophoresis in sodium dodecyl sulfate polyacrylamide gels followed by autoradiography. Isoproterenol stimulated ³²PO₄^3? incorporated into 16 proteins, including 2 phosphoproteins not observed under control conditions. By contrast, prostaglandin E₁ neither caused a measurable change in the protein phosphorylation pattern nor interfered with isoproterenol's capacity to do so. Isoproterenol stimulated myocyte protein phosphorylation in either the presence or absence of extracellular Ca²⁺. The results suggest that the regulation of protein phosphorylation following adenylate cyclase stimulation is: (1) an agonist-specific process and not due solely to a random accumulation of intracellular cycle AMP and activation of protein kinase; (2) the Ca²⁺ mobilization component of β-receptor activation does not account for the paradoxical effects of isoproterenol and prostaglandins E₁; (3) activation of cyclic AMP-dependent protein kinase does not always result in an enhancement of protein phosphorylation.     

Plasma membrane cyclic AMP-dependent protein phosphorylation system in L6 myoblasts

Plasma membranes can be isolated without disruption of cells by the plasma membrane vesiculation technique (Scott, R.E. (1976) Science 194, 743–745). A major advantage of this technique is that it avoids contamination of plasma membranes with intracellular membrane components. Using this method, we prepared plasma membranes from L₆ myoblasts grown in tissue culture and studied the characteristics of the protein phosphorylation system.We found that these plasma membrane preparations contain protein kinase which is tightly bound to the membrane and cannot be removed by washing in EDTA or in high ionic strength salt solutions. This protein kinase activity can catalyze the phosphorylation of several exogenous substrates with decreasing efficiency as acceptors of phosphate: calf thymus histones f₂b, protamine and caseine. Cyclic AMP causes a dose-dependent stimulation of protein kinase activity; the highest stimulation (4-fold) is achieved at concentration 10^?5 M cyclic AMP. Cyclic AMP-dependent stimulation can be completely inhibited by heat-stable protein kinase inhibitor isolated from rabbit skeletal muscle. On the other hand, cyclic GMP does not affect the activity of protein kinase.Plasma membrane-bound protein kinase also catalyzes the phosphorylation of endogenous membrane protein substrates and this is also stimulated by addition of cyclic AMP. Analysis of plasma membrane proteins by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis showed that specific polypeptides are phosphorylated by cyclic AMP-independent and by cyclic AMP-dependent protein kinase systems.The results of these studies demonstrate the presence of endogenous cyclic AMP-dependent and -independent protein phosphorylating systems (enzyme activity and substrates) in purified plasma membrane preparations. These data provide a basis for further investigations on the role of plasma membrane missing data     

Phosphorylation and activation of acetyl-coenzyme A carboxylase kinase by the catalytic subunit of cyclic AMP-dependent protein kinase

The catalytic subunit of cyclic AMP-dependent protein kinase stimulates the inactivation of acetyl-coenzyme A (CoA) carboxylase by acetyl-CoA carboxylase kinase. The stimulated inactivation of carboxylase is due to activation of carboxylase kinase by the catalytic subunit. Activation of carboxylase kinase activity is accompanied by the incorporation of 0.6 mol of phosphate per mole of carboxylase kinase. Addition of the regulatory subunit of cyclic AMP-dependent protein kinase prevents the activation of carboxylase kinase. Phosphorylation and activation of carboxylase kinase has no effect on the K_m for ATP, but decreases the K_m for acetyl-CoA carboxylase from 93 to 45 nm. Inactivation of carboxylase by the carboxylase kinase requires the presence of coenzyme A even when the activated carboxylase kinase is used. Acetyl-CoA carboxylase is not phosphorylated or inactivated by the catalytic subunit of cyclic AMP-dependent protein kinase.     

Comparison of phosphorylation of ribosomal proteins from HeLa and Krebs II ascites-tumour cells by cyclic AMP-dependent and cyclic GMP-dependent protein kinases.

Phosphorylation of eukaryotic ribosomal proteins in vitro by essentially homogeneous preparations of cyclic AMP-dependent protein kinase catalytic subunit and cyclic GMP-dependent protein kinase was compared. Each protein kinase was added at a concentration of 30nM. Ribosomal proteins were identified by two-dimensional gel electrophoresis. Almost identical results were obtained when ribosomal subunits from HeLa or ascites-tumour cells were used. About 50-60% of the total radioactive phosphate incorporated into small-subunit ribosomal proteins by either kinase was associated with protein S6. In 90 min between 0.7 and 1.0 mol of phosphate/mol of protein S6 was incorporated by the catalytic subunit of cyclic AMP-dependent protein kinase. Of the other proteins, S3 and S7 from the small subunit and proteins L6, L18, L19 and L35 from the large subunit were predominantly phosphorylated by the cyclic AMP-dependent enzyme. Between 0.1 and 0.2 mol of phosphate was incorporated/mol of these phosphorylated proteins. With the exception of protein S7, the same proteins were also major substrates for the cyclic GMP-dependent protein kinase. Time courses of the phosphorylation of individual proteins from the small and large ribosomal subunits in the presence of either protein kinase suggested four types of phosphorylation reactions: (1) proteins S2, S10 and L5 were preferably phosphorylated by the cyclic GMP-dependent protein kinase; (2) proteins S3 and L6 were phosphorylated at very similar rates by either kinase; (3) proteins S7 and L29 were almost exclusively phosphorylated by the cyclic AMP-dependent protein kinase; (4) protein S6 and most of the other proteins were phosphorylated about two or three times faster by the cyclic AMP-dependent than by the cyclic GMP-dependent enzyme.     

The activity of protein kinases from hamster fibroblasts towards a synthetic peptide based on a carboxy-terminal portion of ribosomal protein S6

A synthetic decapeptide, S6(231-240), based on a region near the C-terminus of eukaryotic ribosomal protein S6, was used as a substrate for protein kinases (EC 2.7.1.37) from hamster fibroblasts stimulated with fresh medium. Consistent with the results of others using shorter peptides from this region, it was found that the cyclic AMP-dependent protein kinase preferentially phosphorylated the residue corresponding to Ser-235, whereas protein kinase C preferentially phosphorylated the residue corresponding to Ser-236 in this peptide. The peptide did not serve as a substrate for the growth-associated protein kinase from hamster fibroblasts that phosphorylated ribosomal protein S6 in 40S ribosomal subunits, but did serve as a substrate for a previously undetected protein kinase activity that was resolved from the latter by DEAE-cellulose chromatography. This S6(231-240) protein kinase activity did not phosphorylate ribosomal protein S6 in 40S ribosomal subunits, but is possibly a proteolytic fragment of the 40S ribosomal subunit S6 kinase as the latter activity acquired the ability to phosphorylate the decapeptide after partial tryptic proteolysis. The S6(231-240) protein kinase activity preferentially phosphorylated the residue corresponding to Ser-236 with an apparent Km of 15 microM. These results suggest that specific interactions with the ribosome may be required to activate the growth-associated ribosomal protein S6 kinase.     

Inhibition of α-aminoisobutyric acid transport in membrane vesicles from mouse fibroblasts after phosphorylation by cyclic AMP-dependent protein kinase

Cyclic AMP-dependent protein kinases from several mammalian sources inhibit Na⁺-dependent α-aminoisobutyric acid transport by membrane vesicles isolated from 3T3 cells. Evidence is provided that phosphorylation of membrane proteins by the enzyme is responsible for the inhibition. Lysis of the vesicles, or a reduction in the intravesicular volume is not the cause of reduced transport.The cyclic AMP-dependent protein kinase and its catalytic subunit phosphorylate a number of membrane proteins. Most of these proteins are phosphorylated, but to a lesser extent in the absence of protein kinase or cyclic AMP. The phosphorylated proteins remain associated with the membranes during hypotonic lysis treatments, which would be expected to release intra-vesicular contents and loosely associated membrane proteins. ³²P-labeled bands detected on sodium dodecyl sulfate polyacrylamide gels after phosphorylation of membranes by the catalytic subunit of the cyclic AMP-dependent kinase are eliminated by treatment with either pronase or 1 N NaOH, but not by ribonuclease nor by phospholipase C. The stability of the incorporated radioactivity to hot acid and hydroxylamine relative to hot base suggests that most of the ³²P from [γ-³²P]ATP is incorporated into protein phosphomonoester linkages.     

Phosphorylation of filamin and other proteins in cultured fibroblasts.

Incubation of subcellular fractions of fibroblasts with [³²P]ATP demonstrated 10 phosphoproteins whose phosphorylation can be increased by cyclic AMP or cyclic AMP-dependent protein kinase. One of these phosphoproteins, MW 240,000, resembles the actin binding protein, filamin, and can be selectively precipitated by antibodies to chicken gizzard filamin. Furthermore chicken gizzard filamin can be phosphorylated by skeletal muscle protein kinase and cyclic AMP stimulates this reaction.     

Phosphorylation of multiple sites in a 15 000 dalton proteolipid from rat skeletal muscle sarcolemma,catalyzed by adenosine 3′,5′-monophosphate-dependent and calcium / phospholipid-dependent protein kinases

This study reports a partial characterization of a 15 000 dalton (15 kDa) proteolipid present in rat skeletal muscle sarcolemma. The proteolipid is phosphorylated by both cyclic AMP-dependent and calcium / phospholipid-dependent protein kinases, displays an isoelectric point (pI) of 5.9, and can be extracted from sarcolemma by acidified chloroform / methanol (2:1) or non-ionic detergents. Phosphoamino acid analysis and tryptic fingerprinting of the phosphorylated proteolipid indicate that both cyclic AMP- and calcium / phospholipid-dependent protein kinases predominantly phosphorylate serine residue(s) on a single tryptic peptide. Additivity experiments and thermolytic fingerprinting demonstrate a minimum of two distinct phosphorylation sites on the proteolipid, the phosphorylation of which is independently catalyzed by cyclic AMP-dependent and calcium / phospholipid-dependent protein kinases in vitro. This sarcolemma proteolipid, which appears to be identical to a sarcolemma protein previously reported to be phosphorylated upon addition of insulin in a GTP-dependent manner (Walaas, O., Walaas, E., Rye-Alertsen, Å. and Horn, R.S. (1979) Mol. Cell. Endocrinol. 16, 45–55), therefore represents a possible membrane target for those neuronal and hormonal stimuli which can regulate cyclic AMP-dependent or calcium / phospholipid-dependent protein kinase activities in skeletal muscle.     

Phosphorylation of Purified Rat Striatal Tyrosine Hydroxylase by Ca2+/Calmodulin-Dependent Protein Kinase II: Effect of an Activator Protein

The phosphorylation of tyrosine hydroxylase, purified from rat striatum, was investigated using purified Ca2+/calmodulin (CaM)-dependent protein kinase II. This kinase catalyzed the Ca2+-dependent incorporation of up to 0.8 mol 32PO4/mol tyrosine hydroxylase subunit (62 kilodaltons). Reverse-phase high-performance liquid chromatography mapping of tryptic 32P-peptides established that the Ca2+/CaM-dependent protein kinase II phosphorylated a different serine residue than was phosphorylated by the cyclic AMP-dependent protein kinase. Limited proteolysis sequentially reduced the subunit Mr from 62 to 59 kilodaltons and finally to 57 kilodaltons, resulting in loss of the site phosphorylated by the Ca2+/CaM-dependent protein kinase II, but not the site phosphorylated by the cyclic AMP-dependent protein kinase. Phosphorylation by the Ca2+/CaM-dependent protein kinase II had little direct effect on the kinetic properties of tyrosine hydroxylase, but did convert it to a form that could be activated twofold by addition of an activator protein. This heat-labile activator protein increased the Vmax without affecting the Km for the pterin cofactor. This effect was specific in that the activator protein was without effect on nonphosphorylated tyrosine hydroxylase or on tyrosine hydroxylase phosphorylated by the cyclic AMP-dependent protein kinase. These results are consistent with the hypothesis that the "Vmax-type" activation of tyrosine hydroxylase observed upon depolarization of neural and adrenal tissues may be mediated by the Ca2+/CaM-dependent protein kinase II.     

Localization of the catalytic subunit of a cyclic AMP-dependent protein kinase(S) and acceptor proteins on the external surface of the fat cell membrane.

Cyclic AMP in μM concentrations increases the labeling of a membrane component of approximately 53,000 daltons as well as the labeling of a minor peptide of 18,000 daltons when isolated, intact rat fat cells are incubated with μM concentrations of (γ-³²P)ATP. Controlled tryptic digestion of intact cells followed by incubation with (γ-³²P)ATP results in diminution of labeling of both of these phosphopeptides indicating susceptibility, hence, access of either the catalytic site or the substrates to trypsin action. Addition of the catalytic subunit of the cyclic AMP-dependent protein kinase from beef heart to cells previously treated with trypsin results in the labeling of both phosphopeptides comparable to untreated cells. These findings indicate the catalytic subunit(s) of the cyclic AMP-dependent protein kinase(s) as well as these two phosphopeptides of the intact rat fat cell must be located on the external surface of the plasma membrane. Further, the catalytic subunit(s) of the membrane-located cyclic AMP-dependent protein kinase(s) is susceptible to trypsin action whereas the membrane peptides serving as substrates are not.     

Amino acid sequence at the phosphorylated site of rat liver fructose-1,6-diphosphatase and phosphorylation of a corresponding synthetic peptide.

Rat liver fructose-1,6-diphosphatase was phosphorylated with (³²P)ATP and the catalytic subunit of cyclic AMP-dependent protein kinase from pig muscle. After digestion with pepsin, α-chymotrypsin and subtilisin a peptide with the amino-terminal sequence Ser-Arg-Tyr-(³²P)SerP-Leu-Pro-Leu-Pro was isolated. A synthetic unphosphorylated heptapeptide with the same amino acid sequence, ending with leucine, was phosphorylated with an apparent K_m of 400 μM, while the apparent K_m value for fructose-1,6-diphosphatase was 30 μM (subunit concentration). The V_max value was 20 times higher for the peptide than for the enzyme.     

Inactivation of cyclic GMP-dependent protein kinase by N alpha-tosyl-L-lysine chloromethylketone

Cyclic GMP-dependent protein kinase from bovine lung and cyclic AMP-dependent protein kinase from bovine heart are inactivated by N^α-tosyl-L-lysine chloromethylketone (TLCK) in the presence of cyclic GMP and cyclic AMP, respectively. The inactivation of both protein kinases is pseudo-first order, suggesting the rate limiting step is beyond the binding of TLCK. Cyclic GMP-dependent protein kinase is inactivated less than $\text{1}\text{4}$ as rapidly as cyclic AMP-dependent protein kinase, although it shows a higher apparent affinity for TLCK. Cyclic AMP stimulated the rate of inactivation of cyclic AMP-dependent protein kinase 10-fold but cyclic GMP stimulated the rate of inactivation of cyclic GMP-dependent protein kinase only 1.5-fold. The rate of inactivation of cyclic GMP-dependent protein kinase by TLCK is sufficiently rapid (half-time of about 30 min at 37°C with 2 mM TLCK) to account for the effects of TLCK on cell growth observed by others.