Demonstration of cGMP-dependent protein kinase and cGMP-dependent phosphorylation in cell-free extracts of platelets

Homogenates, membranes and cytosol of rat and human platelets were found to contain cGMP-dependent protein kinase immunoreactivity. Specific cGMP-dependent protein kinase immunoreactivity was about 1.7 pmol protein kinase/mg protein for homogenates of human platelets and 0.7 pmol/mg for homogenates of rat platelets; the majority appeared to be associated with the membrane fraction. In membranes of platelets low concentrations of cAMP (0.5-2 microM) stimulated the phosphorylation of five major proteins with apparent relative molecular masses, Mr, of 240 000, 130 000, 50 000, 42 000 and 22 000 while low concentrations of cGMP (0.5-2 microM) stimulated the phosphorylation of three major proteins with apparent Mr of 130 000, 50 000 and 46 000. An affinity-purified antibody against the cGMP-dependent protein kinase was prepared which specifically inhibited the activity of cGMP-dependent protein kinase. In membranes of human platelets this affinity-purified antibody inhibited the cGMP-stimulated phosphorylation of the three proteins with Mr of 130 000, 50 000 and 46 000 while it had no effect on the cAMP-dependent and cyclic-nucleotide-independent protein phosphorylation. The results demonstrate that platelets contain a cGMP-dependent protein kinase and at least three specific substrates for this enzyme. Two of these substrates, the proteins with apparent molecular Mr of 130 000 and 50 000, are substrates for both cAMP- and cGMP-dependent protein kinase. The protein with apparent Mr of 130 000 appears to be closely related to an intrinsic plasma membrane protein of vascular smooth muscle cells which is a substrate for a membrane-associated cGMP-dependent protein kinase. Therefore, cGMP-dependent protein kinase and cGMP-regulated phosphoproteins may mediate in platelets the intracellular effects of those hormones, vasodilators and drugs which elevate the level of cGMP and inhibit platelet aggregation.     

An effect of fibronectin in epidermal growth factor (EGF)-stimulated membrane protein phosphorylation in human carcinoma cells

Membrane components of about 20 kd (kilodalton) which appear weakly phosphorylated in protein kinase assays of A431 human carcinoma plasma membranes to which epidermal growth factor (EGF) is added, increase their relative phosphorylation when human serum fibronectin is also included in the assay. Membranes prepared from ³²PO₄-labelled cells exposed to EGF and fibronectin prior to organelle isolation revealed the presence of an anodic 20 kd component not detected in cells which were not exposed to fibronectin. No comparable effects were apparent with bovine heart protein kinase and with membrane-associated protein kinase preparations which were not responsive to EGF.     

Insulin inhibits the phosphorylation of the membrane cytoskeletal protein spectrin in pig erythrocytes

Incubation of either ghost membranes with 32P- -ATP or intact erythrocytes with 32P-inorganic phosphate led to phosphorylation of the beta-chain of the major membrane-associated protein spectrin. This phosphorylation was reduced by 30% by insulin (10-100 microU/ml) both in membranes and in intact cells. The results show that the membrane-cytoskeleton is responsive to extracellular signals such as hormone receptor activation.     

Phosphorylation of lens intrinsic membrane proteins by protein kinase C

Two intrinsic proteins of bovine lens membranes with apparent relative molecular masses (Mr, app) of 26,000 and 18,000 were phosphorylated in intact membranes by protein kinase C prepared from either bovine brain or lens. The kinase preparations exhibited histone H1 phosphorylation dependent on calcium and phospholipid but not on cAMP. Sodium dodecyl sulfate/polyacrylamide gel electrophoresis of the lens membranes showed a major band at Mr, app = 26,000 (identified as MP26, the main intrinsic protein of lens fiber cells), an intermediate band at Mr, app = 18,000 and several minor bands. Autoradiography of complete assay mixture containing protein kinase C, calcium, magnesium and [gamma-32P]ATP showed major bands at Mr, app = 18,000 and 26,000. Several lines of evidence indicated that the label at Mr, app = 26,000 was associated with MP26, a protein which has been found in lens junctions and which may form cell-cell channels. Treatment of the phosphorylated membranes with chymotrypsin and V8 protease cleaved the major band at Mr, app = 26,000 to fragments of Mr, app .= 22,000 and 24,000. Label was not detected in the resulting Mr, app = 22,000 peptide, but the Mr, app = 24,000 peptide was found to be labeled. Phosphoamino acid analysis of MP26 indicated that approximately 75% of the label was on phosphoserine and 25% was on phosphothreonine. No label was found on phosphotyrosine. These results differ from those reported for cAMP-dependent phosphorylation of lens proteins. Phosphorylation by protein kinase C may account for some of the labeling of MP26 detected in vivo.     

Phosphorylation of 69-kDa choline acetyltransferase at threonine 456 in response to amyloid-beta peptide 1-42

Choline acetyltransferase synthesizes acetylcholine in cholinergic neurons. In the brain, these neurons are especially vulnerable to effects of beta-amyloid (A beta) peptides. Choline acetyltransferase is a substrate for several protein kinases. In the present study, we demonstrate that short term exposure of IMR32 neuroblastoma cells expressing human choline acetyltransferase to A beta-(1-42) changes phosphorylation of the enzyme, resulting in increased activity and alterations in its interaction with other cellular proteins. Using mass spectrometry, we identified threonine 456 as a new phosphorylation site in choline acetyltransferase from A beta-(1-42)-treated cells and in purified recombinant ChAT phosphorylated in vitro by calcium/calmodulin-dependent protein kinase II (CaM kinase II). Whereas phosphorylation of choline acetyltransferase by protein kinase C alone caused a 2-fold increase in enzyme activity, phosphorylation by CaM kinase II alone did not alter enzyme activity. A 3-fold increase in choline acetyltransferase activity was found with coordinate phosphorylation of threonine 456 by CaM kinase II and phosphorylation of serine 440 by protein kinase C. This phosphorylation combination was observed in choline acetyltransferase from A beta-(1-42)-treated cells. Treatment of cells with A beta-(1-42) resulted in two phases of activation of choline acetyltransferase, the first within 30 min and associated with phosphorylation by protein kinase C and the second by 10 h and associated with phosphorylation by both CaM kinase II and protein kinase C. We also show that choline acetyltransferase from A beta-(1-42)-treated cells co-immunoprecipitates with valosin-containing protein, and mutation of threonine 456 to alanine abolished the A beta-(1-42)-induced effects. These studies demonstrate that A beta-(1-42) can acutely regulate the function of choline acetyltransferase, thus potentially altering cholinergic neurotransmission.     

Characterization of the membrane-bound protein kinase C and its substrate proteins in canine cardiac sarcolemma

Cardiac sarcolemma was purified from canine ventricles. Enrichment of the sarcolemmal membranes was demonstrated by the high (Na+ + K+)-ATPase activity of 28.0 +/- 1.5 mumol Pi/mg protein per h and the high concentration of muscarinic receptors with the Bmax of 8.2 +/- 2.5 pmol/mg protein as determined by [3H]QNB binding. The purified sarcolemma also contains significant levels of a membrane-bound Ca2+ and phospholipid-dependent protein kinase (protein kinase C). To elucidate the protein kinase C activity in sarcolemma, a prior incubation of the membranes with EGTA and Triton X-100 was necessary. The specific activity of protein kinase C was found to be 131.4 pmol Pi/mg per min, in the presence of 6.25 micrograms phosphatidylserine and 0.5 mM CaCl2. Treatment of sarcolemma with 12-O-tetradecanoylphorbol 13-acetate (TPA) and phorbol 12,13-dibutyrate (PBu2) resulted in a concentration-dependent activation of protein kinase C activity. The effect of TPA and PBu2 on protein kinase C in sarcolemma was independent of exogenous Ca2+ and phosphatidylserine. Polymyxin B inhibited phorbol-ester-induced activation of protein kinase C activity. The distribution of protein kinase C in the cytosolic fraction was also examined. The specific activity of the kinase in the cytosolic fraction was 59.7 pmol Pi/mg per min. However, the total protein kinase C activity in the cytosol was 213500 pmol Pi/min, compared to that of 1025 pmol Pi/min in the sarcolemma isolated from approx. 100 g of canine ventricular muscle. Several endogenous proteins in cardiac sarcolemma were phosphorylated in the presence of Ca2+ and phosphatidylserine. The major substrates for protein kinase C were proteins of Mr 94 000, 87 000, 78 000, 51 000, 46 000, 11 500 and 10 000. Most of these substrate proteins have not been identified before. Other proteins of Mr 38 000, 31 000 and 15 000 were markedly phosphorylated in the presence of Ca2+ only. Phosphorylation of phospholamban (Mr 27 000 and 11 000) was also stimulated in the presence of Ca2+ and phosphatidylserine, but the low Mr form of phospholamban was distinct from two other low Mr substrate proteins for protein kinase C. Polymyxin B was more selective in inhibiting the protein kinase C dependent phosphorylation. On the other hand, trifluoperazine selectively inhibited the phosphorylation of phospholamban and Mr 15 000 protein. Although the exact function of this kinase is unknown, based on these observations, we believe that protein kinase C in the cardiac sarcolemma may play an important role in the cell-surface-signal regulated cardiac function.     

The beta subunit of the insulin receptor is an insulin-activated protein kinase

In the presence of adenosine 5'-[gamma-32P]triphosphate ([gamma-32P]ATP) and a partially purified human placental insulin receptor preparation, insulin stimulates the phosphorylation of an Mr 94000 protein in a time- and dose-dependent manner. Half-maximal stimulation of 32P incorporation occurs at (2-3) X 10(-9) M insulin, a concentration identical with the Kd for insulin binding in this preparation. Immunoprecipitations with monoclonal anti-insulin receptor antibody demonstrate that the Mr 94000 protein kinase substrate is a component of the insulin receptor, the beta subunit. If the partially purified, soluble placental receptor preparation is immunoprecipitated and then exposed to [gamma-32P]ATP and insulin, phosphorylation of the Mr 94000 protein is maintained. The photoincorporation of 8-azido[alpha-32P]ATP into placental insulin receptor preparations was carried out to identify the ATP binding site responsible for the protein kinase activity. Photoincorporation into numerous proteins was observed, including both subunits of the insulin receptor. However, when photolabeling was performed in the presence of excess adenosine 5'-(beta, gamma-imidotriphosphate), a nonhydrolyzable ATP derivative, the beta subunit of the insulin receptor was the only species protected from label incorporation. These data indicate that the beta subunit of the insulin receptor has insulin-dependent protein kinase activity. Phosphotyrosine formation is the primary result of this activity in placental insulin receptor preparations.     

Renal brush border membranes from mice with X-linked hypophosphatemia: protein composition, phosphate binding capacity, and protein kinase activity

Phosphate uptake by brush-border membrane (BBM) vesicles prepared from hypophosphatemic mice (Hyp) is reduced by half relative to BBM vesicles from normal mice. To investigate this abnormality, we studied the protein composition of BBM, their capacity to bind inorganic phosphate, and their protein kinase activity with and without the addition of exogenous cAMP, in normal and Hyp mice. Gradient polyacrylamide gel electrophoresis of BBM proteins showed 27 bands which were identical in normal and Hyp mice. Incubation of the membranes with ortho[32P]phosphate at 0 degrees C revealed a phosphate binding protein with an apparent molecular weight (Mr) of 79000, which has been previously identified in rats as the monomer of alkaline phosphatase. In normal mice, the Scatchard plot of phosphate binding was not linear, suggesting heterogeneity of the binding sites with two major components. At high substrate concentrations, the affinity (K) was 1.42 mM and maximal binding (Bmax) was 83 pmol/mg protein. At low substrate concentrations, these values were 0.07 mM and 10.9 pmol/mg, respectively. In Hyp mice BBM, only one binding system was found with K and Bmax values of 0.38 mM and 53.8 pmol/mg. Incubation of the membranes with 25 microM[gamma-32P]ATP resulted in the phosphorylation of 11 proteins. The major band (Mr: 79000) corresponded to the inorganic phosphate binding protein, i.e., to the alkaline-phosphatase monomer. The 11 proteins showed maximal phosphorylation at pH 10. The protein of 79000 Mr showed a second peak of phosphorylation at pH 7.5.(ABSTRACT TRUNCATED AT 250 WORDS)     

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) enhances antibody production and protein kinase activity in murine B cells

Treatment of murine spleen cells with 30 nM TCDD resulted in an approximately 3 fold increase in unstimulated antibody production after 3 days in culture. This response was not accompanied by increased cellular proliferation and may represent an effect of TCDD on B cell activation or differentiation. Since PMA is capable of activating B cells, presumably via PKC, we have compared the effects of PMA and TCDD on protein kinase activation and phosphorylation of endogenous proteins in a highly purified preparation of B cells. In contrast to a reduction of cytosolic PKC activity, the expected effect of PMA, TCDD caused an increase in basal kinase activity with no effect on PKC activity. Addition of either PMA or TCDD resulted in enhanced phosphorylation of a similar profile of proteins, including proteins of Mr 12.2, 14.6, 29.2, 52.3 and 62.7 KDa. Addition of TCDD also resulted in the increased phosphorylation of a protein of Mr 45.2, which was unaffected by PMA. Combined treatment with PMA and TCDD resulted in additive responses. The additive effects of PMA and TCDD suggest an interaction at the level of protein phosphorylation which is mediated by different kinases. Therefore, TCDD may be stimulating B cells via an early effect on an unidentified protein kinase.     

Protein kinase activity associated with pancreatic zymogen granules.

Purified zymogen granules were prepared from rat pancreas by using an iso-osmotic Percoll gradient. In the presence of [gamma-32P]ATP, phosphorylation of several granule proteins was induced by Ca2+, most notably a Mr-13 000 protein, whereas addition of cyclic AMP was without effect. When phosphatidylserine was also added, Ca2+ increased the phosphorylation of additional proteins, with the largest effect on a protein of Mr 62 000. Purified granules were also able to phosphorylate exogenous substrates. Ca2+-induced phosphorylation of lysine-rich histone was enhanced over 3-fold in the presence of phosphatidylserine, and cyclic AMP-activated protein kinase activity was revealed with mixed histone as substrate. The concentrations of free Ca2+ and cyclic AMP required for half-maximal phosphorylation of both endogenous and exogenous proteins were 1-3 microM and 57 nM respectively. Treatment of granules with 0.25 M-KCl resulted in the release of phosphatidylserine-dependent kinase activity into a high-speed granule supernatant. In contrast, granule-protein substrates of Ca2+-activated kinase activity were resistant to KCl extraction, and in fact were present in purified granule membranes. Kinase activity activated by cyclic AMP was not extracted by KCl treatment. It is concluded that phosphorylation of integral membrane proteins in the zymogen granule can be induced by one or more Ca2+-activated protein kinases. Such a reaction is a potential mechanism by which exocytosis may be regulated in the exocrine pancreas by Ca2+-mediated secretagogues.     

Phosphorylation of a pancreatic zymogen granule membrane protein by endogenous calcium/phospholipid-dependent protein kinase

The occurrence of phospholipid-sensitive calcium-dependent protein kinase (referred to as C kinase) and its endogenous substrate proteins was examined in a membrane preparation from rat pancreatic zymogen granules. Using exogenous histone H1 as substrate, C kinase activity was found in the membrane fraction. The kinase was solubilized from membranes using Triton X-100 and partially purified using DEAE-cellulose chromatography. An endogenous membrane protein (Mr approximately equal to 18 000) was found to be specifically phosphorylated in the combined presence of Ca2+ and phosphatidylserine. Added diacylglycerol was effective in stimulating phosphorylation of exogenous histone by the partially purified C kinase, but had no effect upon phosphorylation of the endogenous 18 kDa protein by the membrane-associated C kinase. Phosphorylation of the 18 kDa protein was rapid (detectable within 30 s following exposure to Ca2+ and phosphatidylserine), and highly sensitive to Ca2+ (Ka = 4 microM in the presence of phosphatidylserine). These findings suggest a role for this Ca2+-dependent protein phosphorylation system in the regulation of pancreatic exocrine function.     

Epidermal growth factor, but not insulin, stimulates tyrosine phosphorylation of an endogenous protein of Mr 95,000 in triton extracts of human placental syncytiotrophoblast membranes.

1. Triton extracts of syncytiotrophoblast membranes were incubated with [gamma-32P]ATP, MgCl2 and MnCl2. Addition of epidermal growth factor (EGF) resulted in increased phosphorylation not only of the EGF receptor and a Mr-35,000 protein as previously described, but also a protein of Mr 95,000 on both tyrosine and serine residues. In addition, a small increase in the phosphorylation of a protein of Mr 105,000 was observed. Spermine had a similar effect on the phosphorylation of the Mr-95,000 protein, without affecting the phosphorylation of the other proteins. In the absence of MnCl2, the effect of spermine on the phosphorylation of Mr-95,000 protein was still evident, whereas that of EGF was greatly diminished. 2. The Mr-95,000 protein bound poorly to wheat-germ-lectin-Sepharose and was not precipitated by antisera specific for insulin and EGF receptors. The protein continued to exhibit serine and tyrosine phosphorylation on addition of [gamma-32P]ATP, MgCl2 and MnCl2 to a glycoprotein-depleted fraction prepared by chromatography on wheat-germ-lectin-Sepharose. The extent of phosphorylation was no longer increased by spermine or EGF, but was inhibited by heparin. 3. It is suggested that the Mr-95,000 protein not only is a possible direct substrate for the EGF-receptor (but not the insulin receptor) tyrosine kinase but is a substrate for other endogenous kinases, including a protein tyrosine kinase which is probably not a glycoprotein, and a protein serine kinase with properties similar to those of casein kinase II.     

Tyrosine kinase activity in Pseudomonas aeruginosa

Previous evidence showed that b- and a-type flagellins of Pseudomonas aeruginosa are modified in vivo by phosphorylation at tyrosine. This research was designed to demonstrate phosphorylation of flageliin at tyrosine in vitro. Evidence presented showed that flageilin is labelled by [γ-³²P]-ATP, but not by [α-³²P]-ATP, when incubated with cell envelope fractions. Results suggested that autophosphoryiation of a 42 kDa membrane protein occurred. No activity was detected in cytoplasmic fractions. Flagellin protein was identified by flagella-specific monoclonal antibody (mAb) and was labelled with anti-phosphotyrosine mAb. Confirmation of tyrosine kinase activity was shown by labelling of synthetic poly (Glu:Tyr) as a substrate with [γ-³²P]-ATP. Labelling of poly (Glu:Tyr) was heat sensitive and time dependent. Labelled phosphotyrosine was observed in partial acid hydrolysates of substrates. Using poly (Glu:Tyr) as substrate, tyrosine kinase activity was shown to be inhibited by sulphydryl reagents. It appears that tyrosine kinase and flagellin phosphorylation occur in several Pseudomonas spp. Location of phosphotyrosine in a conserved region of flagellin may serve as a cell signal so that intact flagellin is appropriately exported.     

Cyclic nucleotide-dependent protein phosphorylation in vitellogenic follicles of Hyalophora cecropia

(1) In homogenates of vitellogenic follicles from Hyalophora cecropia, cyclic nucleotides promoted the transfer of label from [gamma32P]-ATP to at least four polypeptides. PKI (6-20) amide, an inhibitor of PKA (cAMP-dependent protein kinase), prevented all four reactions. Quantitative tests using kemptide as a substrate indicated that 80% of the total follicular PKA activity was localized in the follicle cells; labeling at 45, 32, and 27 kDa was particle-associated and also restricted to the follicle cells, while a 58 kDa substrate was labeled only in homogenates of the oocyte. (2) When intact follicles were incubated in [32P]-phosphate and okadaic acid, a protein phosphatase inhibitor, the 32 kDa substrate again exhibited cAMP-dependent labeling. There was thus a physiological relationship between PKA activation and 32 kDa protein phosphorylation, while exposure of at least two of the other three substrates to appropriate kinases required homogenization. The latter was illustrated by phosphorylation of the 42 kDa small subunit of vitellogenin, which occurred only when homogenization mixed the proteins of the yolk bodies with cytoplasmic kinases. (3) PKA activation is known to promote the termination of vitellogenesis, even in the absence of detectable labeling of the 32 kDa substrate. The possibility remains that phosphorylation at 32 kDa concerns later aspects of postvellogenic development that were not tested by the assay system used here.     

Extracellular phosphorylation in the parasite, Leishmania major

Intact promastigotes or cell-free extracts of the parasite Leishmania major were labelled with adenosine 5'[gamma-32P]-triphosphate (ATP). This resulted in the identification of eleven phosphoproteins. [gamma-32P]ATP incorporation into endogenous and exogenous substrates was insensitive to most of the commonly used protein kinase inhibitors and activators indicating that the leishmanial enzyme(s) may represent a new class of kinase(s). In addition, exogenous substrate specificity was inconsistent with the preferences of second messenger-dependent protein kinases. Cyclic AMP had differential effects on phosphorylation in intact cells and lysates. The majority of kinase activity could be attributed to an externally oriented membrane-associated protein kinase(s), as no specific cytosolic phosphoproteins were found and intact cells phosphorylated exogenous substrates. Labelled ATP did not cross the membrane and [alpha-32P]ATP was an unsuitable substrate for the phosphorylation activity. The ectokinase activity on live Leishmania exhibited a different substrate preference when compared to the protein kinase activity in the particulate fraction, suggesting that more than one protein kinase may be present in L. major. Three serine-labelled phosphoproteins were specifically released into the medium. The presence of an ecto-kinase and these released phosphoproteins may play a significant role in host-parasite interactions.     

Plasma membrane protein kinase activity in normal and Rous sarcoma virus-transformed chick embryo fibroblasts.

A preliminary study has been carried out to investigate the effect of Rous sarcoma virus transformation on plasma membrane protein kinase activity in chick embryo fibroblasts. Enzyme activity was measured using an in vitro phosphorylation method employing [gamma-32P]ATP with isolated plasma membranes serving as the source of both protein kinase and protein substrate. In general, the enzymatic properties observed were similar to those of other known protein kinases. However, for maximal activity a marked dependence on high Mg2+ concentrations was noted. Evidence was obtained which showed that cyclic nucleotide-dependent protein kinases were present in membranes from normal cells, but none could be measured in preparations from transformed cells. In addition, transformation appeared to result in a slight increase in basal plasma membrane protein kinase activity.     

Acidic and basic fibroblast growth factors stimulate tyrosine kinase activity in vivo

We assessed the ability of acidic and basic fibroblast growth factor (FGF) to stimulate tyrosine kinase activity in intact cells. Immunoblot with polyclonal antiphosphotyrosine antibodies detected a 90-kDa phosphotyrosine-bearing protein in lysates of Swiss 3T3 cells exposed to pituitary-derived FGF, recombinant acidic FGF, or recombinant basic FGF, but not from unstimulated cells or cells exposed to epidermal growth factor or platelet-derived growth factor. Phosphotyrosine and its analogue phenyl phosphate, but not phosphoserine, phosphothreonine, or tyrosine itself, blocked recognition of the 90-kDa protein by antiphosphotyrosine antiserum. A monoclonal antiphosphotyrosine antibody also recognized the 90-kDa protein and was used to partially purify the protein by immunoaffinity chromatography. Phosphoamino acid analysis of the 90 kDa band revealed that it contained 20% phosphotyrosine, 35% phosphothreonine, and 45% phosphoserine. Tyrosine phosphorylation of the 90-kDa protein was detectable within 30 s and reached a plateau within 10 min of FGF addition. The addition of suramin, which blocks the interaction of FGF with its receptor, caused rapid disappearance of the 90 kDa band. Cell fractionation experiments were consistent with the 90-kDa protein being membrane-associated, but cross-linking studies revealed that the FGF receptor had an Mr between 145 and 210 kDa in Swiss 3T3 cells, distinct from the 90-kDa major substrate for tyrosine phosphorylation. These data demonstrate that both acidic and basic FGF activate a tyrosine kinase in vivo leading to phosphorylation of a unique 90-kDa substrate, and they suggest that protein modification by phosphorylation at tyrosine is involved in eliciting the mitogenic effect of FGF.     

Correlation of protein kinase C translocation, P-glycoprotein phosphorylation and reduced drug accumulation in multidrug resistant human KB cells

Treatment of drug-resistant human KB carcinoma cells (KB-V1) with 0.2 microM phorbol 12-myristate 13-acetate (PMA) resulted in increases of 4-fold in both membrane-associated protein kinase C activity and phosphorylation of P-glycoprotein. The response was essentially complete after 30 min and was relatively stable, since both of these parameters remained elevated above basal levels in cells exposed to PMA for 24 hours. In contrast, long-term PMA treatment of drug-sensitive KB-3 cells caused complete depletion of protein kinase C. The rate of accumulation of [3H]vinblastine in KB-V1 cells was 0.8 +/- 0.1 pmol/mg/30 min in the absence, and 1.9 +/- 0.2 pmol/mg/30 min in the presence, of 20 microM verapamil. Preincubation of cells with PMA resulted in a time-dependent decrease, up to 60% after 24 hours, in both of these values. These results suggest that protein kinase C mediated phosphorylation stimulates the drug transport activity of P-glycoprotein.     

Inhibition and stimulation of rat luteal protein phosphorylation by protein kinase effectors

Estradiol-17 beta (E2) predetermined protein phosphorylation systems have been identified recently in midpregnant rat corpus luteum. Major type protein kinase activities in these systems were explored here using as probes protein kinase inhibitors. Luteal nuclear, mitochondrial, microsomal and cytosolic fractions were obtained from rats hysterectomized and hypophysectomized on day 12 of pregnancy and then treated for 72 h with E2. In vitro phosphate transfer from [gamma-32P]ATP was monitored by SDS-PAGE followed by autoradiography. Polymyxin B (PMB), 1-200 microM, a PKC inhibitor, completely blocked, in a dose dependent manner, the Ca2+ phospholipid (PL) stimulated radiolabeling of nuclear fraction Mr 79,000 substrate(s) as expected. Similarly, the calmodulin (CaM) antagonist compound 48/80, 1-20 micrograms/ml, inhibited the Ca2+/CaM-dependent phosphorylation of the microsomal fraction Mr 60,000 and Mr 56,000 proteins. The Ca2+ PL-enhanced labeling of mitochondrial fraction Mr 76,000 substrate(s) was only partially susceptible to inhibition by PMB or compound 48/80. Studies of microsomal fraction phosphoprotein bands not stimulated by added cofactors indicated that the radiolabeling of Mr 75,000 protein(s) was partially blocked by compound 48/80 but not by PMB. Phosphate transfer to Mr 41,000 protein(s) was inhibited by the cAMP-dependent kinase protein inhibitor (PKI), while the phosphorylation of Mr 31,000 protein(s) was refractory to all inhibitors employed here. Surprisingly, regardless of hormonal pretreatment, PMB and compound 48/80 activated in every subcellular fraction the cofactor independent appearance of at least one phosphoprotein band, between Mr 87,000-99,000. This novel observation should be instrumental in understanding the actions of these compounds towards living cells.     

Phosphorylation of the Postsynaptic Density Glycoprotein gp 180 by Ca2+/Calmodulin-Dependent Protein Kinase

Postsynaptic densities (PSDs) were prepared by the aqueous two-phase extraction of synaptic membranes in the presence of n-octyl glucoside. Incubation of postsynaptic densities with [gamma-32P]ATP resulted in the incorporation of 32P into a range of proteins. Isolation of glycoproteins from 32P-labelled PSDs by affinity chromatography on concanavalin A-agarose identified the postsynaptic glycoprotein of apparent Mr 180,000 (gp180) as a substrate for endogenous protein kinase(s). When the phosphorylation reaction was performed in the presence of Ca2+ and calmodulin, there was an overall 13-fold increase in the phosphorylation of PSD proteins. The largest effects of calmodulin were associated with two proteins of molecular weights 51,000 and 60,000, which showed average calmodulin-dependent increases in phosphorylation of 68-fold. The phosphorylation of gp180 was increased 7.5-fold in the presence of calmodulin. Fifty percent of maximum phosphorylation of proteins and glycoproteins occurred with a free Ca2+ concentration of 0.3 X 10(-6) M. The amounts 12.6 micrograms/ml and 9.1 micrograms/ml of calmodulin were required for 50% of maximum phosphorylation of proteins and glycoproteins, respectively. Peptide mapping experiments identified three major phosphorylation sites in gp180. The phosphorylation of all three sites was increased in the presence of calmodulin. Phosphoamino acid analysis of gp180 revealed that [32P]phosphoserine and [32P]phosphothreonine were both produced during the phosphorylation reaction, with phosphoserine being the predominant product. The phosphorylation of both amino acids was increased in the presence of calmodulin. [32P]phosphotyrosine was also identified as a product of the phosphorylation of gp180.