Isolation and characterization of phosphothioredoxin from Excherichia coli.

Thioredoxin was isolated as a phosphoprotein from actively growing cultures of Escherichia coli. Labeling was performed in vivo by growing cells in the presence of 32P-labeled inorganic phosphate, and phosphothioredoxin was purified in one step by immunoabsorption to a thioredoxin antibody column. The stoichiometry of phosphate bound was 0.7 to 0.8 mol of phosphate/mol of thioredoxin. The phospho-amino acid linkage was identified as a thiol phosphate by several criteria: (a) the maximum lability of the phosphate bond was between pH 2.5 and 3.5 (t1/2 (37 degrees) = 200 h (pH 7 to 8); 0.4 h (pH 3.0); 200 h (pH 1.0)); (b) the phosphate linkage was very labile in the presence of iodine at neutral pH (t1/2 less than 1 min); and the phosphopeptide was identified as Cys32-Gly-Pro-Cys35-Lys, the same sequence previously implicated as the active site for disulfide-linked oxidation-reduction reactions. Phosphate was distributed on either cysteine, with 60% of the phosphate bound to cysteine32. Results are discussed in terms of the possible role of phosphothioredoxin as an intermediate in phosphotransferase reactions.     

Autophosphorylation of Mucor rouxii cAMP-dependent protein kinase and its role in holoenzyme activation.

Two phosphoproteins of 53,000 and 63,000 mol. wt detected in partially purified preparations of Mucor rouxii cAMP-dependent protein kinase submitted to phosphorylation conditions with [gamma-32P]ATP are demonstrated to be the result of the autophosphorylation of its regulatory subunit, according to the following criteria: (1) linearity of phosphate incorporation with enzyme sample; (2) independence of phosphate incorporation on temperature; (3) correlation of the phosphoproteins with enzymatic activity in a DEAE-Sepharose chromatography; (4) specific elution of the phosphorylated proteins from cAMP-agarose; (5) phosphorylation of the purified regulatory subunit. Antibodies specific against Mucor regulatory subunit detected an intact subunit of 72,000 mol. wt in crude extracts. Autophosphorylation of the fungal protein kinase A promotes activation of the holoenzyme by cAMP since: (1) under conditions of partial activation, increase of activity is observed when using the phosphoform of the enzyme; (2) release of free catalytic subunit from cAMP-agarose is enhanced when the holoenzyme is previously phosphorylated.     

Estimation of pyruvate,phosphate dikinase activity in maize leaf tissue by (phosphoenolpyruvate plus pyrophosphate)-dependent phosphorylation of AMP

Crude extracts of maize leaf tissue catalysed the phosphorylation of AMP by ³²PPi in the presence of phosphoenolpyruvate (PEP). The reaction was enhanced by F^? and NH₄⁺. The optimum concentrations of AMP, PEP and PPi were 0.3, 10 and 1 mM, respectively. Under these conditions, ca75% of the AMP phosphorylated by ³²PPi was present as ATP and ca25 % as ADP. The activity was reversibly cold labile. The specific activity of crude extracts in the presence of F^? was proportional to enzyme concentration only at protein concentrations < 25,μg/ml. Partially purified pyruvate, phosphate dikinase (PPD) from maize leaf quantitatively phosphorylated AMP to ATP in a (PEP plus PPi)-dependent reaction with the concomitant production of 0.9 mol of pyruvate per mol of AMP phosphorylated. It was concluded that (PEP plus PPi)-dependent phosphorylation of AMP provides a reliable method for estimating PPD activity in crude extracts of maize. Crude maize extracts also catalysed ³²Pi-ATP and ³²PPi-ATP exchange but these activities were not specific for PPD.     

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.     

The phosphorylation of calmodulin and calmodulin fragments by kinase fractions from bovine brain

The phosphorylation of intact calmodulin and of fragments obtained by trypsin digestion was studied, using a protein kinase partially purified from bovine brain. Brain extracts were made in the presence of the detergent CHAPS (3-[3-cholamidopropyl)-dimethylammonio]-1-propanesulfonate). The protein kinase catalyzed the incorporation of nearly 1 mol of 32P from [gamma-32P]ATP into calmodulin fragment 1-106. Incorporation was exclusively into serine 101. With fragment 78-148, the extent of phosphorylation was somewhat less and 32P appeared mainly in threonine residues. Fragment 1-90 was also a fairly good substrate, but the phosphorylation of intact calmodulin never exceeded 0.01 mol per mol. Little or no phosphorylation was seen with parvalbumin, the brain Ca2+-binding protein (CBP-18) and intestinal calcium-binding protein. The protein kinase had no requirement for cAMP or phospholipids. High levels of Mg2+ (60-70 mM) stimulated phosphorylation of the fragments 20-fold. Millimolar concentrations of Ca2+ were inhibitory. It is suggested that the calmodulin fragments were in a conformation more favorable for phosphorylation than intact soluble calmodulin.     

Insulin action inhibits insulin-like growth factor-II (IGF-II) receptor phosphorylation in H-35 hepatoma cells. IGF-II receptors isolated from insulin-treated cells exhibit enhanced in vitro phosphorylation by casein kinase II

Insulin caused a rapid, dose-dependent increase in the binding of 125I-insulin-like growth factor-II (IGF-II) to the surface of cultured H-35 hepatoma cells. The [32P]phosphate content of the IGF-II receptors, immunoprecipitated from extracts of H-35 cell monolayers previously incubated with [32P]phosphate for 24 h, was decreased after brief exposure of the cells to insulin. Analysis of tryptic digests of labeled IGF-II receptors by bidimensional peptide mapping revealed that the decrease in the content of [32P]phosphate occurred to varying degrees on three tryptic phosphopeptides. Thin layer electrophoresis of an acid hydrolysate of isolated IGF-II receptors revealed the presence of [32P] phosphoserine and [32P]phosphothreonine. Insulin treatment of cells caused a decrease in the labeled phosphoserine and phosphothreonine content of IGF-II receptors. The ability of a number of highly purified protein kinases (cAMP-dependent protein kinase, protein kinase C, phosphorylase kinase, and casein kinase II) to catalyze the phosphorylation of purified IGF-II receptors was examined. Casein kinase II was the only kinase capable of catalyzing the phosphorylation of the IGF-II receptor on serine and threonine residues under the conditions of our assay. Bidimensional peptide mapping revealed that the kinase catalyzed phosphorylation of the IGF-II receptor on a tryptic phosphopeptide which comigrated with the main tryptic phosphopeptide found in receptors obtained from cells labeled in vivo with [32P]phosphate. IGF-II receptors isolated by immunoadsorption from insulin-treated H-35 cells were phosphorylated in vitro by casein kinase II to a greater extent than the receptors isolated from control cells. Similarly, IGF-II receptors from plasma membranes obtained from insulin-treated adipocytes were phosphorylated by casein kinase II to a greater extent than the receptors from control adipocyte plasma membranes. Thus, the insulin-regulated phosphorylation sites on the IGF-II receptor appear to serve as substrates in vivo for casein kinase II or an enzyme with similar substrate specificity.     

Increased phosphorylation of a specific protein in mitogen-stimulated lymphocytes

The phosphorylation of proteins from murine splenic lymphocytes was studied. When the phosphorylation of proteins in extracts from Concanavalin A (ConA)-treated lymphocytes was compared with that of resting lymphocytes, there was only one detectable difference between them. A protein of 135,000 mol. wt was highly phosphorylatable in extracts from ConA-treated cells while phosphate incorporation into this protein was slight in extracts from untreated cells. This effect could be observed 12 h after ConA treatment and was maximal during S phase. This soluble protein was also phosphorylated in intact lymphocytes.     

The structure of O-specific polysaccharide of Proteus mirabilis 027 containing amino acids and phosphoethanolamine

The following structure of the repeating unit of the Proteus mirabilis O27 O-specific polysaccharide was established: (formula; see text) where (formula; see text) is N-glucopyranuronoyl-L-lysine, (formula; see text) is N-galactopyranuronoyl-L-alanine. The polysaccharide was parially solvolysed with anhydrous HF and the resulting dephosphorylated tri- and tetrasaccharide with N-acetylglucosamine at the reducing end were studied by means of 1H and 13C NMR spectroscopy and (for methylated derivative of trisaccharide) mass-spectrometry. Smith degradation of the polysaccharide afforded linear polymer, and its structure was investigated by 13C NMR spectroscopy. The position of the ethanolamine phosphate group was determined by means of the analysis of the phosphorylation effects in the 13C NMR spectra of the linear and branched polysaccharides.     

Evidence for phosphorylation of yeast phosphofructokinase

Radioactively labelled material from yeast cells grown in the presence of [32P]phosphate was specifically recognized by antibodies raised against yeast phosphofructokinase. Purified yeast phosphofructokinase was phosphorylated in a cyclic AMP-independent manner by a protein kinase enriched from yeast extracts. This phosphorylation occurred specifically on the beta-subunit, and 0.56 mol of phosphate/mol of subunit was incorporated. The results indicate the phosphorylation of yeast phosphofructokinase both in vivo and in vitro. Phosphofructokinase phosphorylated in vitro was more stable against proteolytic degradation compared to the non-phosphorylated enzyme.     

Cysteine metabolism in cultured tobacco cells

Transported l-[(35)S]cysteine was rapidly metabolized by cultured tobacco cells when supplied to the cells at 0.02 millimolar or 0.5 millimolar. The internal cysteine pool was expandable to approximately 2400 nmoles per gram fresh weight.The (35)S label derived from cysteine was found in several metabolites. The amount of label in glutathione and sulfate was directly proportional to the internal l-[(35)S]cysteine, while the levels of labeled methionine and protein were apparently independent of internal labeled cysteine. Cysteine was more rapidly metabolized when the external cysteine concentration was low (0.02 millimolar) with up to 90% of the (35)S label present as compounds other than cysteine.The initial step in cysteine degradation yielded pyruvate, sulfide, and presumably NH(4) (+). Stoichiometry studies using extracts prepared from acetone powders of tobacco cells indicated that pyruvate and sulfide were produced in a 1:1 ratio. The catabolic reaction was linear with respect to time and amount of protein and had a pH optimum of 8 in crude extracts. Preliminary kinetic data indicated the K(m) to be approximately 0.2 millimolar. The extractable degradative activity was enhanced 15- to 20-fold by preincubating the cells for 24 hours in 0.5 millimolar cysteine. The extractable specific enzyme activity roughly reflected the growth curve of the cells in culture. Maximal cysteine degradation was observed in extracts prepared from late log phase cultures that were preincubated in cysteine, while little activity was found in similar extracts from stationary phase cultures. These results are consistent with an inducible catabolic enzyme similar to the cysteine desulfhydrase from bacteria.     

Epidermal growth factor stimulates the phosphorylation of the calcium-dependent 35,000-dalton substrate in intact A-431 cells

We have previously reported the isolation of a 35-kDa protein from A-431 cells that, in the presence of Ca2+, is an excellent in vitro substrate for the epidermal growth factor (EGF) receptor/kinase present in membrane preparations (Fava, R. A., and Cohen, S. (1984) J. Biol. Chem. 259, 2636-2645). In this communication we demonstrate that the phosphorylation of the 35-kDa protein is markedly enhanced in intact, 32P-labeled, A-431 cells following exposure of the cells to EGF. The 35-kDa protein immunoprecipitated from cells treated with EGF is phosphorylated to a 20-120-fold greater extent than comparable preparations from control cells. Both phosphotyrosine and phosphoserine residues are detected in the protein after treatment of the cells with EGF. EGF-dependent phosphorylation of the 35-kDa protein is barely detected unless the intact cells are exposed to EGF for periods greater than 5 min. We suggest that endosomes containing internalized EGF X receptor/kinase complexes are primarily responsible for the observed phosphorylation of the 35-kDa protein in intact cells.     

Glucagon-induced phosphorylation of pyruvate kinase (type L) in rat liver slices.

The effect of glucagon on the phosphorylation of pyruvate kinase in ³²P-labelled slices from rat liver was investigated. Pyruvate kinase was isolated by immunoadsorbent chromatography. The enzyme was partially phosphorylated in the absence of added hormone (0.2 mol of phosphate/mol of enzyme subunit). Upon incubation with 10^?7 M glucagon, the incorporation of [³²P]phosphate was 0.6–0.7 mol/mol of enzyme subunit. Concomitantly, the concentration of intracellular cyclic 3′,5′-AMP increased from 0.3 to 3.2 μM. The phosphorylation inhibited the enzyme activity at low concentrations of phosphoenolpyruvate (60% at 0.5 mM). Almost maximal phosphorylation of the enzyme was reached within 2 min after the addition of glucagon. The concentration of hormone giving half maximal effect on the pyruvate kinase phosphorylation was about 7×10^?9M. The inactivation of the enzyme paralleled the increase in phosphorylation. It is concluded that pyruvate kinase is phosphorylated in the intact liver cell.     

The site of phosphorylation of troponin I in the perfused rabbit heart. The effect of adrenaline.

1. On treatment of the perfused rabbit heart with adrenaline, the total covalently bound phosphate of troponin I increased from 1.14 mol of phosphate/mol to 1.86 mol of phosphate/mol. 2. Covalently bound phosphate could be identified only in the region of the molecule of cardiac troponin I consisting of residues 1--48. 3. When 32P-labelled orthophosphate was present in the perfusion medium the phosphate at serine-20 became radioactively labelled. This residue was the only significant site of phosphorylation that could be identified. 4. The addition of adrenaline caused a 4--5-fold increase in covalently bound [32P]phosphate. Virtually all of the 32P was located at serine-20. 5. It was concluded from these studies that the extent of phosphorylation of serine-20 of cardiac troponin I increased from 30--40% in the control perfused heart to about 100% in the presence of adrenaline.     

Phosphorylation state of acetyl-coenzyme A carboxylase. II. Variation with nutritional condition

Acetyl-CoA carboxylase from liver exhibits a linear inverse relationship between the ratio of enzymic activities at 0 and 2 mM citrate and the extent of phosphorylation by its kinase, and this citrate activity ratio method was used to examine the effect of nutritional conditions on the phosphorylation state of the enzyme. This method showed that the calculated phosphorylation state, being the extent of phosphorylation at sites accessible to carboxylase kinase, was highest in the livers of starved rats, lower in those fed normally, and lower still in starved rats which had been refed for 48 h on a fat-free diet. The actual values were 0.44, 0.26, and 0 mol of P/subunit, respectively, provided that liver samples were frozen rapidly to liquid nitrogen temperatures and extracted with stopping buffers at temperatures well below freezing. Normal homogenization with stopping buffers (containing inhibitors for protein kinases and phosphatases) resulted in much higher calculated phosphorylation states. The effect of nutritional conditions on the phosphorylation state as estimated reported above was confirmed by purifying the carboxylase from livers of rats, measuring the amount of phosphate which could be incorporated by carboxylase kinase, and comparing this with the phosphorylation state calculated from the citrate activity ratio method or the specific activity. Furthermore, treatment with protein phosphatase of carboxylase from starved rats resulted in the largest increase in specific activity, that from the starved/refed rats in the least. Finally, the effects of hyperglycemia on carboxylase and phosphorylase characteristics in the livers of intact rats were ascertained by taking liver samples and preparing crude extracts by the rapid freezing method described above. Hyperglycemia caused a rapid increase in the activity of the carboxylase and a rapid decrease in its putative phosphorylation state as measured by the citrate activity ratio method. Phosphorylase was also dephosphorylated, as indicated by a decrease in phosphorylase a activity. We conclude that the citrate activity ratio method is a valid test for the phosphorylation state of acetyl-CoA carboxylase in crude extracts of tissue.     

Protein phosphorylation in normal and neoplastic development. Phosphorylation of proteins endogenous to foetal tissues and tumours.

The abilities of proteins endogenous to normal and neoplastic tissues to serve as substrates in a protein-phosphorylation reaction in vitro were compared. After the tissue extracts were incubated with [gamma-32P]ATP, the phosphorylated proteins were separated by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis and the dried gels were subjected to radioautography. Considerable incorporation of 32P into a protein of mol.wt. 135000 was observed with extracts from foetal tissues and tumours, but only minimal incorporation into this protein occurred when extracts from adult tissues were used. The ability of this protein to become phosphorylated in vitro may be related to cell proliferation. When ascites cells were incubated with [32P]Pi, one of the major phosphoproteins migrated on sodium dodecyl suphate/polyacrylamide gels at mol.wt. 135000, suggesting that this protein can be phosphorylated both in intact cells and broken-cell preparations. A protein of mol.wt. 87000 was highly phosphorylatable in extracts from solid tumours, but was not phosphorylated in extracts from ascites tumours, foetal or adult tissues. The phosphorylation pattern of these two proteins can thus distinguish solid neoplasms and normal adult tissues from ascites tumours and from foetal tissues. A protein of mol.wt. 49000, which was the most labelled protein in adult tissues, was also one of the major phosphoproteins in foetal and neoplastic tissues. Numerous mechanisms are postulated to explain how the extent of 32P incorporation into a protein could vary as a function of biological state.     

Extensive cAMP-dependent and cAMP-independent phosphorylation of microtubule-associated protein 2

Microtubule-associated protein 2 (MAP 2) is the major substrate for phosphorylation in purified preparations of brain microtubules. In earlier work, we showed that phosphorylation is catalyzed by a type II cAMP-dependent protein kinase tightly associated with MAP 2 itself. In the present study, we have examined the extent of MAP 2 phosphorylation by its associated protein kinase. Using an inorganic phosphate assay, we found that MAP 2 contained from 8 to 13 mol of phosphate/mol of protein as isolated. The catalytic subunit of the MAP 2-associated kinase catalyzed the incorporation of additional phosphate to a final level of 20-22 mol/mol of MAP 2. Potato acid phosphatase was used to remove phosphate from MAP 2. Rephosphorylation of acid phosphatase-treated MAP 2 resulted in maximal incorporation of 13 mol of phosphate/mol of MAP 2. The rates and extent of [32P] phosphate incorporation into as isolated and dephosphorylated MAP 2 were found to be identical, and phosphate was incorporated into identical peptides in the two preparations. These results were interpreted to indicate that MAP 2 contains as many as 13 cAMP-dependent phosphorylation sites, and approximately eight phosphates of as yet undetermined origin.     

The enzymatic conversion of 3'-phosphate terminated RNA chains to 2',3'-cyclic phosphate derivatives

The enzyme, RNA cyclase, has been purified from cell-free extracts of HeLa cells approximately 6000-fold. The enzyme catalyzes the conversion of 3'-phosphate ends of RNA chains to the 2',3'-cyclic phosphate derivative in the presence of ATP or adenosine 5'-(gamma-thio)triphosphate (ATP gamma S) and Mg2+. The formation of 1 mol of 2',3'-cyclic phosphate ends is associated with the disappearance of 1 mol of 3'-phosphate termini and the hydrolysis of 1 mol of ATP gamma S to AMP and thiopyrophosphate. No other nucleotides could substitute for ATP or ATP gamma S in the reaction. The reaction catalyzed by RNA cyclase was not reversible and exchange reactions between [32P]pyrophosphate and ATP were not detected. However, an enzyme-AMP intermediate could be identified that was hydrolyzed by the addition of inorganic pyrophosphate or 3'-phosphate terminated RNA chains but not by 3'-OH terminated chains or inorganic phosphate. 3'-[32P](Up)10Gp* could be converted to a form that yielded, (Formula: see text) after degradation with nuclease P1, by the addition of wheat germ RNA ligase, 5'-hydroxylpolynucleotide kinase, RNA cyclase, and ATP. This indicates that the RNA cyclase had catalyzed the formation of the 2',3'-cyclic phosphate derivative, the kinase had phosphorylated the 5'-hydroxyl end of the RNA, and the wheat germ RNA ligase had catalyzed the formation of a 3',5'-phosphodiester linkage concomitant with the conversion of the 2',3'-cyclic end to a 2'-phosphate terminated residue.     

Localization of the N-ethylmaleimide reactive cysteine in the beef heart mitochondrial ADP/ATP carrier protein

Alkylation of the ADP/ATP carrier protein in beef heart mitochondria by N-ethylmaleimide (NEM) results in inactivation of transport. One out of the four cysteinyl residues contained in 1 mol of carrier subunit of Mr 32 000 is alkylated by NEM. The identification of the alkylated residue to Cys-56 has been achieved by chemical and enzymatic cleavages. The chemical cleavages included cleavages at the nonalkylated cysteinyl residues by cyanide at alkaline pH and at methionyl residues by cyanogen bromide. Enzymatic cleavage involved the use of trypsin and chymotrypsin; the resulting peptides were resolved by high-performance liquid chromatography. Analysis of a small size [14C]NEM-labeled peptide obtained by tryptic and chymotryptic digestion of the [14C]NEM-labeled carrier protein yielded the following amino acid sequence: (Formula: see text) where X is probably a substituted lysine.     

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.     

Protein kinase C phosphorylates P-glycoprotein in multidrug resistant human KB carcinoma cells

Studies were undertaken to identify the protein kinase(s) responsible for P-glycoprotein phosphorylation in multidrug-resistant (KB-V1) human carcinoma cells and to elucidate the functional role of phosphorylation. P-glycoprotein migrated on sodium dodecyl sulfate gels with apparent Mr 150,000 and is termed P150. When KB-V1 membrane vesicles were incubated with [gamma-32P] ATP, P150 was phosphorylated by an endogenous kinase that exhibited properties of membrane-inserted protein kinase C (PKC). Both membrane-bound P150 and purified P150 served as effective substrates for highly purified rat brain PKC which incorporated approximately 0.6 mol of phosphate/mol of P150. Enzyme assays showed that KB-V1 cells exhibit 4-fold higher PKC activity compared with the drug-sensitive KB-3 cell line. The basal phosphorylation of P150 observed in 32P-labeled cells was increased 2-fold by phorbol ester (PMA) treatment and reduced 30% by treatment with the isoquinolinsulfonamide H-7. Phosphopeptide maps of partially digested P150, phosphorylated either in vitro with PKC or in intact 32P-labeled control or PMA-stimulated cells, were indistinguishable from one another. Drug accumulation assays revealed that PMA treatment of KB-V1 cells significantly reduced [3H]vinblastine accumulation induced by verapamil or by tetrandrine. The results suggest that PKC is primarily responsible for P150 phosphorylation in KB-V1 cells and that phosphorylation may play a modulatory role in the drug transport process.