Phosphorylation of isocitrate lyase in Escherichia coli

Isocitrate lyase from Escherichia coli becomes phosphorylated in vitro by an endogenous kinase when partially purified extracts are incubated with [gamma-32P]ATP. Treatment of isocitrate lyase with histidine modifying reagents, and alkaline hydrolysis of in vitro phosphorylated enzyme indicated the presence of a phosphohistidine residue. Phosphorylation of isocitrate lyase can also occur in vivo, which indicates a possible regulatory significance of this modification. In addition to phosphorylation, isocitrate lyase is capable of incorporating label from both [alpha-32P]ATP and [14C]ATP suggesting that more than one type of covalent modification occurs on this enzyme. This report reviews the studies which have demonstrated the phosphorylation and modification of isocitrate lyase from Escherichia coli.     

Incorporation of [32P]- and [14C]-ATP intoEscherichia coli isocitrate lyase

InEscherichia coli, isocitrate lyase has been shown to be phosphorylated in vitro by [-³²P]-ATP on histidine residues. This phosphorylation is believed to be necessary for activity of this enzyme. Previous work has shown that treatment of isocitrate lyase with acid phosphatase leads to a decrease in activity as well as a loss of incorporated [³²P]-phosphate in a time-dependent manner. In addition to phosphorylation by [-³²P]ATP, isocitrate lyase has been found to incorporate radioactive label from [-³²P]ATP and from [¹⁴C]ATP. This finding may indicate that more than one type of covalent modification occurs on this enzyme. Isocitrate lyase activity, inE. coli, may be regulated by posttranslational modification in several ways.     

Studies on a rat-liver cell-sap protein yielding 3-[32P]-phosphohistidine after incubation with [32P]ATP and alkaline hydrolysis. Identification of the protein as ATP citrate lyase

1. The rat-liver cell-sap material from which 3-[32P]phosphohistidine was previously isolated after incubation with [gamma-32P]ATP and alkaline hydrolysis, was shown to increase about 6-fold on a high-carbohydrate diet. This increase in 32P labelling corresponded to the increase in ATP citrate lyase activity of livers of rats fed on a high-carbohydrate diet, as reported by others. 2. ATP citrate lyase [ATP:citrate oxaloacetate-lyase (CoA-acetylating and ATP-dephopshorylating), EC 4.1.3.8] was purified from rat liver essentially according to the method of Plowman and Cleland (J. Biol. Chem., 242 (1967) 4239). The purified enzyme was incubated for a short time at 0 degree with [gamma-32P]ATP in the presence of 20 mM magnesium acetate. The phosphorylated protein was hydrolysed in alkali and the main part of the radioactivity was identified as 3-[32P]phosphohistidine. The identity of the phosphorylated amino acid was established by Dowex-1 chromatography, paper electrophoresis, paper chromatography and by analysis of the stability to acid. 3. It is concluded from these and previous results from this laboratory that ATP citrate lyase and nucleoside diphosphate kinase (ATP:nucleoside diphosphate phosphotransferase, EC 2.7.4.6) account for most of the normal rat-liver cell-sap protein which is rapidly phosphorylated by ATP.     

Phosphorylation of histidine in proteins by a nuclear extract of Physarum polycephalum plasmodia

A high salt nuclear extract from the true slime mold Physarum polycephalum was used as a source of kinase activity for the incubation of calf thymus histones with [gamma-32P]ATP. A major proportion of the 32P incorporated into histones was acid-labile and alkali-stable. The nature of the alkali-stable phosphorylated component was analyzed by subjecting the phosphorylated protein to total alkaline hydrolysis and separating the resultant phosphoamino acids by anion exchange chromatography. The 32P-labeled material co-chromatographed with phosphohistidine standards and did not co-chromatograph with phosphoserine, phosphothreonine, or phosphotyrosine standards. In similar experiments using reversed phase high-performance liquid chromatography to separate the phosphoamino acids, the 32P-labeled phosphoamino acid behaved like the 1-isomer of phosphohistidine, in not being retained by the column, and unlike 3-phosphohistidine, phosphoserine, phosphothreonine, phosphotyrosine, and phosphoarginine, which were all retained on the column. Histone H4 was a good substrate for the histidine kinase activity and the location of the phosphorylated histidine residue was probed by peptide mapping using chymotrypsin or V8 protease. Both maps were consistent with labeling of histidine 75 and inconsistent with labeling of histidine 18. The data show that Physarum nuclei contain a major kinase activity which produces phosphohistidine. The methods we have developed for studying this kinase activity provide the basis for a complete characterization of the structure and function of the Physarum enzyme and can be applied to the study of similar kinase activities in other systems.     

Amino acid sequences around the sites of phosphorylation in the pig heart pyruvate dehydrogenase complex.

1. When pig heart pyruvate dehydrogenase complex was phosphorylated to completion with [gamma-32P]ATP by its intrinsic kinase, three phosphorylation sites were observed. The amino acid sequences around these sites were: sequence 1, Tyr-Gly-Met-Gly-Thr-Ser(P)-Val-Glu-Arg; and sequence 2, Tyr-His-Gly-His-Ser(P)-Met-Ser-Asp-Pro-Gly-Val-Ser(P)-Tyr-Arg. 2. When phosphorylated to inactivation by repetitive additions of limiting quantities of [gamma-32P]ATP, phosphate was incorporated mainly (more than 90%) into Ser-5 of sequence 2. Phosphorylation of this site thus results in activation of pyruvate dehydrogenase. 3. If Ser-5 is phosphorylated with ATP and the enzyme then incubated with [gamma-32P]ATP, phosphorylation of the remaining sites occurred. Ser-12 of sequence 2 is phosphorylated about twice as rapidly as Ser-6 of sequence 1. 4. Incubation of pyruvate dehydrogenase with excess [gamma-32P]ATP with termination of phosphorylation at about 50% complete inactivation showed that Ser-5 of sequence 2 was phosphorylated most rapidly, but also that Ser-12 of sequence 2 was significantly (15% of total) phosphorylated. Ser-6 sequence 1 contained about 1% total P. 5. These results suggest that addition of limiting amounts of ATP produces primarily phosphorylation of Ser-5 of sequence 2 (inactivating site). This also occurs during incubation with excess ATP before complete inactivation occurs, but a greater occupancy of other sites also occurs during this treatment.     

Phosphorylation of ATP citrate lyase in response to glucagon.

Incubation of hepatocytes with [32P]orthophosphate resulted in the incorporation of 32P into material that is precipitated by reaction with antibodies to ATP citrate lyase. The amount of radioactivity precipitated was decreased when unlabeled, purified ATP citrate lyase was added to extracts of hepatocytes that had been incubated with [32P]orthophosphate. Addition of glucagon to hepatocytes that had been preincubated with [32P]orthophosphate resulted in a 56% increase in acid-stable 32P in the trichloroacetic acid-insoluble portion of immunoprecipitates. Catalytic phosphate bound to ATP citrate lyase reaction with ATP and Mg2+ is acid-labile; thus, glucagon-dependent phosphorylation is distinguished from the catalytic phosphate. When hepatocytes were incubated in the absence of [32P]orthophosphate and extracted in a medium containing [gamma-32P]ATP, no acid-stable 32P was present in immunoprecipitates. This indicates that the incorporation into ATP citrate lyase of acid-stable phosphate occurs prior to extraction of the enzyme. Preliminary studies, using a procedure that allows for measurement of enzyme activity starting 1 min after beginning the extraction of lyase from hepatocytes, have shown no difference in lyase activity when hepatocytes are treated with or without glucagon.     

In vitro phosphorylation ofEscherichia coli isocitrate lyase

The in vitro phosphorylation of isocitrate lyase was demonstrated in partially purified sonic extracts ofEscherichia coli. Extracts were incubated with [gamma³²P]-ATP and subsequently analyzed by two-dimensional polyacrylamide gel electrophoresis. Isocitrate lyase was determined to be phosphorylated by autoradiography and Western blot analyses of the gels. Purified isocitrate lyase comigrates with the phosphorylated form of the enzyme; this suggests that the enzyme may become catalytically active concomitant with phosphorylation.     

Lipid phosphorylation in chloroplast envelopes. Evidence for galactolipid CTP-dependent kinase activities

Lipid phosphorylation takes place within the chloroplast envelope. In addition to phosphatidic acid, phosphatidylinositol phosphate, and their corresponding lyso-derivatives, we found that two novel lipids underwent phosphorylation in envelopes, particularly in the presence of carrier-free [gamma-(32)P]ATP. These two lipids incorporated radioactive phosphate in chloroplasts in the presence of [gamma-(32)P]ATP or [(32)P]P(i) and light. Interestingly, these two lipids were preferentially phosphorylated in envelope membranes in the presence [gamma-(32)P]CTP, as the phosphoryl donor, or [gamma-(32)P]ATP, when supplemented with CDP and nucleoside diphosphate kinase II. The lipid kinase activity involved in this reaction was specifically inhibited in the presence of cytosine 5'-O-(thiotriphosphate) (CTPgammaS) and sensitive to CTP chase, thereby showing that both lipids are phosphorylated by an envelope CTP-dependent lipid kinase. The lipids were identified as phosphorylated galactolipids by using an acid hydrolysis procedure that generated galactose 6-phosphate. CTPgammaS did not affect the import of the small ribulose-bisphosphate carboxylase/oxygenase subunit into chloroplasts, the possible physiological role of this novel CTP-dependent galactolipid kinase activity in the chloroplast envelope is discussed.     

The enzymatic preparation of [alpha-(32)P]nucleoside triphosphates, cyclic [32P] AMP, and cyclic [32P] GMP.

A method has been developed for the enzymatic preparation of alpha-(32)P-labeled ribo- and deoxyribonucleoside triphosphates, cyclic [(32)P]AMP, and cyclic [(32)P]GMP of high specific radioactivity and in high yield from (32)Pi. The method also enables the preparation of [gamma-(32)P]ATP, [gamma-(32)P]GTP, [gamma-(32)P]ITP, and [gamma-(32)P]-dATP of very high specific activity and in high yield. The preparation of the various [alpha-(32)P]nucleoside triphosphates relies on the phosphorylation of the respective 3'-nucleoside monophosphates with [gamma-(32)P]ATP by polynucleotide kinase and a subsequent nuclease reaction to form [5'-(32)P]nucleoside monophosphates. The [5'-(32)P]nucleoside monophosphates are then converted enzymatically to the respective triphosphates. All of the reactions leading to the formation of [alpha-(32)P]nucleoside triphosphates are carried out in the same reaction vessel, without intermediate purification steps, by the use of sequential reactions with the respective enzymes. Cyclic [(32)P]AMP and cyclic [(32)P]GMP are also prepared enzymatically from [alpha-(32)P]ATP or [alpha-(32)P]GTP by partially purified preparations of adenylate or guanylate cyclases. With the exception of the cyclases, all enzymes used are commerically available. The specific activity of (32)P-labeled ATP made by this method ranged from 200 to 1000 Ci/mmol for [alpha-(32)P]ATP and from 5800 to 6500 Ci/mmol for [gamma-(32)P]ATP. Minor modifications of the method should permit higher specific activities, especially for the [alpha-(32)P]nucleoside triphosphates. Methods for the use of the [alpha-(32)P]nucleoside phosphates are described for the study of adenylate and guanylate cyclases, cyclic AMP- and cyclic GMP phosphodiesterase, cyclic nucleotide binding proteins, and as precursors for the synthesis of other (32)P-labeled compounds of biological interest. Moreover, the [alpha-(32)P]nucleoside triphosphates prepared by this method should be very useful in studies on nucleic acid structure and metabolism and the [gamma-(32)P]nucleoside triphosphates should be useful in the study of phosphate transfer systems.     

Bovine brain Na+, K+-stimulated ATP phosphohydrolase studied by a rapid-mixing technique. Detection of a transient [32P]phosphoenzyme formed in the presence of potassium ions.

1. Conditions for binding of [gamma-32P]ATP to bovine brain Na+,K+-stimulated ATPase were investigated by the indirect technique of measuring the initial rate of 32P-labelling of the active site of the enzyme. 2. At 100 muM [gamma-32P]ATP in the presence of 3 mM MgCl2, approximately the same very high rate of formation of [32P]phosphoenzyme was obtained irrespective of whether [gamma-32P]ATP was added to the enzyme simultaneously with, or 70 ms in advance of the addition of NaCl. A comparatively slow rate of phosphorylation was obtained at 5 muM[gamma-32P]ATP without preincubation. However, on preincubation of the enzyme with 5 muM[gamma-32P]ATP a rate of formation of [32P]phosphoenzyme almost as rapid as at 100 muM[gamma-32P]ATP was observed. 3. A transient [32P]phosphoenzyme was discovered. It appeared in the presence of K+, under conditions which allowed extensive binding of [gamma-32P]-ATP. The amount of [gamma-32P]ATP that could be bound to the enzyme seemed to equal the amount of [32P] phosphorylatable sites. 4. The formation of the transient [32P] phosphoenzyme was inhibited by ADP. The transient [32P] phosphoenzyme was concluded mainly to represent the K+-insensitive and ADP-sensitive E1-32P. 5. When KCl was present in the enzyme solution before the addition of NaCl only a comparatively slow rate of phosphorylation was observed. On preincubation of the enzyme with [gamma-32]ATP an increase in the rate of formation of [32P] phosphoenzyme was obtained, but there was no transient [32P]-phosphoenzyme. The transient [32P]phosphoenzyme was, however, detected when the enzyme solution contained NaCl in addition to KCl and the phosphorylation was started by the addition of [gamma-32P]ATP.     

Phosphorylation of rat liver mitochondrial glycerol-3-phosphate acyltransferase by casein kinase 2

We have previously shown rat liver mitochondrial glycerol-3-phosphate acyltransferase (mtGAT), which catalyzes the first step in de novo glycerolipid biosynthesis, is stimulated by casein kinase 2 (CK2) and that a phosphorylated protein of approximately 85 kDa is present in CK2-treated mitochondria. In this paper, we have identified the (32)P-labeled 85-kDa protein as mtGAT. We have also investigated whether the phosphorylation of mtGAT is because of CK2. Mitochondria were treated with CK2 and [gamma-(32)P]GTP as the phosphate donor. Autoradiography, Western blot, and immunoprecipitation results showed mtGAT was phosphorylated by CK2. Next, we incubated mitochondria with CK2 and either ATP or GTP, in the presence of heparin, a known inhibitor of CK2. Heparin inhibited CK2-induced stimulation of mtGAT activity; this inhibition resulted in decreased (32)P-labeling of mtGAT. Additionally, mitochondria were treated with CK2 and [gamma-(32)P]ATP in the presence of staurosporine (a serine/threonine protein kinase inhibitor), genistein (a tyrosine kinase inhibitor), and 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole (DRB, a CK2 inhibitor). Only DRB, the CK2 inhibitor, greatly reduced the amount of (32)P-incorporation into mtGAT by CK2. Finally, isolated mitochondrial outer membrane was incubated with cytosol in the presence of [gamma-(32)P]GTP; (32)P-labeled mtGAT was detected. Collectively, these data suggest that CK2 phosphorylates mtGAT. The impact of our results in the regulation of mtGAT and other anabolic processes is discussed.     

Phosphorylation of calf thymus RNA polymerase II by nuclear cyclic 3',5'-AMP-independent protein kinase.

Nucleoplasmic RNA polymerase II (nucleosidetriphosphate:RNA nucleotidyltransferase, EC 2.7.7.6) from calfthymus is phosphorylated by homologous cyclic AMP-independent protein kinase (ATP:protein phosphotransferase, EC 2.7.1.37). Polyacrylamide gel electrophoresis of the 32P-labeled RNA polymerase II under non-denaturing conditions revealed that both forms of the enzyme were phosphorylated. Polyacrylamide gel electrophoresis of the 32P-labeled RNA polymerase II under denaturing conditions showed that the 25 000 dalton subunit was the phosphate acceptor subunit. Partial acid hydrolysis of the 32P-labeled RNA polymerase II followed by ion-exchange chromatography revealed serine and threonine as the [32P]phosphate acceptor amino acids. Phosphorylation of the RNA polymerase II was accompanied by a stimulation of enzymatic activity and was dependent upon the presence of ATP.     

The VirA protein of Agrobacterium tumefaciens is autophosphorylated and is essential for vir gene regulation.

The virA and virG gene products are required for the regulation of the vir regulon on the tumor-inducing (Ti) plasmid of Agrobacterium tumefaciens. VirA is a membrane-associated protein which is homologous to the sensor molecules of other two-component regulatory systems. We overproduced truncated VirA proteins in Escherichia coli by deleting different lengths of the 5'-coding region of the virA gene and placing these genes under lacZ control. These proteins were purified from polyacrylamide gels and renatured. The renatured proteins became radiolabeled when they were incubated with [gamma-32P]ATP but not with [gamma-32P]GTP or [alpha-32P]ATP, which suggests an ATP gamma-phosphate-specific autophosphorylation. The smallest VirA protein, which retained only the C-terminal half of the protein, gave the strongest autophosphorylation signal, which demonstrates that the C-terminal domain has the autophosphorylation site. The phosphorylated amino acid was identified as phosphohistidine, and a highly conserved histidine was found in all of the VirA homologs. When this histidine was changed to glutamine, which cannot be phosphorylated, the resulting VirA protein lost both its ability to autophosphorylate and its biological function as a vir gene regulator. Results of this study indicate that VirA autophosphorylation is required for the induction of the vir regulon and subsequent tumor induction on plants by A. tumefaciens.     

Direct evidence for an ADP-sensitive phosphointermediate of (K+ + H+)-ATPase

Direct evidence for the occurrence of an ADP-sensitive phosphoenzyme of (K+ + H+)-ATPase, the proton-pumping system of the gastric parietal cell is presented. The enzyme is phosphorylated with 5 microM [gamma-32P]ATP in 50 mM imidazole-HCl (pH 7.0) and in the presence of 7-15 microM Mg2+. Addition of 5 mM ADP to this preparation greatly accelerates its hydrolysis. We have been able to establish this by stopping the phosphorylation with radioactive ATP, by adding 1 mM non-radioactive ATP, which leads to a slow monoexponential process of dephosphorylation of 32P-labeled enzyme. The relative proportion of the ADP-sensitive phosphoenzyme is 22% of the total phosphoenzyme. Values for the rate constants of breakdown and interconversion of the two phosphoenzyme forms have been determined.     

Inhibition of alpha-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 alpha-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 intravesicular 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 [gamma-32P]ATP is incorporated into protein phosphomonoester linkages.     

Purification and phosphorylation of the Arc regulatory components of Escherichia coli.

In Escherichia coli, a two-component signal transduction system, consisting of the transmembrane sensor protein ArcB and its cognate cytoplasmic regulatory protein ArcA, controls the expression of genes encoding enzymes involved in aerobic respiration. ArcB belongs to a subclass of sensors that have not only a conserved histidine-containing transmitter domain but also a conserved aspartate-containing receiver domain of the regulator family. 'ArcB (a genetically truncated ArcB missing the two transmembrane segments on the N-terminal end) and ArcA were purified from overproducing cells. Autophosphorylation of 'ArcB was revealed when the protein was incubated with [gamma-32P]ATP but not with [alpha-32P]ATP or [gamma-32P]GTP. When ArcA was incubated in the presence of 'ArcB and [gamma-32P]ATP, ArcA acquired radioactivity at the expense of the phosphorylated protein 'ArcB-32P. When a limited amount of 'ArcB was incubated with excess ArcA and [gamma-32P]ATP, ArcA-32P increased linearly with time. Under such conditions, for a given time period the amount of ArcA phosphorylated was proportional to the concentration of 'ArcB. Thus, 'ArcB acted as a kinase for ArcA. Chemical stabilities of the phosphorylated proteins suggested that 'ArcB-32P contained both a histidyl phosphate and an aspartyl phosphate(s) and that ArcA-32P contained only an aspartyl phosphate(s).     

Metabolic regulation during early frog development: flow of glycolytic carbon into phospholipids in Xenopus oocytes and fertilized eggs

32P-labeled glucose 6-phosphate, [32P]phosphoenolpyruvate, and [gamma-32P]ATP were injected into oocytes and fertilized eggs of Xenopus laevis, and the incorporation of the 32P label was followed into phospholipids. Several classes of phospholipids incorporated 32P label from the injected glycolytic intermediates, including lysophosphatidic acid, phosphatidic acid, phosphatidylinositol, and phosphatidylinositol phosphates, inferring de novo synthesis of these lipids from dihydroxyacetone phosphate or glycerol 3-phosphate. Injection of [gamma-32P]ATP into oocytes and fertilized eggs led to labeling of phosphatidylinositol phosphate and phosphatidylinositol bisphosphate, indicating an active phosphatidylinositol cycle in resting oocytes and fertilized eggs. Maturation and fertilization of the oocyte led to a qualitative change in phosphatidylinositol metabolism, increased labeling of phosphatidylinositol phosphate compared to phosphatidylinositol bisphosphate (either from glycerol 3-phosphate or from ATP). This change occurs late in the maturation process, and the new pattern of phosphatidylinositol metabolism is maintained during the rapid cleavage stages of early embryogenesis.     

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.     

Protein phosphorylation during 1-methyladenine-induced maturation of Asterias oocytes

Maturation was induced in Asterias oocytes with 1-methyladenine (1-MA) at a final concentration of 2 microM. At 5, 10, and 30 min of treatment, oocytes were homogenized and the cytosolic fraction was prepared. The cytosol was incubated with [gamma-32P]ATP and [gamma-32P]GTP. The phosphorylated proteins were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and the radioactivity in the gels was determined by autoradiography. The cytosol prepared from 1-MA-treated oocytes incubated with [gamma-32P]ATP showed a marked increase in the radiolabeling of proteins with estimated molecular weights of 70,000 and 62,000 Da. With [gamma-32P]GTP a 56,000-Da protein showed increased radiolabeling. The present finding suggests that an early biochemical event of 1-MA-induced oocyte maturation in Asterias is the stimulation of phosphorylation of specific proteins.     

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.