Protein kinase C phosphorylates the carboxyl terminus of the plasma membrane Ca(2+)-ATPase from human erythrocytes.

Purified Ca(2+)-stimulated, Mg(2+)-dependent ATPase (Ca(2+)-ATPase) from human erythrocytes was phosphorylated with a stoichiometry of about 1 mol of phosphate/mol of ATPase at both threonine and serine residues by purified rat brain type III protein kinase C. In the presence of calmodulin, the phosphorylation was markedly reduced. Labeled phosphate from [gamma-32P]ATP was retained on an 86-kDa calmodulin-binding tryptic fragment of Ca(2+)-ATPase but not on 82- and 77-kDa non-calmodulin-binding fragments. Similarly, fragmentation of the phosphorylated Ca(2+)-ATPase by calpain I revealed that calmodulin-binding fragments (127 and 125 kDa) retained phosphate label whereas a non-calmodulin-binding fragment (124 kDa) did not. The calmodulin-binding domain, located about 12 kDa from the carboxyl terminus of the Ca(2+)-ATPase, was thus located as a site of protein kinase C phosphorylation. A synthetic peptide corresponding to a segment of the calmodulin-binding domain (H2 N-R-G-L-N-R-I-Q-T-Q-I-K-V-V-N-COOH) was indeed phosphorylated at the single threonine residue within this sequence. The additional serine phosphorylation site was carboxyl terminal to the calmodulin domain. Phosphorylation by purified type III protein kinase C (canine heart) antagonized the calmodulin activation of the Ca(2+)-ATPase, particularly at lower Ca2+ concentrations (0.2-1.0 microM). By contrast, a purified but unresolved protein kinase C isoenzyme mixture from rat brain stimulated the activity of Ca(2+)-ATPase prepared in asolectin, but not glycerol, by more than 2-fold in the presence of the ionophore A23187, without increasing its Ca2+ sensitivity. The results clearly indicate that human erythrocyte Ca(2+)-ATPase is a substrate of protein kinase C, but the effect of phosphorylation on the activity of the enzyme depends on the isoenzyme form of protein kinase C used and on the lipid associated with the Ca(2+)-ATPase.     

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.     

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

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

Antigen-induced secretion of histamine and the phosphorylation of myosin by protein kinase C in rat basophilic leukemia cells

IgE-mediated stimulation of rat basophilic leukemia (RBL-2H3) cells results in the secretion of histamine. Myosin immunoprecipitated from these cells shows an increase in the amount of radioactive phosphate incorporated into its heavy (200 kDa) and light (20 kDa) chains. In unstimulated cells two-dimensional mapping of tryptic peptides of the myosin light chain reveals one phosphopeptide containing the serine residue phosphorylated by myosin light chain kinase. Following stimulation a second phosphopeptide appears containing a serine residue phosphorylated by protein kinase C. Tryptic phosphopeptide maps derived from myosin heavy chains show that unstimulated cells contain three major phosphopeptides. Following stimulation a new tryptic phosphopeptide appears containing a serine site phosphorylated by protein kinase C. The stoichiometry of phosphorylation of the myosin light and heavy chains was determined before and after antigenic stimulation. Before stimulation, myosin light chains contained 0.4 mol of phosphate/mol of light chain all confined to a serine not phosphorylated by protein kinase C. Cells that secreted 44% of their total histamine in 10 min exhibited an increase in phosphate content at sites phosphorylated by protein kinase C from 0 mol of phosphate/mol of myosin subunit to 0.7 mol of phosphate/mol of light chain and to 1 mol of phosphate/mol of heavy chain. When RBL-2H3 cells were made permeable with streptolysin O they still showed a qualitatively similar pattern of secretion and phosphorylation. Our results show that the time course of histamine secretion from stimulated RBL-2H3 cells parallels that of myosin heavy and light chain phosphorylation by protein kinase C.     

Autophosphorylation and autoactivation of spleen protein tyrosine kinase

Incubation of a highly purified bovine spleen protein tyrosine kinase with [gamma-32P]ATP and Mg2+ resulted in a gradual radioactive labeling of the protein kinase (50 kDa) with no change in the protein kinase activity toward angiotensin II. On the other hand, treatment of the protein tyrosine kinase with an immobilized alkaline phosphatase caused essentially complete loss in the kinase activity, which could be restored by incubation of the enzyme with ATP and Mg2+. By using the alkaline phosphatase-treated kinase, time courses of the protein phosphorylation and the enzyme activation were demonstrated to correlate closely. These results indicate that this protein tyrosine kinase relies on autophosphorylation for activity and that the purified enzyme usually exists in a fully phosphorylated state. The radioactive labeling of the purified kinase during incubation with [gamma-32P]ATP resulted from a phosphate exchange reaction: the exchange of [gamma-32P]phosphate of ATP with the protein bound phosphate as previously suggested (Kong, S.K., and Wang, J.H. (1987) J. Biol. Chem. 262, 2597-2603). It could be shown that the autophosphorylation of phosphatase-treated tyrosine kinase was strongly inhibited by the substrate angiotensin II, whereas the exchange reaction carried out with untreated tyrosine kinase was not. Autophosphorylation is suggested to be an intermolecular reaction since its initial rate is proportional to the square of the protein concentration.     

Phosphorylation of partially purified 1-O-alkyl-2-lyso-sn-glycero-3-phosphocholine:acetyl-CoA acetyltransferase from rat spleen.

A new improved method for purification of the enzyme 1-O-alkyl-2-lyso-sn-glycero-3-phosphocholine: acetyl-CoA acetyltransferase (EC 2.3.1.67) from rat spleen is described. The catalytic subunit of cyclic AMP-dependent protein kinase in the presence of MgATP stimulated about 3-fold the activity of this partially purified enzyme activity. When [gamma-32P]ATP was included in the assay mixture, the analysis of phosphoprotein products by SDS/polyacrylamide-gel electrophoresis and autoradiography showed the incorporation of [32P]phosphate into a single protein band of about 30 kDa. Analysis of the phosphorylated amino acids indicated that the phosphate was incorporated into a serine residue. Activation of the acetylation reaction by the protein kinase was reversible. The reversal of the activation was coincident with the loss of the [32P]phosphate incorporated into the 30 kDa protein band, which suggests that the acetyltransferase is regulated by a phosphorylation-dephosphorylation mechanism dependent on cyclic AMP.     

In vitro phosphorylation of human complement factor C3 by protein kinase A and protein kinase C. Effects on the classical and alternative pathways

Complement factor C3, recently found to contain covalently bound phosphate, was phosphorylated in vitro by cyclic AMP-dependent protein kinase (protein kinase A) and Ca2(+)-activated, phospholipid-dependent protein kinase (protein kinase C). Both protein kinases phosphorylated the same serine residue(s) located in the C3a portion of the alpha-chain. In addition, protein kinase C phosphorylated the beta-chain to a lesser extent. Protein kinase A gave a maximal incorporation of 1 mol of phosphate/mol of C3 while that value with protein kinase C was 1.5 mol of phosphate/mol of C3. The velocity in pmol of [32P]phosphate/(min x unit kinase) was 20 times higher for protein kinase C than for protein kinase A although a 10 times lower ratio of protein kinase to C3 was used in the former case. The apparent Km for C3 was 2.6 microM when protein kinase C was used. The phosphorylated C3 was found to be more resistant to partial degradation by trypsin than unphosphorylated C3. It was also found that phosphorylation of C3 in the C3a portion of the alpha-chain inhibited both the classical and alternative complement activation pathways on an approximately stoichiometric basis.     

Phosphorylation of smooth muscle heavy meromyosin by calcium-activated, phospholipid-dependent protein kinase. The effect on actin-activated MgATPase activity

Smooth muscle heavy meromyosin (HMM) can serve as a substrate for the Ca2+-activated, phospholipid-dependent protein kinase (protein kinase C) as well as for the Ca2+/calmodulin-dependent kinase, myosin light chain kinase. When turkey gizzard HMM is incubated with protein kinase C, 1.7-2.2 mol of phosphate are incorporated per mol of HMM, all of it into the 20,000-Da light chain of HMM. Two-dimensional peptide mapping following tryptic hydrolysis revealed that protein kinase C phosphorylated a different site on the 20,000-Da HMM light chain than did myosin light chain kinase. Moreover, sequential phosphorylation of HMM by myosin light chain kinase and protein kinase C resulted in the incorporation of 4 mol of phosphate/mol of HMM, i.e. 2 mol of phosphate into each 20,000-Da light chain. When unphosphorylated HMM was phosphorylated by myosin light chain kinase, its actin-activated MgATPase activity increased from 4 nmol to 156 nmol of phosphate released/mg of HMM/min. Subsequent phosphorylation of this phosphorylated HMM by protein kinase C decreased the actin-activated MgATPase activity of HMM to 75 nmol of phosphate released/mg of HMM/min.     

Growth stimulation by serum in Entamoeba histolytica is associated with protein tyrosine dephosphorylation

Very little protein tyrosine phosphorylation was observed in growing (exponential-phase) Entamoeba histolytica cells by immunoblotting and quantitative immunofluorescence. After 1 h of serum deprivation, two proteins (42 and 38 kDa in SDS-PAGE) were tyrosine phosphorylated and two more proteins (96 and 63 kDa) also showed tyrosine phosphorylation when examined after 4 h of serum deprivation. Intense enhancements of anti-phosphotyrosine immunofluorescence levels were observed during this period of serum withdrawal. Membrane-associated tyrosine kinase activity reached a peak (3.5-fold increase) 1 h after serum deprivation and decreased thereafter reaching a basal level by 2 h of serum deprivation. Interestingly, tyrosine kinase activities remained unaffected by serum stimulation (2-60 min) of serum-deprived cells. Also, during this period of serum stimulation tyrosine phosphorylated proteins of serum-deprived cells were dephosphorylated. Tyrosine phosphatase activities were suppressed during serum deprivation and on serum addition to serum-deprived cells tyrosine phosphatase activities increased significantly. Our data attest that protein tyrosine phosphorylation was associated with growth inhibition of E. histolytica and serum stimulation of E. histolytica produced tyrosine phosphatase activation and protein tyrosine dephosphorylation.     

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.     

CD45, an integral membrane protein tyrosine phosphatase. Characterization of enzyme activity

Although CD45 resembles the low Mr protein tyrosine phosphatases (PTPases) from human placenta in its specificity for phosphotyrosyl residues and absolute dependence on sulfhydryl compounds for activity, it also exhibits a number of distinguishing features. Most notably, it displayed substrate specificity in vitro, preferentially dephosphorylating myelin basic protein, over the other substrates tested, with high specific activity. Limited trypsinization of CD45 generated active fragments of approximately 65 kDa that were apparently derived exclusively from the intracellular segment of the molecule. These retained high activity against myelin basic protein, suggesting that this is an intrinsic feature of the PTPase domains and not the result of secondary interactions between the substrate and the putative ligand binding structure. With reduced carboxamidomethylated and maleylated lysozyme as substrate, CD45 was stimulated up to 12-fold by basic compounds such as spermine; divalent metal ions were also stimulatory, most notably Zn2+, which was previously identified as a potent inhibitor of the low Mr PTPases. CD45 was phosphorylated to high stoichiometry by casein kinase-2 (up to 1.5 mol/mol) and also by glycogen synthase kinase 3 (approximately 0.3 mol/mol) and protein kinase C (approximately 0.1 mol/mol); in all cases, no alteration in enzyme activity was detected following these modifications. Autophosphorylated preparations of epidermal growth factor receptor, insulin receptor, and p56lck protein tyrosine kinases were also substrates for CD45 in vitro.     

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.     

Regulation of the epidermal growth factor receptor by phosphorylation

The receptor for epidermal growth factor (EGF) is a glycosylated transmembrane phosphoprotein that exhibits EGF-stimulable protein tyrosine kinase activity. On EGF stimulation, the receptor undergoes a self-phosphorylation reaction at tyrosine residues located primarily in the extreme carboxyl-terminal region of the protein. Using enzymatically active EGF receptor purified by immunoaffinity chromatography from A431 human epidermoid carcinoma cells, the self-phosphorylation reaction has been characterized as a rapid, intramolecular process which is maximal at 30-37 degrees C and exhibits a very low Km for ATP (0.2 microM). When phosphorylation of exogenous peptide substrates was measured as a function of receptor self-phosphorylation, tyrosine kinase activity was found to be enhanced two to threefold at 1-2 mol of phosphate per mol of receptor. Analysis of the dependence of the tyrosine kinase activity on ATP concentration yielded hyperbolic kinetics when plotted in double-reciprocal fashion, indicating that ATP can serve as an activator of the enzyme. Higher concentrations of peptide substrates were found to inhibit both the self- and peptide phosphorylation, but this inhibition could be overcome by first self-phosphorylating the enzyme. These results suggest that self-phosphorylation can remove a competitive/inhibitory constraint so that certain exogenous substrates can have greater access to the enzyme active site. In addition to self-phosphorylation, the EGF receptor can be phosphorylated on threonine residues by the calcium- and phospholipid-dependent protein kinase C. The sites on the EGF receptor phosphorylated in vitro by protein kinase C are identical to the sites phosphorylated on the receptor isolated from A431 cells exposed to the tumor promoters 12-O-tetradecanoylphorbol 13-acetate or teleocidin. This phosphorylation of the EGF receptor results in a suppression of its tyrosine kinase and EGF binding activities both in vivo and in vitro. The EGF receptor can thus be variably regulated by phosphorylation: self-phosphorylation can enhance tyrosine kinase activity whereas protein kinase C-catalyzed phosphorylation can depress enzyme activity. Because these two phosphorylations account for only a fraction of the phosphate present in the EGF receptor in vivo, other protein kinases can apparently phosphorylate the receptor and these may exert additional controls on EGF receptor/kinase function.     

Purification and characterization of a protein-tyrosine kinase encoded by the Abelson murine leukemia virus

Sequences termed v-abl, which encode the protein-tyrosine kinase activity of Abelson murine leukemia virus, have been expressed in Escherichia coli as a fusion product (ptabl50 kinase). This fusion protein contains 80 amino acids of SV40 small t and the 403 amino acid protein kinase domain of v-abl. We report here the purification and characterization of this kinase. The purified material contains two proteins (Mr = 59,800 and 57,200), both of which possess sequences derived from v-abl. Overall purification was 3,750-fold, with a 31% yield, such that 117 micrograms of kinase could be obtained from 40 g of E. coli within 6-7 days. The specific kinase activity is over 170 mumol of phosphate min-1 mumol-1, comparable to the most active protein-serine kinases. Kinase activity is insensitive to K+, Na+, Ca2+, Ca2+-calmodulin, cAMP, or cAMP-dependent protein kinase inhibitor. The Km for ATP is dependent on the concentration of the second substrate. GTP can also be used as a phosphate donor. The enzyme can phosphorylate peptides consisting of as few as two amino acids and, at a very low rate, free tyrosine. Incubation of the kinase with [gamma-32P]ATP results in incorporation of 1.0 mol of phosphate/mol of protein. This reaction, however, cannot be blocked by prior incubation with unlabeled ATP. Incubation of 32P-labeled kinase with either ADP or ATP results in the synthesis of [32P]ATP. This suggests the phosphotyrosine residue on the Abelson kinase contains a high energy phosphate bond.     

Regulation of a rat lung protein tyrosine kinase activity by reversible phosphorylation/dephosphorylation

Incubation of a partially purified protein tyrosine kinase from rat lung with Mg2+ and ATP resulted in about 10-15-fold activation of the enzyme activity as judged by the phosphorylation of poly(Glu:Tyr,4:1), an exogenous substrate. The activation was time dependent and was associated with the phosphorylation of a single protein band of 50 kDa. Phosphoamino acid analysis of the phosphorylated protein indicated that tyrosine was the amino acid being phosphorylated. Upon gel filtration on a Sephacryl S-200 column, the phosphorylated protein co-eluted with protein tyrosine kinase and ATP-binding activities, suggesting that all three activities are part of the same protein. In addition, pretreatment of the partially purified protein tyrosine kinase with alkaline phosphatase inhibited its enzyme activity which could be restored by reincubation with Mg2+ and ATP. These data suggest that a temporal relationship exists between the phosphorylation and the activation states of rat lung protein tyrosine kinase, and that the phospho- and dephospho- forms represent the active and inactive (or less active) forms, respectively, of the enzyme.     

Phosphorylation of liver gap junction protein by protein kinase C

The 27 kDa protein, a major component of rat liver gap junctions, was shown to be phosphorylated in vitro by protein kinase C. The stoichiometry of the phosphorylation indicated that approx. 0.33 mol phosphate was incorporated per mol 27 kDa protein. Phosphorylation was entirely dependent on the presence of calcium and was virtually specific for serine residues. For comparison, the gap junction protein was also examined for its phosphorylation by cAMP-dependent protein kinase, the extent of phosphorylation being one-tenth that exerted by protein kinase C.     

Autophosphorylation of rat brain Ca2+-activated and phospholipid-dependent protein kinase

Ca2+-activated and phospholipid-dependent protein kinase (protein kinase C) isolated from rat brain cytosol undergoes autophosphorylation in the presence of Mg2+, ATP, Ca2+, phosphatidylserine, and diolein. Approximately 2-2.5 mol of phosphate were incorporated per mol of the kinase. After sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography, the phosphorylated kinase showed a single protein band of Mr = 82,000 compared to the Mr = 80,000 of the nonphosphorylated enzyme. Analysis of the 32P-labeled tryptic peptides derived from the autophosphorylated kinase by peptide mapping revealed that multiple sites were phosphorylated. Both serine and threonine residues were found to be labeled with 32P. Limited proteolysis of the autophosphorylated kinase with trypsin resulted in the conversion of the kinase into a phospholipid- and Ca2+-independent form. Two major 32P-labeled fragments, Mr = 48,000 and 38,000, were formed as a result of proteolysis, suggesting that the catalytic domain and possibly the Ca2+- and phospholipid-binding region were both phosphorylated. Protein kinase C autophosphorylation has a Km for ATP (1.5 microM) about 10-fold lower than that for phosphorylation of exogenous substrates. The kinetically preferred autophosphorylation appears to be an intramolecular reaction. The autophosphorylated protein kinase C, unlike the protease-degraded enzyme, still depends on Ca2+ and phospholipid for maximal activity. However, the autophosphorylated form of the kinase has a lower Ka for Ca2+ and a higher affinity for the binding of [3H]phorbol-12, 13-dibutyrate. These findings suggest that autophosphorylation of protein kinase C may be important in the regulation of the enzymic activity subsequent to signal transduction.     

Phosphorylation of the purified receptor for calcium channel blockers by cAMP kinase and protein kinase C

The dihydropyridine receptor purified from rabbit skeletal muscle contains three proteins of 165, 55 and 32 kDa. cAMP kinase and protein kinase C phosphorylate the 165-kDa and the 55-kDa proteins. At identical concentrations of each protein kinase, cAMP kinase phosphorylates the 165-kDa protein faster than the 55-kDa protein. Protein kinase C phosphorylates preferentially the 55-kDa protein. cAMP kinase incorporates up to 1.6 mol phosphate/mol protein into the 165-kDa protein and 1 mol/mol into the 55-kDa protein upon prolonged incubation. At a physiological concentration of cAMP kinase 1 mol phosphate is incorporated/mol 165-kDa protein within 10 min, suggesting a physiological role of this phosphorylation. Protein kinase C incorporates up to 1 mol phosphate/mol into the 55-kDa protein and less than 1 mol/mol into the 165-kDa protein. Tryptic phosphopeptide analysis reveals that cAMP kinase phosphorylates two distinct peptides in the 165-kDa protein, whereas protein kinase C phosphorylates a single peptide in the 165-kDa protein. cAMP kinase and protein kinase C phosphorylate three and two peptides in the 55-kDa protein, respectively. Mixtures of the tryptic phosphopeptides derived from the 165-kDa and 55-kDa proteins elute according to the composite of the two elution profiles. These results suggest that the 165-kDa protein, which contains the binding sites for each class of calcium channel blockers and the basic calcium-conducting structure, is a specific substrate for cAMP kinase. The 55-kDa protein apparently contains sites preferentially phosphorylated by protein kinase C.     

A model for the regulation of the activity of L-asparaginase/ kinase enzyme of Tetrahymena pyriformis

L-Asparaginase of Tetrahymena pyriformis is a lipoprotein with relative M(r) approximately 200 kDa and one subunit size of 39 kDa. This enzyme also exhibits protein kinase activity and it is autophosphorylated in tyrosine residues. Phosphorylation-dephosphorylation of L-asparaginase resulted in complete loss or activation by more than 10-fold of its catalytic activity. Both native and dephosphorylated forms of L-asparaginase are inactivated by phospholipase C and this inactivation can be reversed by the addition of lipids. Based on these results a working hypothesis is suggested that L-asparaginase of T. pyriformis exists in four interconvertible forms: Form A, phosphorylated complexed with lipids, form HA, dephosphorylated (highly active), form I, free of lipids, (inactive) and form B, free of lipids and phosphate.     

Endogenous protein kinase-C activity and phosphorylated proteins in messenger ribonucleoprotein complexes of developing embryos of alfalfa.

Developing somatic and zygotic embryos of alfalfa (Medicago sativa L.) exhibited endogenous protein kinase activity and protein acceptors of phosphate groups using both cell-free translational extracts and oligo(dT)-cellulose-column-purified mRNPs. The cell-free-translation extracts from pre-cotyledonary-stage somatic embryos had approximately 50- and 100-fold more protein kinase activity than cotyledonary-stage somatic and zygotic embryos. Several polypeptides were phosphorylated; some of them were unique to the early stage and some to the late-stage developing embryos. A 65 kDa protein was phosphorylated heavily in pre-cotyledonary-stage somatic embryos. This phosphorylated protein was comprised of three main components, two of which were phosphorylated heavily. Heat-shock treated-embryos lost their exitant kinase activity and at the same time another form of protein kinase activity was activated which phosphorylated a novel 28 kDa protein. Endogenous protein kinase activity was also observed within the mRNPs of polysomal and non-polysomal fractions of developing embryos, and this phosphorylated only 65, 43 and 30 kDa proteins within these fractions. A 30 kDa protein from the pre-cotyledonary-stage somatic embryos showed a higher affinity for accepting phosphate groups than the proteins from cotyledonary-stage somatic or zygotic embryos. The activity of protein kinase was largely c-AMP-independent, but was dependent on Ca2+, phospholipid and phorbol ester. The enzyme belongs to the protein kinase-C family; the 65 kDa protein cross-reacts with antibodies made against protein kinase-C (alpha- and beta-isoforms) and it may be an autophosphorylated protein.