Regulation of erythrocyte Ca2+ pump activity by protein kinase C.

Using either inside-out vesicles (IOV) prepared from human erythrocytes or purified Ca2+-ATPase from the same source, the effects of protein kinase C (Ca2+/phospholipid-dependent enzyme) on Ca2+ transport and Ca2+-ATPase activity were measured. Incubation of IOV with protein kinase C in the presence, but not absence, of either 12-O-tetradecanoylphorbol-13-acetate or diolein led to a Ca2+-dependent stimulation of ATP-dependent calcium uptake. The effect was a 5-7-fold increase of Vmax without a significant change in the apparent Km for Ca2+. By comparison, the effect of calmodulin was a 14-fold stimulation of Vmax and a 4-fold reduction in apparent Km. The effect of protein kinase C and calmodulin on Ca2+ uptake were nearly additive. Stimulation of IOV Ca2+ transport by protein kinase C was entirely reversible by treatment of activated IOV with alkaline phosphatase. Incubation of purified Ca2+-ATPase with protein kinase C in the presence of 12-O-tetradecanoylphorbol-13-acetate or diolein led to a stimulation of Ca2+-dependent ATPase activity. These results indicate that protein kinase C stimulates the activity of the plasma membrane Ca2+ pump by a direct effect on the pump protein.

fold reduction in apparent K,. The effect of protein kinase C and calmodulin on Ca2+ uptake were nearly additive. Stimulation of IOV Ca2+ transport by protein kinase C was entirely reversible by treatment of activated IOV with alkaline phosphatase. Incubation of purified Ca2+-ATPase with protein kinase C in the presence of 12-0-tetradecanoylphorbol-13-acetate or diolein led to a stimulation of Ca2+-dependent ATPase activity. These results indicate that protein kinase C stimulates the activity of the plasma membrane Ca2+ pump by a direct effect on the pump protein.
The activity of the plasma membrane Caz+ pump is of critical importance to the maintenance of cellular Ca2+ homeostasis (1)(2)(3). This pump catalyzes the ATP-dependent exchange of internal Ca2+ for external H+ (4, 5) and is responsible for the maintenance of a 5,000-10,000-fold Ca2+ concentration gradient across the piasma membrane. A major mechanism by which hormones and neurotransmitters regulate cell function is by altering Ca2+ influx rates across the plasma membrane of target cells (1,(6)(7)(8). In nearly all instances in which an extracellular messenger induces a sustained increase in Ca2+ influx rate, there is a compensatory increase in Ca2+ efflux rate so that during the sustained response, there is an increase in Ca2+ cycling across the plasma membrane without a net accumulation of Ca2+ by the cell (6,8). The biochemical and cellular basis for the homeostatic regulation of Ca2+ pump activity is not completely known, but * This work was supported by Grant DK-19813 from the National Institute of Diabetes and Digestive and Kidney Diseases of the National Institutes of Health and by National Research Service Award Training Grant GM-07223 to the Department of Cell Biology. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
$ This work was performed in partial fulfillment of a Ph.D. thesis requirement.
To whom correspondence should be addressed: Dept. of Internal Medicine, Yale University School of Medicine, 333 Cedar St., New Haven, CT 06510. one very important feature is the direct allosteric activation of the pump by calmodulin (9)(10)(11). A rise in intracellular Ca2+ concentration leads to Ca2+-dependent association of CaM' with the pump. As a consequence, the VmaX increases and the K , for Ca2+ decreases (12).
In recent years, a second major pathway by which Ca2+ regulates cell function has been recognized the protein kinase C pathway (13,14). There is evidence in several different cellular systems that the activity of the plasma membraneassociated protein kinase C is controlled by the rate of Ca'+' influx across this membrane (6,8). Also, there is evidence which suggests that phorbol ester activators of protein kinase C stimulate Ca2+ efflux from certain cells (15-21) and in high concentrations stimulate the ATP-dependent uptake of Ca2+ into inside-out vesicles prepared from neutrophils (22). However, there is no direct evidence that under physiologic circumstances protein kinase C is an activator of the plasma membrane Ca'+ pump itself.
The present experiments were undertaken to determine whether or not protein kinase C has any effect upon Ca2+ pump activity. The systems chosen for this examination were IOV prepared from human erythrocytes and purified Ca2+-ATPase from the same source. This erythrocyte system was chosen because it is the most extensively studied of the plasma membrane Ca2+-ATPases (2,12) and has been traditionally viewed as a model for other plasmalemma calcium pumps. Furthermore, the human erythrocyte has been shown to possess protein kinase C (23), and this enzyme binds to red cell IOV in a Ca2+-and diacylglycerol (or phorbol ester-)-dependent manner (24).

MATERIALS AND METHODS
The 'SCaC12 and [32P]ATP were obtained from Amersham Corp. DEAE-cellulose, phenyl-Sepharose, cyanogen bromide-activated Sepharose 4B, TPA, histone, and calmodulin were purchased from Sigma. Fractogel HW55S was obtained from Rainin Instrument Co. Inc. and AH-Sepharose from Pharmacia LKB Biotechnology Inc. The 1,2-diolein was purchased from Avanti Polar Lipids. "Bakerflex" DEAE-cellulose TLC plates were obtained from J. T. Baker Chemical Co. Alkaline phosphatase (calf intestine, molecular biology grade) was purchased from Boehringer Mannheim. Coomassie. Blue staining of SDS-polyacrylamide gel electrophoresis gels showed the phosphatase preparation to be composed of a single polypeptide of about 55,000 daltons.
The calmodulin used for purification of the Ca2'-ATPase was prepared from bovine brain according to the method of Gopalakrishna and Anderson (25). SDS-polyacrylamide gel electrophoresis analysis of the final calmodulin preparation showed it to be absolutely pure as judged by Coomassie Blue staining.

2195
Purification of Protein Kinase C-Protein kinase C was purified from bovine brain by modifications of the procedure of Kikkawa et al. (26). All steps were performed at 4 "C. Freshly excised tissue was immediately homogenized by a Polytron apparatus in 20 mM Tris-HC1, pH 7.5,250 mM sucrose, 10 mM EGTA, 2 mM EDTA, 10 mM 2mercaptoethanol, 1 mM phenylmethylsulfonyl fluoride, and 50 p~ leupeptin. The homogenate was spun at 100,000 X g for 25 min, and the supernatant was added to 250 ml of DEAE-cellulose preequilibrated with buffer A (20 mM Tris-HC1, pH 7.5, 0.5 mM EGTA, 0.5 mM EDTA, 10 mM 2-mercaptoethanol). The mixture was stirred for 1 h; the resin was filtered on a scintered glass funnel and washed with 8 volumes of buffer A and 8 volumes of buffer A containing 20 mM NaCl. Enzyme was then eluted with 3 volumes of buffer A, 90 mM NaCI.
The eluent was stirred for 1 h with 60 ml of threonine-Sepharose suspended in buffer A. (Threonine was coupled to AH-Sepharose by the method of Kikkawa et al. (26).) The resin was then filtered on a scintered glass funnel and washed with 6-7 volumes of buffer A and 4 volumes of buffer A, 400 mM NaC1. Protein kinase C activity was then eluted with 250 ml of buffer A, 800 mM NaCI.
This eluent was adjusted to 1.5 M NaCl and applied to a 10-ml phenyl-Sepharose column. This column was washed with 5 volumes of buffer A, 1.5 M NaCl and 5 volumes of buffer A, 0.6 M NaCl. Protein kinase C activity was eluted by a 100-ml gradient of 0.6-0 M NaCl in buffer A, followed by 30 ml of buffer A.
Pooled kinase activity was concentrated by overnight dialysis against a solution of 7.5% polyethylene glycol in buffer A. The final step of preparation was gel filtration on Fractogel HW55S with buffer A, 100 mM NaCl. Pooled protein kinase C was stored at -80 "C in buffer A, 100 mM NaCl, 10% glycerol (v/v), and 0.05% Triton X-100. The enzyme remained active for more than 6 months.
Assay of Protein Kinase C-Kinase activity was assessed by Ca2+/ phospholipid-dependent phosphorylation of histone 1 (Sigma histone fraction 111s). Ten microliters of enzyme was assayed in buffer containing 20 mM Tris-HC1, pH 7.5,1 mM EGTA, 1.2 mM CaC1, (free Caz+, 200 p~) , 20 pg/ml phosphatidylserine (PS), 2 pg/ml diolein, and 200 pg/ml histone in a total volume of 0.1 ml. PS and diolein, dissolved in chloroform and dried under nitrogen, were suspended by sonication in H,O. Samples were prewarmed for 1 min at 30 "C, and [32P]ATP (2-5 pCi/pmol) was added to 20 p~ to initiate the reaction. After 5 min at 30 "C, the reaction was stopped with 0.8 ml of ice-cold 20% trichloroacetic acid. Samples were filtered on 0.45-pm Millipore filters and were washed with 5 ml of 5% trichloroacetic acid. Filters were counted in H,O in a scintillation counter. Activity in the presence of Ca2+ alone was subtracted from that obtained with Caz+ plus PS and diolein. One unit of protein kinase C activity was defined as that amount transferring 1 pmol of phosphate to histone per min under these conditions. Preparation ofZOV-Inside-out vesicles were prepared from freshly drawn heparinized blood with some modification of the procedure described elsewhere (5). To rid the membranes of endogenous calmodulin and protein kinase C, intact cells were washed three times in 2 volumes of 155 mM NaCI, 5 mM sodium phosphate, pH. 7.4 (phosphate-buffered saline), containing 10 mM EGTA. Cells were then lysed in 12 volumes of 5 mM sodium phosphate, pH 8.2, 10 mM EGTA. Membranes were washed three times in the same phosphate buffer with decreasing amounts of EGTA 5, 1, and 0 mM. They were allowed to endovesiculate for 1 h in 0.5 mM sodium phosphate, pH 8.2, and all further steps were as outlined in Ref. 5. Dextran was used at 0.06 g/ml. All steps were performed at 0-4 "C.
Final suspensions of IOV were rapidly frozen in liquid nitrogen and stored at -80 'C. They were slowly thawed at room temperature as needed and were used within 3 weeks of preparation. The percentage of vesicles obtained in the inside-out orientation was calculated from measurements of the latency of acetylcholinesterase activity (27).
Preparation of ZOV for Caz+ Transport Studies-In the initial phase of this study, it was found that, at a given concentration of free Caz+, Caz+ uptake rates varied widely from one IOV preparation to another. In addition, prewarming of vesicles at 37 "C in the absence of ATP before initiation of Ca2+ uptake led to a progressive decline in subsequent Ca2+ transport rates in most IOV preparations. The observed decline was limited, however; eventually, a low but consistent basal rate of transport was reached, beyond which further incubation yielded little change. The rates at which this final rate was achieved were quite variable between IOV preparations.
Based on the possibility that differences in the extent of phosphorylation of plasma membrane proteins (including possibly the Ca2+ pump) might contribute to the observed variability in basal uptake rates, pretreatment of IOV with alkaline phosphate was explored. Preincubation of IOV with alkaline phosphatase was found to induce a more rapid decline in subsequently measured Ca" transport rates when compared with prewarming in the absence of phosphatase. Again, a low basal rate of Ca2+ uptake was achieved, beyond which continued incubation with phosphatase had no effect. In addition, such treatment totally eliminated the deactivating effect of further prewarming once phosphatase was removed. Hence, a standard protocol of alkaline phosphatase pretreatment was developed, which yielded extremely consistent results with all IOV preparations.
Thawed vesicles were treated with alkaline phosphatase prior to ca2+ uptake experiments. IOV in 40 mM NaPIPES, pH 7.2 (1-3 IOV units of acetylcholinesterase activity/ml), were suspended in 2 volumes of 40 mM NaPIPES to obtain a final pH of 7.7. Alkaline phosphatase (19-24 units/pl) was added to 60-80 units/ml. Vesicles were incubated at 37 "C for 20 min, cooled on ice, and then spun in a tabletop microcentrifuge. Pellets were washed three times in 10 volumes of ice-cold NaPIPES, pH 7.2, and were then resuspended in the same buffer for Ca2+ uptake measurements. Acetylcholinesterase assays were performed to assess IOV recovery. Phosphatase treatment did not significantly affect acetylcholinesterase activity.
4sCa2+ Uptake-ATP-dependent Ca" transport was determined in buffer containing 40 mM NaPIPES, pH 7. Purified protein kinase C was added to 40-370 units/ml (total volume, 300-500 pl). TPA dissolved in ethanol was added to vesicles at 0-100 nM (final concentration of ethanol 51%). Diolein, dissolved in chloroform, was dried under nitrogen and redissolved in ethanol; it was added to a final concentration of 10-100 pg/ml. Equivalent volumes of protein kinase C storage buffer and ethanol were added to controls as appropriate.
Samples were prewarmed to 37 "C for 2 min, and ATP (vanadatefree) was added to 0.9 mM to initiate transport. At appropriate time points, 50-pl aliquots were vacuum-filtered on 0.45-pm Millipore filters presoaked in 250 mM sucrose, 40 mM NaPIPES, pH 7.2. Filters were washed with 6 ml of ice-cold 250 mM sucrose, 40 mM NaPIPES. Uptake of "Ca by IOV was then determined by liquid scintillation spectrometry.
Purification of Erythrocyte Caz+-ATPase-A modification of the procedure developed by Carafoli and co-workers (10,28-30) was used. Recently outdated red blood cells from the blood bank (150 ml packed volume) were washed twice in 150 mM NaCI, 5 mM sodium phosphate, pH 7.5, containing 10 mM EGTA. They were then lysed in 5 mM sodium phosphate, pH 8.2 (5P8), 10 mM EGTA as for preparation of IOV. Membranes were washed three times in 5P8 without EGTA to rid them of soluble proteins, including hemoglobin, and then once more in 5P8, 10 mM EGTA to deplete them of bound calmodulin. Finally, membranes were washed three times in 10 mM HEPES, pH 7.5,130 mM NaCI, 0.5 mM MgCl,, 5 mM 2-mercaptoethanol (HEPES buffer). CaCI, to 50 p~ was included in the last wash (HEPES/Ca2+ buffer).
To extract the CaZ+-ATPase, packed membranes were resuspended in HEPES/CaZ+ buffer in a total volume of 30 ml; Triton X-100 was added to 0.4% (v/v), and the solution was incubated on ice for 15 min. After centrifugation at 40,000 X g for 6 min, the supernatant was aspirated and membranes were reextracted in the same manner. PS was added to the combined supernatants to stabilize the ATPase (final concentration, 0.4 mg/ml PS in 34 ml).
The membrane extract was applied to a 0.7-ml calmodulin-Sepharose affinity column, preequilibrated in HEPES/CaZ+ buffer with 0.4 mg/ml PS, 0.4% Triton X-100. (Purified calmodulin was coupled to cyanogen bromide-activated Sepharose 4B as described in Ref. 10.) The column was washed with 10 volumes of HEPES/Ca2+ buffer with 0.4 mg/ml phosphatidylcholine (PC) and 0.1% Triton X-100. Subsequently, a series of reductions in free Ca2+ was used to elute ATPase activity from the CaM column. First, free Ca" was reduced to 5 pM in HEPES buffer containing 0.4 mg/ml PC, 0.05% Triton X-100 (1 mM total EGTA). Next, a 10-volume gradient of free Caz+ was applied (5 p~ free Ca2+ to 1 mM EGTA with no added Caz+). Finally, the column was washed with 1 mM EGTA in HEPES buffer, 0.4 mg/ml PC, 0.05% Triton X-100.  Collected fractions were assayed for ATPase activity as described below. The Calcon program was used to calculate amounts of EGTA and Ca2+ needed to obtain desired free Ca" concentrations. Peaks of activity were pooled, 10% glycerol (v/\) was added, and enzyme was dialyzed by micro-flow dialysis against HEPES buffer, 0.4 mg/ml PC, 0.05% Triton X-100, 10% glycerol, and 50 p M free Ca" (EGTA, 0.1 mM total). Ca" and glycerol were included to stabilize the activity during dialysis and storage. The ATPase was frozen and stored at -80 "C.
ATPase Assay-For the purified Ca2+-ATPase, 10-30 p1 of enzyme were incubated at 37 "C in 40 mM NaPIPES, pH 7.2,5 mM MgPIPES, 5 mM sodium phosphate, 100 pM free Ca", and 4.2 p M A23187 to avoid possible sequestering of Ca2+ in lipid micelles (total volume, 60 pl). Calmodulin, protein kinase C/phosphatidylserine, TPA, or diolein were added to assay mixtures as needed. The reaction was started with addition of [32P]ATP, 100 p~. At various time points (5-25 min), 15-p1 aliquots were withdrawn, and the reaction was stopped by addition of SDS and EDTA to 2% and 8 mM, respectively. Onemicroliter samples were spotted in triplicate on DEAE-cellulose TLC plates (20 X 5 cm). The plates were developed in 0.1 M sodium acetate buffer, pH 4.75, and then exposed to Kodak X-AR5 x-ray film for 2- of ATP-dependent Ca2+ uptake by IOV at 0.6 p~ free Ca2+, with 100 nM TPA present in all samples. IOV were incubated with no added protein kinase C (PKC) (nl, A) or with various doses of purified protein kinase C as indicated (15-147 units (u) of kinase activity/sample (0)). HT, V, incubation with 147 units of heat-treatedprotein kinase C. After 40 min of Caz+ uptake, 5 pM A23187 was added to release accumulated Ca2+. Retained Caz+ was measured 15 min later. ATP-independent binding of Caz+ by IOV was subtracted from all points. UACE, units of acetylcholinesterase activity. B, stimulated Ca2+ uptake rates (during the linear, post-lag phase) plotted as a function of added protein kinase C. The ordinate represents the -fold increase over the rate measured in the absence of protein kinase C. C, time course of ATP-dependent Ca2+ uptake conducted at 0.6 p~ free Ca2+ and varying concentrations of TPA. IOV were present at the same concentration as above, and 147 units of protein kinase C (370 units/ml) were added to all samples. A, no added T P A 0, TPA added in amounts indicated. A23187 was added as described above. Before addition to the Caz+ uptake incubation medium, these IOV were stored on ice for approximately 6 h. D, stimulated rates of Ca2+ uptake plotted against the added concentration of TPA. The ordinate represents -fold stimulation of the rate measured in the absence of TPA. Half-maximal stimulation was found at approximately 10 nM TPA.
Protein Kinase C Regulation of the ea2+ Pump

RESULTS
The results of time course experiments performed on alkaline phosphatase pretreated vesicles are shown in Fig. l. The data represent the combined results from eight separate experiments performed on three different IOV preparations, after 1-15 days of storage at -80 "C. At 100 p~ free Ca2+, the mean basal Ca2+ uptake rate with alkaline phosphatasetreated IOV was 1.92 nmol/min/IOV units of acetylcholinesterase activity (range 1.5-2.4), and uptake was always linear for at least 40 min. In the presence of maximal protein kinase C and 25 nM TPA, the mean initial Ca2+ transport rate was 5.26 nmol/min/IOV units of acetylcholinesterase activity (range 4.7-5.5), representing approximately a 2.7-fold stimulation. Uptake of Ca2+ under these conditions was not linear after 20 min.
Dose Dependence of Protein Kinase C Stimulation-The effect of various doses of protein kinase C upon time courses of IOV Ca2+ uptake is shown in Fig. 2  At 0.6 p~ free ca2+, a brief delay in the protein kinase C-induced stimulation was noted, and this lag time was also dependent upon the dose of kinase added. Initial stimulated rates of Ca2+-uptake were calculated at these and other doses of protein kinase C, and the dose-response relationship is shown in Fig. 2B. The rate of Ca2+ transport in the presence of TPA but no added protein kinase C was indistinguishable from the basal rate (no additions).
When protein kinase C was heat-treated prior to addition (70 "C, 10 min), a previously maximal dose was completely ineffective in stimulating Ca2+ transport (Fig. 2 A ) . Because calmodulin is heat stable, this result eliminates the possibility that the observed stimulation might be due to calmodulin contamination in the kinase preparation.
In all cases, addition of ionophore A23187 at 40 min released Ca2+ to approximately the same basal levels. Hence, the measured increases in Ca2+ accumulation resulted from increased transport across the membrane and not simply from enhanced binding of Ca2+ to the vesicles.
TPA Dependence of Protein Kinase C Stimulation-Protein kinase C stimulation of Ca2+ uptake was dependent upon the dose of phorbol ester (TPA) added, as shown by the time courses in Fig. 2C. A delay in activation was observed in these experiments as well. At 0.6 p~ free Ca2+ and at 147 units (-370 units/ml) of protein kinase C, half-maximal activation of transport rate occurred at approximately 10 nM TPA (Fig.  20), which is consistent with other reports of its potency as an activator of protein kinase C (31)(32)(33).
Initial stimulated Ca2+ transport rates in the presence of maximal protein kinase C and phorbol ester were quite similar as measured in several different experiments. However, a late phase deactivation of Ca2+ uptake often occurred. This deactivation was not observed with IOV in the absence of protein kinase C and TPA. The timing and extent of this inhibition was quite variable; occasionally it was not observed at all within the time course studied (compare Fig. 2, A and C). Storage of vesicles on ice for several hours after phosphatase pretreatment reduced or eliminated the late phase deactivation.
Diacylglycerol as a Protein Kinase C Activator-The diacylglycerol, 1,2-diolein, was as effective as TPA in supporting protein kinase C stimulation of Ca2+ uptake at 0.6 p M free Ca2+ (Fig. 3). Maximal stimulation at this Ca2+ concentration was obtained with 20 pg/ml diolein. As with TPA, diolein did not stimulate Ca2+ transport in the absence of added protein kinase C. Eventual inhibition of the protein kinase C stimu- latory effect was observed with diolein as with TPA.
Reuersal of Protein Kinase C Stimulation with Alkaline Phosphatase-To determine whether activation of Caz+ uptake by protein kinase C could be reversed by phosphatase treatment, alkaline phosphatase-pretreated IOV were incubated at 37 "C for 5 min with 10 pM free Caz+ and 0.9 mM ATP, in the presence of maximal protein kinase C and TPA. Control IOV were similarly incubated, but with no protein kinase C or TPA added. All vesicles were then centrifuged and washed, and ATP-dependent Ca2+ uptake was measured at 0.6 p~ free Ca2+ on a portion of both protein kinase Cstimulated and control IOV. The remainder of both samples was then split into 2 aliquots which were incubated for 10 min at 37 "C, pH 7.7, with or without alkaline phosphatase. No ATP was present. Then, the resulting Ca2+ transport rates were measured.
As shown in Fig. 4, incubation of protein kinase C-stimulated IOV without alkaline phosphatase led to a decrease in the protein kinase C-stimulated Ca2+ uptake rate when ATP and 0.6 p~ free Ca2+ were reintroduced. However, incubation with alkaline phosphatase further reduced the uptake rate to a value indistinguishable from that of control IOV that had not been preactivated with protein kinase C/TPA. Ca2+ uptake by control IOV before the 10-min, pH 7.7 incubation did not differ from that shown for control IOV after such treatment.
Ca2+ Dependence of Protein Kinase C Stimulation-The effects of protein kinase C and TPA upon Ca2+ uptake rates were examined over a wide Ca2+ concentration range. IOV samples were incubated with Caz+, with or without maximal protein kinase C/TPA. Aliquots were filtered 15 min after addition of ATP, and Caz+ binding in the absence of ATP was subtracted from each determination.
As shown in Fig. 5A, IOV in the absence of protein kinase C exhibited two phases of Ca2+ transport activity. A high affinity but low velocity phase was observed up to 10 PM free Ca2+, and a second phase of low affinity but higher velocity was observed above 10 pM ca2+. These two Ca2+ "transport sites" have been reported previously, although the estimates of K,,, and Vmax in each phase have varied (12,34).
Protein kinase C in the presence of TPA significantly increased initial Ca2+ transport rates above 0.1 FM free ca2+. Relative to basal rates in the absence of kinase, the greatest effect of protein kinase C was found at 2-5 p~ Ca2+. Thereafter, stimulated transport rates increased roughly in parallel with basal rates, indicating that the activating effect of protein kinase C was primarily upon the low velocity, high affinity Ca2+ transport phase. In the absence of phorbol ester, protein kinase C did not stimulate Ca2+ transport at any concentration of Ca2+ tested.
Ca2+ Dependence of Calmodulin-stimulated Ca2+ Uptak-In a similar set of experiments, the effect of calmodulin upon Ca2+ transport rates in alkaline phosphatase pretreated IOV was examined. In the presence of 7.5 pg/ml CaM, stimulation of Ca2+ transport occurred above 50 nM free Ca2+, reaching a maximum at 2-5 p~ (Fig. 5B). At higher concentrations of free Ca2+, uptake rates progressively declined. Some investigators have reported this phenomenon (27,35,36), while others have found no such inhibition (11,37). Under these conditions, CaM was a more potent activator of the Ca2+ pump than was protein kinase C.
Addition of maximal protein kinase C/TPA to CaM-IOV further stimulated Ca2+ uptake at free Ca2+ concentrations above 0.2 PM. Since the effects of the two activators were approximately additive at maximally effective doses of each, CaM and protein kinase C appear to activate Ca2+ transport FIG. 6. Lineweaver-Burk analysis of initial Ca2+ uptake rates. Concentrations of free Caz+ were set between 0.2-10 pM (high affinity phase shown in Fig. 5). Reciprocal rates are plotted as (nmol/ min/IOV units of acetylcholinesterase activity)". 0, alkaline phosphatase-pretreated IOV with no added activators and A, with 7.5 pg/ ml CaM; 0, protein kinase C-activated IOV. In the latter case, IOV were preactivated with 370 units/ml protein kinase C and 100 nM TPA at 10 p~ '"Ca2' before addition of buffered %a2+ to the desired final free concentration. Caz+ uptake rates were then determined from samples taken at five different time points during a total of 15 min. All uptakes were found to be linear, and no lag phase was observed. through different mechanisms. No inhibition of Ca2+ transport above 10 pM free Caz+ was observed when protein kinase C/TPA was added in combination with CaM.

-
Kinetic Effects of CaM and Protein Kinase C-A detailed kinetic analysis of the effects of either CaM or protein kinase C on high affinity Ca2+ transport was examined. Lineweaver-Burk plots of initial ATP-dependent Ca2+ uptake rates within the range of 0.2-10 p~ free Ca2+ are shown in Fig. 6. Normal and CaM-IOV data are derived from Fig. 5; all Ca2+ uptakes at all Ca2+ concentrations in such vesicles were previously found to be linear when alkaline phosphatase-treated IOV were used.
To ensure that the Ca2+-dependence of protein kinase Cstimulated Ca2+ transport reflected only the activity of a fully activated Ca2+ pump, and did not also involve a Ca2+-dependence in the activation of protein kinase C itself, IOV were preactivated by exposure to protein kinase C/TPA, ATP, and 10 p~ unbuffered 40Ca2+ for 5 min at 37 "C. The concentration of free Ca2+ was then adjusted as appropriate by addition of EGTA-buffered, radiolabeled Ca2+. Subsequent uptake of Ca2+ was found to be linear for at least 15 min at all concentrations of free Ca2+. Protein kinase C/TPA did not significantly alter the apparent K,,, for Ca2+ of IOV ca2+ transport  ca2+ uersus 1.2 f 0.4 p M caz+ for control IOV). However, the apparent Vmax was increased from 0.6 to 3.3 nmol/min/IOV units of acetylcholinesterase activity by prior protein kinase C-dependent activation. In contrast, CaM not only increased V,, (derived to be 10.0 nmol/min/IOV units of acetylcholinesterase activity) but also significantly decreased the apparent K , for Ca2+ to 0.30 f 0.05 p~ free Ca2+.

Effects of Protein Kinase C at Low Ca" Concentrations-
The results presented above suggest that at free Ca2+ concentrations below 0.1 p~, protein kinase C in the presence of TPA does not significantly activate IOV Ca2+ transport. However, extended time courses were conducted to measure Caz+ uptake at 80 nM Ca2+, a concentration approximating basal erythrocyte intracellular free Ca2+ (36). In fact, after a prolonged lag phase (20-35 min in different experiments), protein kinase C/TPA eventually stimulated Ca'+ uptake almost as much as calmodulin did (Fig. 7A). When protein kinase C/ TPA and CaM were added together, Ca2+ transport was further stimulated. The enhanced rate of uptake remained constant for a more prolonged period than that observed in the presence of CaM alone.
A similar experiment using diolein instead of TPA to activate protein kinase C showed that this diacylglycerol did not support protein kinase C stimulation of Ca2+ transport at such low Ca2+ concentrations (Fig. 7B). However, in another experiment, IOV were first exposed to protein kinase C/diolein for 8 min in the presence of ATP and 0.6 pM Caz+; EGTA was subsequently added to reduce the free Ca2+ concentration to 80 nM, and Ca2+ uptake rates were then measured during the next 20 min. This procedure was designed to mimic the Ca2+ transients observed in a stimulated cell or in one permeabilized to Ca2+. Whereas diolein was ineffective in supporting protein kinase C stimulation at constant exposure to 80 nM free Ca2+, brief exposure to 0.6 p~ Ca2+ enabled diolein to activate the transport system when Ca2+ was then reduced to 80 nM (Fig. 8). Preexposure of IOV to elevated Ca2+ did not significantly affect rates of Ca2+ transport observed in the  presence of CaM alone. Effect of Activators upon the Purified Ca2+-ATPase-Theoretically, stimulation of IOV Ca2+ transport by protein kinase C could occur either by direct action of the kinase upon the Ca2+ pump ATPase or by an indirect mechanism involving protein kinase C-induced modification of other membrane components. Therefore, the erythrocyte Ca2+-ATPase was solubilized and purified t o determine whether protein kinase C can directly activate the Ca2+-dependent ATPase. The major peak of Ca2+-dependent ATPase was eluted from the calmodulin affinity column during the gradient of decreasing free Ca2+ concentration (see "Materials and Methods"). This activity represented the calcium pump enzyme, for the major protein band migrated with an apparent molecular mass of 125-130 kD on SDS-polyacrylamide gels. In addition, this protein showed a Ca2+-dependent autophosphorylation that was acid-stable but labile to hydroxylamine, thus representing the phosphorylated intermediate of the pump's catalytic cycle.
As shown in Fig. 9, addition of calmodulin at 7.5 pg/ml stimulated Ca2+-dependent ATPase activity at 100 p~ free Ca2+. Alternatively, the Ca*+-ATPase was stimulated by addition of protein kinase C and either 100 nM TPA or 0.5 pg/ ml diolein. (Small amounts of phosphatidylserine were added to maximally activate the kinase.) Enhancement of ATPase activity by the combined addition of calmodulin and protein kinase C/TPA or protein kinase C/diolein revealed an additive effect, just as in IOV Ca2+ transport experiments.

DISCUSSION
In this investigation, the activated form of purified protein kinase C was found to stimulate Ca2+ transport in alkaline phosphatase-pretreated IOV (Figs. 1-8). Significant effects were observed at physiologically relevant concentrations of free Ca2+: 0.08-5 p~ (Figs. 2-5 and 8). Protein kinase C stimulated Ca2+ transport only in the presence of the specific activators phorbol ester or diacylglycerol. These properties reflect the activation characteristics of purified protein kinase C reported previously by others. Treatment of protein kinase C-activated IOV with alkaline phosphatase completely re-versed stimulation of Ca2+ uptake, consistent with a mechanism involving protein phosphorylation.
Protein kinase C and phorbol eswr increased the maximum velocity of erythrocyte IOV Ca" transport but had no significant effect upon the apparent K , for Ca2+ (Figs. 5 and 6). In contrast, calmodulin increased bot,h the maximum velocity and the Ca2+ sensitivity of the pump (Fig. 6). A combination of the two activators (protein kinase C/TPA or protein kinase C/diolein, plus CaM) showed additive effects at maximal doses of each (Fig. 5), and therefore calmodulin and protein kinase C may be said to exert their effects through different mechanisms.
Either protein kinase C/TPA or protein kinase C/diolein were found to stimulate the activity of the purified calcium pump ATPase (Fig. 9), just as they stimulated Ca2+ transport in intact vesicles. Therefore, it appears that protein kinase C acts directly upon the calcium pump and not indirectly through intermediate effectors. Preliminary results indicate that protein kinase C does, in fact, phosphorylate the purified calcium pump (not shown); this is now being investigated further.
Alkaline phosphatase-pretreated IOV were found to exhibit far more consistent Ca2+ transport rates than nontreated vesicles, and their stimulation by protein kinase C/TPA was more consistently observed. This result suggests that the calcium pump may already be activated to some extent by phosphorylation in vesicles as normally prepared. (Incubation of protein kinase C-activated IOV at 37 "C in the absence of ATP leads to a slow reversal to basal Ca2+ uptake rates, suggesting that the erythrocyte membrane contains an endogenous phosphatase which may act upon the Ca2+ pump). In IOV never treated with phosphatase, inherent Ca2+ uptake rates never exceeded those obtained with in vitro addition of maximal protein kinase C/TPA to phosphatase-treated IOV. However, the rates were occasionally equal, in which case no stimulation by protein kinase C/TPA was observed. This suggests that protein kinase C may have been the endogenous activating factor in normal membranes. In fact, human erythrocytes do contain protein kinase C (23) and can produce diacylglycerol by Ca2+-stimulated breakdown of phosphatidylinositol (38, 39). Erythrocytes have been shown to be permeabilized to Ca2+ by sheer stress (40), and thus blooddrawing and handling procedures may induce activation of endogenous protein kinase C.
In the present experiments on alkaline phosphatase-treated IOV, CaM was found to maximally stimulate Ca2+ transport about 14-fold. This would also suggest that the basal activity of phosphatase-treated IOV is lower than that of conventionally prepared membranes, in which a 2-6-fold stimulation of transport by CaM has been reported (11,12). In contrast with other reports (41,42), CaM was definitely found to activate IOV Ca2+ transport at the very low free Ca2+ concentration of 80 nM (Fig. 7). No "Ca" transient" was required to induce activation. Although this CaM-stimulated uptake rate was only a fraction of maximal rates observed at about 1 pM free Ca2+, it represented a significant increase over basal rates in phosphatase-treated vesicles. Possibly, CaM remains bound to at least a fraction of the calcium pump proteins in intact cells at resting Ca" concentrations.
Above 0.1 p~ free Ca2+, stimulation of Caz+ transport by protein kinase C in the presence of TPA was significant within minutes; this level of Ca2+ would represent only a very slight elevation of cytosolic Ca2+ in intact resting cells (36). At the very low Ca2+ concentration of 80 nM, after a long delay, TPA plus protein kinase C eventually stimulated IOV Ca2+ transport to the same degree as CaM (Fig. 7), and the effects of combined protein kinase C/TPA and CaM were additive. This The Caz+ pump drives exchange of Ca2+ for protons by the hydrolysis of ATP. The resulting transmembrane pH gradient is discharged by exchange of inorganic anion ( A -) for OH-(or HC03-) through band 111 protein (Ref. 5). CaM regulates pump activity by a Ca2+-dependent direct binding to the ATPase protein. In the presence of elevated cytosolic free Ca2+ and diacylglycerol (DG) produced in the membrane, soluble protein kinase C ( P K C ) binds to the membrane and becomes activated. The protein kinase C then also activates the Ca2+ pump, probably by direct phosphorylation of the ATPase protein.

Protein Kinase C Reg1
indicates that treatment of intact cells with phorbol ester to activate protein kinase C may alter basal Ca2+ transport activity.
Diolein was as effective as TPA in supporting protein kinase C stimulation of ca2+ transport at 0.6 pM free Ca2+ (Fig. 3). However, diolein was ineffective in supportingprotein kinase C stimulation at 80 nM free Ca2+, unless IOV in the presence of protein kinase C/diolein were first exposed to a transient elevation of Ca2+ to 0.6 p~ (Fig. 8). If the red cell Ca2+-ATPase is truly representative of the plasmalemma1 Ca2+ pump in other cell types, these results are important for two major reasons. First, as diacylglycerol is the natural agent stimulating protein kinase C in intact cells, the calcium pump is probably not stimulated by protein kinase C in resting cells. Second, the concentration of diacylglycerol in the plasma membrane rises in hormonally stimulated cells during and after a cytosolic Ca2+ transient (6,43). As shown by the experiments reported here, diolein can induce protein kinase C stimulation of erythrocyte plasma membrane Ca2+ transport after brief exposure of the membrane to elevated Ca2+, even when the Ca2+ concentration then returns to basal levels (Fig.  8). Together, these observations suggest that in an intact cell, protein kinase C activation of Ca2+ extrusion occurs only during the posttransient phase of cell stimulation. Because the effects of CaM and protein kinase C are then additive, the activity of the calcium pump would be higher in the post-Caz+ transient phase than in a resting cell. A new model of the erythrocyte plasma membrane calcium pump is thus presented in Fig. 10. In an intact cell in its basal ztion of the Ca2+ Pump state, calmodulin may bind to and activate the Ca2+ pump to some degree. When intracellular Ca2+ is elevated, CaM further stimulates pump activity. Elevation of cytosolic Ca2+, in combination with production of diacylglycerol, enables endogenous protein kinase C to bind to the plasma membrane. This membrane-associated protein kinase C, in turn, phosphorylates the Caz+ pump protein and further activates the pump. Excess cytosolic Ca2+ may then be extruded, and as the free Ca2+ falls toward basal levels both CaM and protein kinase C enable the pump to remain in an activated state. One would predict that upon removal of agonist, when influx of Ca2+ ceases and diacylglycerol is no longer produced, the Ca2+-ATPase would be dephosphorylated by a n endogenous phosphatase as yet uncharacterized. Efflux of Ca2+ through the calcium pump would return to its original slow rate.