site at which phosphorylation occurs is disputed (Rosa et al., 1992b; Varro and ..... gland (Brooks, 1989), which is consistent with our data sug- gesting the ...
Journal of Cell Science 106, 1221-1226 (1993) Printed in Great Britain © The Company of Biologists Limited 1993
1221
Differential effect of brefeldin A on phosphorylation of the caseins in lactating mouse mammary epithelial cells Mark D. Turner1, Susan E. Handel1, Colin J. Wilde2 and Robert D. Burgoyne1,* 1The
Physiological Laboratory, University of Liverpool, P O Box 147 Liverpool L69 3BX, UK 2Hannah Research Institute, Ayr KA6 5HL, UK
*Author for correspondence
SUMMARY The major milk proteins, the caseins, contain multiple phosphorylation sites. Phosphorylation of the caseins is necessary to allow Ca2+ binding and aggregation of the caseins to form micelles. We have followed the phosphorylation of the caseins in isolated acini from lactating mouse mammary gland. Incubation of mammary cells with [32P]orthophosphate revealed that phosphorylation of newly synthesised caseins was complete within 20 minutes of synthesis. Extensive secretion of -, - and - caseins occurred over a 2 hour period. Activation of the regulated secretory pathway using ionomycin over the last hour resulted in a preferential increase in secretion of - and -caseins. Brefeldin A (BFA) inhibited protein secretion and synthesis in mam-
mary cells in prolonged incubations. An examination of short-term treatments with BFA on 32P incorporation into the caseins revealed a differential effect of BFA in which the drug inhibited phosphorylation of - and but not -caseins. These results suggest that phosphorylation of -casein normally occurs in Golgi cisternae whereas that of - and - caseins occurs in the transGolgi network. Phosphorylation of specific secretory proteins may, therefore, occur in different Golgi compartments.
INTRODUCTION
established. Caseins contain multiple phosphorylation sites and the phosphorylation of these allows the caseins to bind Ca2+, which in turn leads to their aggregation to form the casein micelle (Aoki et al., 1992; Holt and Sawyer, 1988). Mammary epithelial cells secrete caseins by both constitutive and regulated pathways (Turner et al., 1992) but it is not clear whether casein aggregation and micelle formation has any role in sorting into the regulated pathway. Since phosphorylation is of crucial importance for casein micelle formation in the secretory pathway, we have investigated the site of casein phosphorylation using the fungal metabolite brefeldin A (BFA). The effects of BFA on many cell types has been well documented. BFA treatment results in the retrograde transport of cis-, medial- and trans-Golgi but not TGN enzymes back to the ER, with a resulting inhibition of protein flow through the secretory pathway (Chege and Pfeffer, 1990; Doms et al., 1989; Lippincott-Schwartz et al., 1989, 1991; Misumi et al., 1986). BFA, therefore, results in a functional dissection of Golgi cisternae from the TGN. Examination of the effects of BFA on phosphorylation should indicate whether phosphorylation of newly synthesised proteins occurs in the Golgi cisternae or in the TGN. Recent studies on two other secretory proteins resulted in different conclusions (Rosa et al., 1992b; Varro
Many secretory proteins become phosphorylated during their movement through the secretory pathway. Examples of phosphorylated secretory proteins include the caseins (Bingham and Farrell, 1974; Bingham et al., 1972; Capasso et al., 1989; West and Clegg, 1984), the enkephalin precursor (Watkinson et al., 1989), progastrin (Dockray et al., 1987; Varro et al., 1988) and the chromogranins (Rosa et al., 1992b). Post-translational phosphorylation of secretory proteins is believed to occur within the Golgi complex (Bingham et al., 1972; West and Clegg, 1984) but the exact site at which phosphorylation occurs is disputed (Rosa et al., 1992b; Varro and Dockray, 1993). The role of secretory protein phosphorylation is not clear in all cases. It has been suggested that phosphorylation regulates subsequent proteolytic processing of prohormones (Dockray et al., 1987; Watkinson et al., 1989). Phosphorylation could also be important for protein aggregation, which is believed to play a role in sorting into the regulated pathway within the trans-Golgi network (TGN) (Burgess and Kelly 1987; Chanat and Huttner, 1991). In the case of the caseins, the major milk proteins secreted by the lactating mammary epithelial cells, the function of phosphorylation is well
Key words: Golgi, phosphorylation, casein, brefeldin A, mammary cells
1222 M. D. Turner and others and Dockray, 1993). We show here that BFA has differential effects on the phosphorylation of the caseins, suggesting that secretory protein phosphorylation can specifically occur in different Golgi compartments.
polyacrylamide gel electrophoresis (SDS-PAGE) and fluorography or direct autoradiography for 35S and 32P, respectively. The autoradiograms were quantified using a Bio-Rad video densitometer.
Electron microscopy MATERIALS AND METHODS Materials L-[35S]methionine and [32P]orthophosphate were obtained from Amersham plc, Amersham, UK. Unless stated otherwise, tissue culture media were obtained from Gibco Laboratories and other reagents from Sigma.
Preparation of mammary epithelial cell acini from lactating mammary gland Mammary tissue was removed from Tuck’s no. 1 mice at midlactation (10 days post-partum) and dissociated by incubation with 0.14% collagenase (Worthington) for up to 90 minutes at 37°C (Turner et al., 1992). Digestion was stopped when the majority of acini were reduced to clumps of 10-30 cells and the cell suspension was filtered through a 150 µm pore nylon mesh (Lockertex). Cells were harvested by centrifugation at 80 g for 5 minutes and resuspended in HBSS supplemented with 5 µg/ml insulin, 0.01 µg/ml cortisol, 0.04 mg/ml DNase I, 0.1 mg/ml trypsin inhibitor, centrifuged and filtered through a 53 µm pore nylon mesh. The filtrate was centrifuged as above and cells resuspended in culture medium (50% M199, 50% Ham’s F12, 5 µg/ml insulin, 0.01 mg/ml cortisol, 0.01 µg/ml prolactin, 0.01 µg/ml EGF and 0.001 µg/ml T 3). The cells were kept in tissue culture flasks for 1 hour at 37°C in an atmosphere containing 5% CO2 to remove fibroblasts and macrophages, which attach to the flasks. The resulting cell suspension showed 90-95% cell viability as assessed by trypan blue exclusion and approximately 85% of the cells were present in acini (Turner et al., 1992).
Incorporation of [35S]methionine into cellular proteins and assay of protein synthesis and secretion Freshly isolated acini were incubated in culture medium for 1 hour with 25 µCi/ml L-[ 35S]methionine (cell-labelling grade, specific activity >1300 Ci/mmol) at a density of 6×106 cells per ml in the presence or absence of brefeldin A at 1 or 5 µg/ml. The cells were washed by centrifugation at 80 g for 5 minutes and the cell pellet were resuspended in culture medium with the appropriate concentration of BFA and incubated for a further hour in the absence of [35S]methionine. Incubations were terminated by centrifugation at 2,500 g in a microcentrifuge for 1 minute. The incorporation of [35S]methionine into cell-associated or released protein was determined by TCA precipitation and scintillation counting as previously described (Turner et al., 1992), to determine the extent of protein synthesis and secretion.
SDS-polyacrylamide gel electrophoresis and autoradiography for the detection of incorporation of [35S]methionine and [32PO4] into specific caseins Isolated mammary cells were incubated with 25 µCi/ml of [35S]methionine or 75 µCi/ml of [ 32P] orthophosphate and incubated for the times indicated, in the presence or absence of 5 µg/ml BFA. In some cases 10 µM ionomycin or 100 µg/ml cyclohex imide was also present. Incubations were terminated by centrifugation at 2,500 g in a microcentrifuge for 1 minute. Samples were processed as previously described (Turner et al., 1992) by SDS-
Cells at a density of 108/ml were incubated for 1 hour with or without 5 µg/ml BFA. The cells were fixed and processed for electron microscopy as described (Turner et al., 1992) and viewed using a Philips EM300 at 60KV.
RESULTS The caseins are by far the major phosphoproteins synthesised in lactating mammary epithelial cells, amounting to around 80% of total synthesised protein, and their synthesis and secretion can be readily followed using isolated acini from lactating mouse mammary gland (Rennison et al., 1992; Turner et al., 1992). Using [32P]orthophosphate incorporation, and analysis by SDS-polyacrylamide gel electrophoresis and autoradiography, we initially examined the time course of casein phosphorylation within the cells. In order to develop a pulse-chase type protocol to allow determination of the kinetics of phosphorylation of synthesised caseins, the cells were incubated with 32PO4 for 5 minutes, further protein synthesis was blocked by addition of 100 µg/ml cycloheximide and then the increase in 32P incorporation over time was followed. The major caseins, α, β and γ, were, as expected, the most abundant cellular phosphoproteins. The major phosphoproteins visualised in Fig. 1A comigrated with authentic mouse caseins and were secreted from the cells as expected for the caseins. Similar levels of 32PO4 were incorporated into the α- and β-caseins. Since these caseins are synthesised to similar extents, this suggests that these two caseins attain similar levels of phosphorylation. As shown in Fig. 1 (which is representative of two separate experiments), cell associated α-caseins were maximally phosphorylated within 20 minutes and the amount of detectable phosphorylated casein declined after the peak at 20 minutes, consistent with constitutive secretion of the caseins; but note that substantial amounts of phosphorylated caseins remain in the cells even at the latest (60 minutes) time point examined in this experiment. In fact, most casein secretion only occurs after a lag period of 60 minutes after synthesis (Turner et al., 1992) and therefore some loss of label, seen in Fig. 1, could be due to intracellular casein proteolysis. The time course of phosphorylation is as expected if phosphorylation occurs while newly synthesised caseins are transported through the Golgi complex. As shown in Fig. 2A, the major phosphorylated proteins secreted into the medium are the α-, β- and γ-caseins. Constitutive secretion of these phosphoproteins was detectable within 1 hour but considerably higher levels were seen after a 2-hour incubation period (Fig. 2A, lane b) as we have previously reported for [35S]methionine-labelled caseins (Turner et al., 1992). Since lactating mammary cells also possess a regulatory pathway for casein secretion, we examined the secretion of phosphorylated caseins in the presence of the Ca2+ ionophore, ionomycin (Fig. 2A, lane c), to activate this pathway. Activation of regulated secre-
Casein phosphorylation 1223
A
A
B B
Fig. 1. The time course of casein phosphorylation in mammary cells. Isolated mammary acini were incubated with 32PO4 for 5 minutes and then cycloheximide was added to prevent further protein synthesis. Samples were taken at the times indicated after cycloheximide addition, and analysed by SDS-PAGE and autoradiography (A). The positions of migration of molecular mass standards are indicated by arrows on the left (from top: 67 kDa, 43 kDa, 29 kDa). The extent of phosphorylation was quantified for each casein by densitometry and expressed as absorbence (A) (B).
tion resulted in an overall increase in the release of [32P]labelled caseins, which was most marked in the case of the α- and γ-caseins (Fig. 2A, lane c), indicating that these may be preferentially routed to the regulated secretory pathway. Similar cell numbers were loaded on each gel track but, to rule out differences due to variation in gel loading, the extent of secretion of α-, β- and γ-caseins was determined from three independent experiments (Fig. 2B). β-Casein secretion was hardly affected by ionomycin, suggesting that this casein goes preferentially into the constitutive pathway. In order to determine where phosphorylation of the caseins occurs within the Golgi complex we made use of BFA to dissociate the Golgi cisternae from the TGN. The effects of BFA on mammary epithelial cells had not previously been reported; we determined that these cells are BFA-sensitive. Fig. 3 shows an experiment in which cells were treated with BFA during an initial 1 hour of [35S]methionine labelling. Casein synthesis and secretion
Fig. 2. Secretion of 32P-labelled caseins by consitutive and regulated secretory pathways. Isolated mammary acini were incubated with 32PO4 for 1 or 2 hours, and 32P-labelled proteins secreted into the medium were analysed by SDS-PAGE and autoradiography. (A) Autoradiogram showing 32P-labelled proteins in the medium after incubation for 1 hour (lane a), 2 hours (lane b) or for 1 hour followed by 1 hour with 10 µM ionomycin present (lane c). The positions of α-, β- and γ-caseins are shown. (B) Quantification of the change in 32P-labelled casein secretion in response to ionomycin. Data are derived from three experiments. The intensity of labelling in the presence of ionomycin was expressed as a percentage of the respective control casein value and shown as mean ± s.e.m. from three experiments.
over a further 1 hour incubation were then assayed. [35S]methionine was only present during the first hour and BFA was present in both incubation steps. This treatment with BFA resulted in a marked inhibition of secretion and in addition the extent of synthesis was reduced, as described for various other cell types (Fishman and Curran, 1992). Treatment with BFA for 1 hour resulted in marked morphological changes to the ER of mammary cells, consistent with inhibition of protein transport out of the ER and swelling of the ER due to secretory protein accumulation (Fig. 4). Due to the finding that long-term treatment with BFA inhibited protein synthesis we examined the effect of short BFA treatments on 32P incorporation into casein. Addition of 5 µg/ml BFA simultaneously with 32PO4 or preincubation for 5 minutes prior to a 20 minutes labelling period had little effect on casein phosphorylation (Fig. 5). In contrast, preincubation with 5 µg/ml BFA for 10 minutes markedly affected casein phosphorylation over the subse-
1224 M. D. Turner and others demonstrated by a parallel experiment in which the effect of BFA on [35S]methionine incorporation was monitored using the same protocol (Fig. 6B). The extent of incorporation of [35S]methionine was similar for each of the caseins and was little affected by BFA treatment under these conditions. DISCUSSION
[Brefeldin A] (µg/ml)
Fig. 3. Effect of BFA on protein synthesis and secretion from mammary cells. Isolated mammary acini were incubated with [35S]methionine for 1 hour in the presence or absence of BFA. The cells were washed and incubated for a further hour with or without BFA. The extent of incorporation of [35S]methionine in the cell pellet and the medium was determined by TCA precipitation and scintillation counting to allow determination of protein secretion or synthesis (total incorporation). The extent of secretion was expressed as a percentage of total cpm incorporated and data are shown as mean ± s.e.m. (n=4).
quent 20-minute labelling period. These results show that any effect of BFA was time-dependent, as expected if its effects required reorganisation of the Golgi cisternae rather than some direct inhibition of phosphorylation. Preincubation in BFA had a differential effect on 32P incorporation into the caseins (Fig. 6A). Phosphorylation of α-casein was essentially unaffected but the phosphorylation of the β- and γ-caseins was markedly reduced by BFA. This differential effect of BFA on 32P incorporation into caseins was not due to a rapid differential effect on casein synthesis as
We have followed the phosphorylation of caseins, which are the major proteins synthesised in isolated mammary cells, and the secretion of the phosphorylated caseins. Brefeldin A was found to inhibit casein secretion from mammary cells as expected from its effects on many cell types. The most significant finding, however, was that BFA had a differential effect on phosphorylation of the caseins, with the phosphorylation of the β- and γ-caseins being substantially inhibited in BFA-treated cells but the phosphorylation of α-caseins being essentially unaffected. The effect of BFA on many cell types has been documented in detail (Klausner et al., 1992) and it appears to have a specific action leading to disruption of membrane traffic. No other actions of BFA are known and the time-dependent effect of BFA on casein phosphorylation in mammary cells would be consistent with this being a consequence of organelle reorganisation rather than resulting from a direct effect on protein kinases. BFA has been reported to result in retrograde transport of enzymes of the cis-, medial- and transcisternae of the Golgi back to the ER. In contrast, TGN markers are segregated into alternative structures (Chege and Pfeffer, 1990; Doms et al., 1989; Lippincott-Swartz et al., 1989, 1991; Reaves and Banting, 1992) and secretory proteins are prevented from exiting the TGN (Miller et al., 1992; Rosa et al., 1992a). The differential effects of BFA on casein phosphorylation suggests that phosphorylation of
Fig. 4. Effect of BFA on ER morphology in mammary cells. Isolated mammary acini were incubated without (A) or with (B,C) 5 µg/ml BFA for 1 hour. (B) and (C) The range of morphology of the ER seen in BFA-treated cells. Bar, 200 nm.
Casein phosphorylation 1225
A
Fig. 5. Lack of effect of short BFA treatment times on 32PO4 incorporation into the caseins. Isolated mammary acini were incubated without (control) or with 5 µg/ml BFA during a 20 minute 32PO4 labelling period (BFA1) or for a prior 5 minute preincubation in addition to the 20 minute labelling period (BFA2). The incorporation of label at the end of these times was analysed by SDS-PAGE, autoradiography and densitometry. In this experiment (representative of two experiments) the β- and γcaseins were analysed together, as they were not well resolved, and the data were expressed as a percentage of the control value.
α-casein is mediated primarily by a casein kinase, normally located within the Golgi cisternae, so that phosphorylation can proceed even in the presence of BFA following collapse of cisternal enzymes to the ER, where they can continue to modify newly synthesised proteins. In contrast, since BFA would prevent newly synthesised proteins gaining access to the TGN, the inhibition of β- and γ-casein phosphorylation in BFA-treated cells suggests that these caseins are phosphorylated by a casein kinase located in the TGN. Some residual phosphorylation of β- and γ-caseins (around 35%) occurred in BFA-treated cells. This could represent phosphorylation of caseins that had already reached the TGN or, alternatively, could be due to a low level of phosphorylation of these caseins by casein kinase in Golgi cisternae. This latter interprepation may be consistent with earlier studies on mammary casein kinases (Brooks, 1989). Previous work has shown that casein kinases are located within the Golgi fractions of mammary tissue (Bingham and Farrell, 1974; Bingham et al., 1972; Capasso et al., 1989; West and Clegg, 1984) but the precise localisation of the kinases within the Golgi complex is unknown. Interestingly, enzymic studies have demonstrated the presence of two distinct casein kinases, with differing substrate specificity for α- compared to β-caseins, in bovine mammary gland (Brooks, 1989), which is consistent with our data suggesting the presence of two casein kinases with distinct localisations within the Golgi complex. One of these kinases phosphorylated both α- and β-caseins (the cisternal enzyme?) and the other could potentially be the TGNlocalised enzyme, since it phosphorylated only β-casein (Brooks, 1989). A definitive demonstration of the differing localisation of the two distinct casein kinases will require immunocytochemical data. At present, however, specific antisera for the two kinases are not available. Previous studies on the effects of BFA on secretory pro-
B
Fig. 6. Effect of BFA on 32PO4 and [35S]methionine incorporation into the caseins following a 10-minute pretreatment period. Isolated mammary acini were incubated without (control) or with 5 µg/ml BFA for 10 minutes and then 32PO4 (A) or [35S]methionine (B) was added for a further 20-minutes incubation. The incorporation of label was analysed by SDSPAGE, autoradiography and densitometery of individual casein bands. The data for each casein were expressed as a percentage of the control value and are shown as mean ± s.e.m. from three experiments.
tein phosphorylation resulted in different conclusions. Phosphorylation of chromogranin B was unaffected by BFA treatment of PC12 cells, suggesting a Golgi cisternal localisation for the kinase (Rosa et al., 1992b). In contrast, phosphorylation of progastrin in gastric mucosal segments was inhibited by BFA, implicating a kinase located in the TGN (Varro and Dockray, 1993). These studies together with our results suggest that phosphorylation of different secretory proteins, or particular phosphorylation sites, may specifically occur at distinct sites within the Golgi complex. Phosphorylation of the caseins leads to Ca2+ binding and initiates casein aggregation and micelle formation. Consistent with the suggestion that α-casein phosphorylation occurs in the Golgi cisternae, it has recently been shown that casein sub-micelles are already detectable in the cisGolgi cisternae (Clermont et al., 1993). It is likely, therefore, that casein phosphorylation begins in this compartment. Since β- and γ-casein phosphorylation apparently occurs in the TGN, association of these caseins with the micelles will occur at a later stage in micelle formation. In support of this, we have seen by immunogold labelling that
1226 M. D. Turner and others not all casein micelles contain β-casein and that substantial amounts of β-casein are present in mammary cell secretory vesicles outside of the micelle (Handel et al., unpublished observations). An additional observation in this study was that the regulated phase of casein secretion activated by the Ca2+ ionophore ionomycin contained a higher ratio of α- and γcasein to β-casein than the constitutive phase. The significance of this is unclear, and the relative abundance of casein species in each pathway could not be fully explained on the basis of phosphorylation-dependent aggregation affecting sorting between the pathways, since β- and γ- caseins were both apparently phosphorylated in the TGN but are differentially distributed between constitutive and regulated pathways. This work was supported by a Link Research Grant from the Agricultural and Food Research Council to R.D.B. and C.J.W. and in part by the Scottish Office of the Agricultural Fisheries Department (C.J.W.). M.D.T. was supported by a research studentship from the Agricultural and Food Research Council.
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