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Biology of Reproduction 63, 12-20 (2000)
© 2000 Society for the Study of Reproduction, Inc.

Minireview

Phosphorylation of Myristoylated Alanine-Rich C Kinase Substrate (MARCKS) Protein Is Associated with Bovine Luteal Oxytocin Exocytosis1

Ugur Sallia, Sara Supancica, and Fredrick Stormshak2,a

a Departments of Biochemistry/Biophysics and Animal Sciences, Oregon State University, Corvallis, Oregon 97331


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 ELEMENTS OF EXOCYTOSIS
 MARCKS PROTEIN: STRUCTURAL AND...
 IS MARCKS INVOLVED IN...
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The ruminant corpus luteum, in addition to producing progesterone, synthesizes and secretes oxytocin (OT) during the estrous cycle. Secretion of oxytocin occurs by exocytosis of membrane-encapsulated granules of this hormone. Exocytosis of oxytocin involves transport of granules through a cytoskeletal matrix including an actin cortex closely associated with the plasma membrane (PM). Actin filaments crosslinked by various proteins give rise to the structural integrity of the cortex. Myristoylated alanine-rich C kinase substrate (MARCKS), a protein specifically phosphorylated by protein kinase C (PKC), crosslinks actin filaments and anchors the actin network to the inner leaflet of the PM. There is evidence that the intact actin cortex may serve as a barrier, precluding fusion of transport vesicles with the PM. In some secretory cells, phosphorylation of MARCKS has resulted in its translocation from the PM to the cytoplasm with an associated disassembly of the actin cortex. Prostaglandin F2{alpha} (PGF2{alpha}) stimulation of the bovine corpus luteum during the midluteal phase of the estrous cycle activates PKC, which is associated with an increase in OT secretion in vivo and in vitro. Data are presented demonstrating that stimulation of bovine luteal cells with PGF2{alpha} on Day 8 of the cycle promotes rapid phosphorylation of MARCKS protein and causes its translocation from the PM to the cytoplasm and concomitant, enhanced exocytosis of OT. These data are consistent with the premise that MARCKS plays a role in the exocytotic process.

corpus luteum, oxytocin


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
ELEMENTS OF EXOCYTOSIS
 MARCKS PROTEIN: STRUCTURAL AND...
 IS MARCKS INVOLVED IN...
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Extensive reviews covering the subject of ovarian production of oxytocin (OT) in ruminant species have been published since the late 1980s [1–5]. In domestic ruminants, OT is produced by granulosa cells of the preovulatory follicle and by the corpus luteum. These previous reviews have described, in general terms, the characteristics of oxytocin production by these ovarian tissues and defined the factors that regulate its synthesis during various reproductive states of cows and ewes. These broad aspects of ovarian OT production will not be covered here, except as deemed necessary to provide the reader with adequate background information. Rather, this minireview will focus on the molecular aspects of exocytosis of OT from bovine luteal cells and will include recent data on the role of myristoylated alanine-rich C kinase substrate (MARCKS) protein in this phenomenon.

Luteal Concentrations of Oxytocin During the Estrous Cycle

Concentrations of OT in bovine and ovine corpora lutea are maximal during the midluteal phase of the estrous cycle and gradually decrease thereafter [6–8]. Bovine and ovine corpora lutea consist of large and small steroidogenic cells producing progesterone; however, the large luteal cell has been identified as the sole source of oxytocin [9]. This large luteal cell is endowed with the majority of receptors for prostaglandin F2{alpha} (PGF2{alpha}) [10]. The fact that luteal concentration of OT is maximal during the midluteal phase of the cycle is somewhat unique because it occurs days after the peak in luteal concentrations of the mRNA for this nanopeptide. In cows and ewes, OT-neurophysin-I mRNA increases early after luteinization of granulosa cells and attains maximal concentrations by approximately Day 3 of the cycle, then declines to low concentrations during the midluteal phase [11, 12]. Administration of PGF2{alpha} to cows or ewes during the midluteal phase of the estrous cycle causes an immediate increase in luteal OT secretion [13–15]. This response to exogenous PGF2{alpha} is consistent with the postulated functional relationship between these two hormones in causing luteolysis [16]. It should be noted that infusions of norepinephrine into heifers during the midluteal phase of the cycle also stimulate an immediate release of luteal OT [17], but the mechanism of action of this catecholamine is unknown. In vitro exposure of luteal slices [7] or cells [18] to PGF2{alpha} also stimulates release of OT.

Stimulus-Secretion Coupling in Luteal Oxytocin Secretion

In the mature bovine and ovine corpora lutea, the binding of PGF2{alpha} to its receptor results in the activation of phospholipase Cß (PLC) through coupling with a Gq protein [19, 20]. Activation of PLC is the initial step of the phosphoinositide cascade that generates the second messengers diacylglycerol (DAG) and inositol trisphosphate [21]. Inositol trisphosphate (IP3) binding to receptors in the endoplasmic reticulum stimulates the release of a pool of Ca2+, thus increasing intracellular concentrations of this ion ([Ca2+]i). Bovine and ovine large luteal cells respond to PGF2{alpha} with a rapid transient increase in [Ca2+]i, as a result of IP3 action, followed by a secondary sustained increase in [Ca2+]i, apparently due to influx of extracellular Ca2+ [22, 23]. The increase in intracellular Ca2+ not only promotes translocation of some protein kinase C (PKC) isozymes to the plasma membrane, but in concert with DAG, is essential in activating the conventional isoforms of PKC [24]. Calcium activation of kinases as well as phosphatases (to strike a balance in protein phosphorylation and dephosphorylation) is requisite for stimulus-coupled secretion [25]. Because of its multiple protein targets, Ca2+ plays an important role in regulating several steps in exocytosis, such as size of vesicle pools and membrane fusion events [26]. On the basis of its central role, Ca2+ may be regarded as a primary stimulus for exocytosis.


   ELEMENTS OF EXOCYTOSIS
TOP
 ABSTRACT
 INTRODUCTION
 ELEMENTS OF EXOCYTOSIS
MARCKS PROTEIN: STRUCTURAL AND...
 IS MARCKS INVOLVED IN...
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Transport Vesicles

Considerably more is known about how proteins are packaged and delivered from the endoplasmic reticulum to the Golgi than is known about the transport of proteins from the latter organelle to the plasma membrane (PM). In fact, the molecular mechanisms underlying the budding of transport vesicles from the Golgi, the mechanics of vesicle transport, and the fusion of the vesicle with the PM (exocytosis) have until recently been a mystery. It has now been determined that the budding of a transport vesicle from the trans-Golgi region involves attachment of a small GTP-binding protein called ADP-ribosylation factor (ARF). This protein controls the budding process and triggers the laying down of a set of coat proteins on the surface of the emerging vesicle (a coatmer shell). Vesicles containing neurophysin-oxytocin that are destined for transport to the PM are most likely coated with clathrin molecules and a core complex of proteins referred to as vesicle (v) soluble NSF attachment protein receptors (v-SNAREs) that binds to cognate target membrane (t) soluble NSF attachment protein receptors (t-SNAREs) in the PM [27]. After budding, the bound GTP of ARF is hydrolyzed to GDP, which may facilitate dissociation of the coat proteins exposing the v-SNAREs. The journey of vesicles across the cytoplasm to the PM is facilitated by movement along microtubules [28], but many aspects of this enhanced transport remain an enigma. Docking or fusion of the transport vesicle requires that the v-SNAREs (synaptobrevins) interact with specific t-SNAREs (syntaxin and SNAP-25). Additionally, fusion is mediated by an ATPase N-ethylmaleimide-sensitive fusion protein (NSF) and three to six soluble NSF attachment proteins (SNAPs) that bind to the SNARE (SNAP receptor) complex after interaction of v- and t-SNAREs. Hydrolysis of ATP by NSF protein generates energy that causes dissociation of the v- and t-SNAREs and releases the SNAP proteins. This event presumably creates a state that represents an irreversible step toward bilayer fusion. After hydrolysis of ATP, but before bilayer fusion is complete, the vesicular Ca2+-binding protein synaptotagmin binds to the PM. Synaptotagmin is an integral membrane protein localized to both synaptic and neurohormone secretory vesicles [26]. The large cytoplasmic domain of this protein contains two conserved C2 motifs, each similar to the Ca2+- and phospholipid-binding domain of PKC [29], that permit interaction with the PM. Binding of Ca2+ to synaptotagmin might be the switch that promotes rapid completion of the fusion process [27].

Bovine large luteal cells contain maximal quantities of transport vesicles carrying dense granules of neurophysin/oxytocin between Days 7 and 14 of the estrous cycle [30]. Administration of PGF2{alpha} to cows during the midluteal phase of the cycle causes rapid degranulation of large luteal cells [31], consistent with the increase in systemic concentrations of oxytocin detected after injection of this eicosanoid [15]. These secretory granules in luteal cells exist as a large cluster in a paranuclear position with the size of the cluster often exceeding that of the adjacent nucleus. The clustered pattern of secretory granules in the bovine luteal cell differs from the more diffuse distribution of granules observed in luteal cells of the sow [32] and ewe [33]. In pregnant cows, the population of oxytocin secretory granules is apparently maintained at a high level beyond Day 20 of gestation, but is near depletion by Day 30 [5]. The how and why of the observed differences in oxytocin secretory granule maintenance between the cyclic and pregnant cow are unknown but the presence of the embryo somehow impacts large luteal cell function in this species.

The Actin Cortex: A Potential Barrier to Exocytosis

The cytoskeleton consists of three major types of protein filaments: microtubules, microfilaments, and intermediate filaments [34]. Microfilaments are polymers of actin that together with numerous actin-binding and associated proteins constitute the actin cytoskeleton. Actin exists either in a monomeric (G-actin) or polymeric (F-actin) form. Among its many functions in mammalian cells, the actin cytoskeleton is vital for transmembrane signaling, endocytosis, and exocytosis [35]. The array of F-actin filaments that underlies and interacts with the plasma membrane forms what is referred to as the actin cortex. Actin filaments are not generated randomly or uniformly throughout the cell, but rather at discrete nucleation sites at the PM. Focal adhesions and adherens junctions are membrane-associated multimolecular complexes that control actin nucleation. Organization of the actin cortex is controlled by accessory proteins, which include filament crosslinking proteins (actin-binding protein [ABP]-280, Spectrin, ABP-120, {alpha}-actinin, myristoylated alanine-rich C kinase substrate [MARCKS]), bundling proteins, Ca2+-dependent filament severing proteins, and proteins that stabilize filaments [34, 35]. Some of the crosslinking proteins such as ABP-280, Spectrin, and MARCKS may help link actin to the PM. It is noteworthy that phosphatidylinositol-4,5-bisphosphate and Ca2+ play a role in promoting the function of several proteins associated with the assembly and disassembly of actin filaments [34, 36]. These proteins appear to be specific targets of the phosphoinositide signaling pathway that is activated upon exposure of luteal cells to PGF2{alpha}.

It has been proposed that the actin cortex acts as a barrier to the secretory granules, blocking their access to the plasma membrane. Early evidence that the actin cortex can block exocytosis was provided by Orci et al. [37], who demonstrated that disruption of the cortex in pancreatic ß cells with cytochalasin B enhanced glucose-induced secretion of insulin. Subsequently, Vitale et al. [38] reported that treatment of cultured bovine chromaffin cells with phorbol ester, a PKC activator, caused cortical filamentous actin disassembly within 6 min and potentiated nicotine-induced catecholamine exocytosis. These authors proposed that activation of PKC facilitated exocytosis via a destabilization of the actin cortex. Similarly, Muallem et al. [39] exposed permeabilized rat pancreatic acinar cells to ß-thymosin and gelsolin S1 fragment, which are actin monomer-binding proteins that cause actin depolymerization. These proteins reduced actin filaments at the apical surface of the cell with a resultant increase in exocytosis of amylase. Pretreatment of the permeabilized cells with phalloidin, an inhibitor of actin depolymerization, suppressed exocytosis of amylase. As emphasized by Muallem et al. [39], disruption of the actin filamentous cortical network must be minimal in order for exocytosis to occur. In their studies, extensive actin depolymerization by ß-thymosin inhibited exocytosis.


   MARCKS PROTEIN: STRUCTURAL AND FUNCTIONAL ASPECTS
TOP
 ABSTRACT
 INTRODUCTION
 ELEMENTS OF EXOCYTOSIS
 MARCKS PROTEIN: STRUCTURAL AND...
IS MARCKS INVOLVED IN...
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The MARCKS protein is a unique protein both structurally and functionally. It is a heat stable protein and its molecular mass ranges from 28 kDa (chicken) to 32 kDa (bovine) [40]. However, MARCKS migrates differently on SDS-PAGE with its molecular mass ranging from 60 kDa (chicken) to 87 kDa (bovine). Elaborate studies conducted by Albert et al. [41] and Hartwig et al. [42] revealed that this anomalous migration of MARCKS is due to its rod-shaped structure, with a dimension of 33 nm x 2.5 nm. The murine protein acquires its rod-shaped structure from short peptide motifs repeated a number of times in its overall amino acid sequence [43]. The MARCKS protein consists of an abundance of alanine, which constitutes 31% of 335 amino acids in the bovine MARCKS and 27% of 281 amino acids in the chicken MARCKS [43].

The MARCKS protein possesses two highly conserved regions: the N-terminal domain (amino acids 1–70) and the phosphorylation site domain (PSD, also called the effector domain; amino acids 130–180) [44, 45]. The N-terminus of MARCKS is myristoylated, which may occur cotranslationally or posttranslationally by covalently attaching myristic acid, a 14-carbon saturated fatty acid, to the N-terminal glycine residue of MARCKS [46]. This linkage is mediated by N-myristoyl transferase. Myristoylation of MARCKS is necessary for the membrane attachment of the protein [47]. Myristate anchors the protein to the PM by simply penetrating into the hydrophobic monolayer.

The phosphorylation site domain of MARCKS is a highly basic region containing 12 to 13 positively charged Lys/Arg residues [48]. This basic region also electrostatically interacts with the membrane, providing additional support to myristic acid-mediated binding of MARCKS to the PM. Phosphorylation of MARCKS by PKC occurs in this region at three or four serine residues, depending on the species [45, 49]. Protein kinase C and its active subunit, protein kinase M (PKM), have high specificity for phosphorylation of this region as shown by the use of a recombinant peptide identical to the PSD [50]. It has also been shown that other kinases, including cAMP-dependent protein kinase and calmodulin-dependent protein kinases, do not phosphorylate MARCKS or recombinant PSD [49]. The phosphorylation site domain of MARCKS also binds calmodulin (CaM) [45] and actin [42]. In its unphosphorylated state and in the presence of Ca2+, MARCKS binds to calmodulin; however, phosphorylated MARCKS does not bind calmodulin [45, 51]. The phosphorylation domain of murine MARCKS (approximately 50 amino acids) is proposed to be {alpha}-helical, containing the phosphorylation site on one side of the helix and five lysine residues that are known to be CaM/actin binding sites on the other side [52]. Binding of CaM to MARCKS results in decreased affinity of MARCKS to unilamellar phospholipid vesicles in vitro [53]. The ability of MARCKS to bind and crosslink filamentous actin (F-actin) is closely related to its phosphorylation status. Unphosphorylated MARCKS binds to F-actin and crosslinks two or more actin filaments together [42, 47]. In contrast, phosphorylation of MARCKS results in weaker binding of the protein to F-actin and disrupts its ability to crosslink actin filaments, as shown in direct binding studies [42]. In fact, in studies using cell cultures, MARCKS translocates from the membrane to cytoplasm in response to activation of PKC [47, 54]. This process of translocation leads to disassembly of the F-actin cytoskeleton [55, 56].


   IS MARCKS INVOLVED IN EXOCYTOSIS OF OXYTOCIN GRANULES IN THE BOVINE CORPUS LUTEUM?
TOP
 ABSTRACT
 INTRODUCTION
 ELEMENTS OF EXOCYTOSIS
 MARCKS PROTEIN: STRUCTURAL AND...
 IS MARCKS INVOLVED IN...
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
To address this question the following study was conducted with two objectives: 1) to determine whether exposure of dispersed bovine luteal cells to PGF2{alpha}, phorbol ester (12-O-tetradecanoylphorbol-13-acetate [TPA]), and Ca2+ ionophore A23187 would stimulate phosphorylation of MARCKS, and 2) to examine the temporal relationship between the subcellular distribution of MARCKS protein and oxytocin secretion in corpora lutea of PGF2{alpha}-treated heifers on Day 8 of the estrous cycle.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 ELEMENTS OF EXOCYTOSIS
 MARCKS PROTEIN: STRUCTURAL AND...
 IS MARCKS INVOLVED IN...
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Animals

Fourteen beef heifers were checked for behavioral estrus (Day 0) twice daily, using a vasectomized bull. On Day 8 of the cycle, heifers were prepared for removal of the corpus luteum (CL) and collection of jugular blood. The CL was removed per vaginum under lidocaine (2%)-induced caudal epidural anesthesia as described by Orwig et al. [15]. Upon removal, the CL was transported to the laboratory in a phosphate-deficient medium (Dulbecco Modified Eagle Medium, DMEM; Gibco BRL, Grand Island, NY) containing gentamicin (30 µg/ml; Gibco BRL) at 4°C. All experimental procedures were performed in accordance with the institutional guidelines for the care and use of animals.

All chemicals were purchased from Sigma (St. Louis, MO), Bio-Rad (Hercules, CA), or Boehringer Mannheim (Indianapolis, IN) unless otherwise noted. Radioisotopes were purchased from Dupont New England Nuclear (Boston, MA).

Experiment 1. Phosphorylation of MARCKS Protein In Vitro

Corpora lutea removed from four heifers were maintained at 4°C, freed of connective tissue, weighed, sliced (0.3 mm thick), and minced with a razor blade. Luteal tissue was dissociated with collagenase (3000 U/g of tissue) [57] in 25 ml of DMEM containing DNase I (1.4 U/ml). An aliquot of dispersed cells was exposed to 0.04% trypan blue and examined by light microscopy to determine cell viability. The percentage of live cells ranged from 70 to 80. Aliquots of 10 x 106 luteal cells were placed into four flasks, gassed with 95%O2 :5% CO2, and incubated in 2 ml of phosphate-deficient DMEM for 1.5 h at 37°C. After "phosphate starvation," cells were washed twice with 5 ml of medium, then resuspended in 2 ml of DMEM containing [32P]orthophosphate (100 µCi/ml), and incubated for 1.5 h (37°C) under an atmosphere of 95% O2:5% CO2. Flasks containing cells were incubated an additional 30 min after receiving the following treatments dissolved in absolute ethanol to achieve the indicated final concentrations: control (ethanol, 5 µl/ml), PGF2{alpha} (56 nM cloprostenol; Bayer Corp., Shawnee Mission, KS), TPA (1 µM), and A23187 (Ca2+ ionophore; 5 µM). After stopping the reaction by placing the flasks on ice, cells were homogenized with a glass Dounce tissue grinder (Wheaton Scientific, Millville, NJ) in 1 ml Buffer A (50 mM Tris-HCl, pH 8.3, 5 mM EDTA, 0.15 M NaCl) containing enzyme inhibitors (5 µM microcystin, 1 µM calpeptin, 1x protease inhibitor cocktail set I; Calbiochem, La Jolla, CA). In order to acquire cytosolic and membrane fractions, homogenates were first centrifuged at 1000 x g at 4°C for 10 min to eliminate nuclei and then centrifuged at 100 000 x g to separate the cytosolic fraction (supernatant) from the membrane (pellet). The cytosolic fraction was transferred to another tube and the pellet containing membrane proteins was resuspended in 0.5 ml of Buffer A containing 1% nonidet P-40. Solubilized membrane proteins were recovered by centrifugation at 100 000 x g for 1 h (4°C) and the supernatant was transferred to another tube. Both cytosolic and membrane proteins were placed into a boiling water bath for 10 min and subsequently centrifuged at 10 000 x g for 10 min to obtain heat stable MARCKS protein. Protein concentrations of cytosolic and membrane fractions were determined by use of the Pierce BCA protein assay (Pierce, Rockford, IL). Protein from each cytosolic (90 µg/lane) and each membrane (35 µg/lane) fraction for control and treated cells were subjected to 7.5% SDS-PAGE overnight at 10 mA, 200 V and then transferred to a nylon membrane in a 5-hr period at 210 mA, 60 V. Autoradiography of the membrane was performed to detect the bands of phosphorylated proteins. After obtaining an adequate autoradiograph image, nylon membranes were processed for Western blotting to confirm the precise location of phosphorylated MARCKS on the gel and to permit densitometric quantitation of the transferred protein. In order to reduce nonspecific binding, membranes were incubated for 2 h (25°C) with 5% nonfat dry milk (NFDM; Fred Meyer, Inc., Portland, OR) dissolved in water. Following this incubation, membranes were immunoblotted with mouse monoclonal MARCKS antibody (1:500 in 2.5% NFDM; antihuman MARCKS; Upstate Biotechnology, Lake Placid, NY) for 1 h (25°C). The protein-antibody complexes were probed with an antimouse IgG-alkaline phosphatase secondary antibody (Santa Cruz, CA). MARCKS bands were visualized after processing the membranes by using an alkaline phosphatase conjugate substrate kit (Bio-Rad). Densitometry readings were performed on MARCKS bands from autoradiographs and corresponding bands from Western blots using a densitometer (Molecular Dynamics, Sunnyvale, CA). Data are expressed as the ratio of densitometric units of 32P-phosphorylated MARCKS to those of the transferred MARCKS protein.

A similar phosphorylation experiment was conducted to determine if dispersed luteal cells would respond to treatments in a shorter exposure period. Dispersed luteal cells (10 x 106) from each of two heifers prepared and exposed to [32P]orthophosphate in DMEM (as described above) were treated with ethanol (control; 10 µl), PGF2{alpha} (56 nM), TPA (1 µM), and A23187 (Ca2+ ionophore; 5 µM) for 1 and 5 min at 37°C. Incubation was stopped immediately and cells were homogenized in 1 ml of Buffer A to extract the proteins. The homogenate was subjected to the centrifugation series as described above and the resulting supernatant (cytosolic fraction) was processed to acquire the heat-stable MARCKS protein. Protein concentrations of the cytosolic fraction were determined by Pierce BCA protein assay. Equal amounts of protein (300 µg) from control and treated cells were immunoprecipitated with 1:100 antibovine polyclonal MARCKS antibody (provided by Dr. Perry Blackshear) as described by Uberall et. al. [58]. Immunoprecipitation products were then subjected to 7.5% SDS-PAGE as described above. After electrophoretic fractionation, gels were dried with a vacuum dryer (Bio-Rad) and subjected to autoradiography to detect phosphorylated MARCKS. Molecular mass of visualized MARCKS bands was confirmed with molecular weight markers (Gibco-BRL).

Experiment 2. Subcellular Distribution of MARCKS Protein Relative to Oxytocin Secretion After PGF2{alpha} Stimulation In Vivo

In experiment 2, eight heifers were assigned randomly in equal numbers to control (saline) or treatment (PGF2{alpha}) groups. Sequential blood samples were collected from the jugular vein of heifers by insertion of a 16-gauge 8.3-cm Angiocath catheter (Becton-Dickinson Deseret Medical, Sandy, UT), which was flushed with heparinized saline to keep the catheter patent during the process of blood collection. A blood sample was drawn and designated as the background sample as soon as the catheter was in place. All blood samples were collected into 10 ml heparinized vacutainer tubes (Becton Dickinson Vacutainer Systems, Franklin Lakes, NJ) followed by immediate addition of EDTA (0.5 M; 20 µl) and 1,10-phenanthroline (5 mg/ml in ethanol; 10 µl) to block endogenous oxytocinase activity. Colpotomy was performed as described above, but the CL was not removed until 5 min after initiating the treatments. Treatments, PGF2{alpha} (500 µg cloprostenol/2 ml) or saline (2 ml), were administered via the catheter and an immediate blood sample was taken at Time 0. Additional blood samples were collected after 2.5, 5, 10, 20, 30, and 40 min. Blood samples were centrifuged at 4°C, and plasma was stored at -20°C until assayed for OT.

After removal, the CL was maintained at 4°C and was freed of connective tissue, weighed, minced, and homogenized with Buffer A (1 g/ml) using a Tekmar Tissuemizer (Tekmar Co., Cincinnati, OH). The homogenate was centrifuged at 1000 x g for 10 min (4°C) and the resulting supernatant was processed as described for experiment 1 to acquire the cytosolic fraction and membrane pellet. The membrane pellet was resuspended in 1 ml of Buffer A containing 1% nonidet P-40. The solubilized membrane proteins were separated from the residual pellet by centrifugation at 100 000 x g for 1 h (4°C). The heat-stable MARCKS protein in the cytosolic and membrane fraction was obtained as described above for experiment. 1. Protein concentrations of the fractions were quantified by Pierce BCA protein assay. In order to determine and compare the subcellular distribution of MARCKS in response to PGF2{alpha} treatment, the same amount of protein (300 µg/lane) from the cytosolic and membrane fractions was subjected to 7.5% SDS-PAGE and transferred to a nylon membrane. Membranes were then immunoblotted with monoclonal MARCKS antibody and stained with alkaline phosphatase (see above) to visualize the MARCKS protein. Densitometry readings were performed on MARCKS bands from Western blots. The data are presented as the ratio of arbitrary densitometric units of membrane MARCKS to cytosolic MARCKS.

Oxytocin RIA

Oxytocin was extracted from 1 ml of plasma and measured by RIA using methods adapted from Schams [59] and Abdelgadir et al. [7], using an OT antibody generously provided by Dr. Dieter Schams, Technical University of Munich, Germany. The mean extraction efficiency was 63.8% as determined by the addition of [3H]OT (4000 cpm/ml; 44.5 Ci/mmol). Plasma concentrations of OT determined by RIA were corrected for losses due to extraction. Plasma sample volumes used in the RIA were 100 µl/tube. Intra-assay coefficient of variation, determined from quality controls with a known amount of OT that was near the midpoint of the standard curve, was 3.14%. Sensitivity of the assay was 1 pg/tube.

Statistical Analysis

The data obtained from experiment 1 were analyzed by analysis of variance for an experiment of complete randomized block design. The differences in membrane to cytosol ratio between saline and PGF2{alpha} treatments in experiment 2 were tested for significance using Student's t-test. Data on plasma concentrations of OT were subjected to repeated measures analysis of variance.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 ELEMENTS OF EXOCYTOSIS
 MARCKS PROTEIN: STRUCTURAL AND...
 IS MARCKS INVOLVED IN...
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Phosphorylation of MARCKS Protein In Vitro

Figure 1A shows an autoradiograph of MARCKS protein in cytosolic and membrane fractions of control and treated cells that were electrophoretically fractionated and transferred to a nylon membrane for subsequent Western blotting. Treatments of the dispersed luteal cells with PGF2{alpha}, TPA, and A23187 for 30 min resulted in increased phosphorylation of MARCKS protein found in the 87-kDa region of both cytosol and membrane fractions (arrow). Figure 1B depicts the Western blot used to obtain the autoradiographic image shown in Figure 1A. The Western blot indicates a single band in the 87-kDa region that matches with the bands on the autoradiograph.



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  		FIG. 1. Effects of PGF2{alpha}, TPA, and A23187 on A) phosphorylation of cytosolic and membrane MARCKS determined by autoradiography of Western blots. Arrow indicates phosphorylated MARCKS protein (87 kDa). C (Control; ethanol, 10 µl), PGF2{alpha} (56 nM), TPA (1 µM), A23187 (5 µM). B) Western blot of the same nylon membrane displaying a single band and an equal amount of protein per each lane within cell fraction

The ratios of densitometric units of phosphorylated MARCKS to that of MARCKS protein present in the corresponding lane are shown in Figure 2. Prostaglandin F2{alpha}, TPA, and A23187 increased phosphorylated MARCKS in cytosol by three-, four-, and threefold, respectively (P < 0.01) while it was increased seven-, nine-, and sixfold in the membrane fraction (P < 0.01).



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  		FIG. 2. Changes in mean (±SEM) concentrations of phosphorylated MARCKS in luteal cells exposed to control vehicle (C), PGF2{alpha}, TPA, or A23187. Each bar represents the mean of four animals. Values represent arbitrary densitometric units (DU). **P < 0.01

Additionally, exposing cells 1 or 5 min to treatments increased phosphorylation of MARCKS in the cytosolic fraction. These data indicate that treatments were able to evoke a response in dispersed luteal cells in an incubation period shorter than 30 min (Fig. 3).



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  		FIG. 3. Autoradiography of phosphorylated immunoprecipitated MARCKS after exposure of luteal cells to control vehicle (C), PGF2{alpha}, TPA, and A23187 for 1 or 5 min. Equal amounts of protein samples were immunoprecipitated

Subcellular Distribution of MARCKS Protein Relative to Oxytocin Secretion after PGF2{alpha} Stimulation In Vivo

Corpora lutea of saline-treated control animals contained relatively greater quantities of MARCKS associated with the PM (Fig. 4A). In contrast, administration of PGF2{alpha} to heifers caused translocation of MARCKS protein from membrane to cytoplasm within 5 min of treatment (Fig. 4B). Relative ratios of membrane to cytosolic MARCKS in saline and PGF2{alpha}-treated animals differed (P < 0 .01; Fig. 5). Greater concentrations of MARCKS were found in the cytosol when luteal cells were stimulated with PGF2{alpha}. The residual MARCKS protein associated with the membrane fraction of corpora lutea of PGF2{alpha}-treated heifers (Fig. 4B) is believed to be partially phosphorylated (upper band) and the unphosphorylated form of MARCKS (lower band) that remains bound to membrane and actin filaments. Translocation of MARCKS protein from membrane to cytoplasm in luteal cells of PGF2{alpha}-treated heifers was associated with a concurrent increase in OT secretion. In PGF2{alpha}-treated heifers, mean plasma concentrations of OT (Fig. 6) were maximal by 5 min posttreatment, remained elevated up to 20 min, and then declined to basal concentrations by 40 min (35 min after CL removal). Mean plasma concentrations of oxytocin in control heifers were low throughout the entire treatment and sampling regimen.



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  		FIG. 4. A representative Western blot of luteal samples from four saline-treated (A) and four PGF2{alpha}-treated (B) heifers depicting the subcellular distribution of MARCKS protein (arrow). Equal amounts of protein (300 µg) were loaded from both cytosol and membrane fractions. Samples were applied in duplicate



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  		FIG. 5. Effect of PGF2{alpha} stimulation on luteal subcellular distribution of MARCKS. The data are expressed as the mean (±SEM) ratio of densitometric units of membrane-bound MARCKS to that of cytosolic MARCKS representing the immunoreactive MARCKS bands displayed in Figure 4. **P < 0.01



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  		FIG. 6. Mean (±SEM) plasma oxytocin concentrations of both saline and PGF2{alpha}-treated heifers (n = 4 each). Corpora lutea were removed 5 min after saline or PGF2{alpha} injection. Time (min) axis represents the intervals that blood samples were taken. Prostaglandin F2{alpha} induced oxytocin secretion by the CL within 5 min of the injection (P < 0.05). After removal of the CL, oxytocin levels gradually decreased to baseline (at 40 min)


   DISCUSSION
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 ABSTRACT
 INTRODUCTION
 ELEMENTS OF EXOCYTOSIS
 MARCKS PROTEIN: STRUCTURAL AND...
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The results of this study indicate that in vitro treatments of bovine luteal cells with PGF2{alpha}, TPA, or A23187 cause an immediate increase in phosphorylation of cytosolic and membrane-associated MARCKS protein, which is evident even as late as 30 min after exposure to the stimulant. The time course of this response is consistent with that observed relative to luteal secretion of OT induced by administration of PGF2{alpha} to heifers on Day 8 of the cycle (experiment 2 and Orwig et al. [15]). The observed phosphorylation of MARCKS protein is similar to the data of Quarles et al. [60] who reported that PGF2{alpha} and TPA stimulated phosphorylation of this protein in cultured MC-3T3-E1 osteoblasts. The mechanism by which PGF2{alpha}, TPA, and A23187 promote phosphorylation of MARCKS is believed to occur via activation of PKC. The way in which PGF2{alpha} activates PKC in luteal cells has been previously discussed in this review.

The bovine corpus luteum contains all known conventional (c) PKC isoforms ({alpha}, ßI, ßII, and {gamma}) and the novel (n) PKC, {epsilon} [20, 61]. Although increased intracellular concentration of Ca2+ along with DAG, phosphatidylserine (PS), and fatty acids (FA) is necessary to activate cPKC isoforms, nPKC and atypical PKC isoforms do not require Ca2+ [24, 62]. In other tissues, it appears that the isozymes of PKC found in the bovine CL are capable of recognizing and phosphorylating MARCKS with differing specificity [58, 63, 64]. Because of its structural similarity to DAG, TPA simply inserts into the plasma membrane and mimics the action of DAG, thus, directly activating PKC without increasing intracellular Ca2+ [65]. Previous studies demonstrated that TPA and A23187 [66] are as effective as PGF2{alpha} [7] in inducing OT secretion from bovine luteal slices in vitro. Vitale et al. [67] demonstrated that TPA treatment disrupted the cortical F-actin network and promoted the movement of secretory vesicles toward the submembrane zone in chromaffin cells. On this basis, it may be speculated that inducing PKC activation in luteal cells would evoke a similar response resulting in fusion and exocytosis of oxytocin granules. In the present study, in addition to PGF2{alpha} and TPA, we investigated the effects of increased intracellular Ca2+, without stimulating increased production of DAG, on MARCKS phosphorylation. Incubation of cells with A23187 increased the phosphorylation of this protein presumably activating one or more Ca2+-dependent PKC isoforms (i.e., cPKCs). Although there is no direct evidence, the latter finding suggests that luteal PKC {epsilon} is probably not responsble for phosphorylation of MARCKS.

Although phosphorylation of MARCKS occurs when the protein is associated with the plasma membrane, we found that both membrane and cytosolic fractions of cells exposed to PGF2{alpha}, TPA, and A23187 contained greater concentrations of phosphorylated MARCKS compared with those of controls. Immediately after phosphorylation, MARCKS is released from the membrane, which results in an accumulation of phosphorylated MARCKS in the cytoplasm [47, 68]. This phosphorylated MARCKS is either dephosphorylated by phosphatases and returns to the membrane [47, 68] or undergoes proteolytic cleavage regulated by a lysosomal protease, cathepsin B [69]. However, PKC-phosphorylated MARCKS is thought to be a poor substrate for this protease [70]; thus, MARCKS lingers in the cytoplasm until it is dephosphorylated. This phenomenon may explain why MARCKS is still detectable 30 min after treatment.

Because dispersed luteal cells may behave differently from cells in the intact tissue, further experiments were conducted in vivo. Prostaglandin F2{alpha} treatment of heifers promoted translocation of MARCKS protein from membrane to cytoplasm within 5 min in the CL. The amount of MARCKS protein was significantly greater in the luteal membrane fraction of control heifers compared with the amount in PGF2{alpha}-treated heifers. However, in PGF2{alpha}-injected heifers, the amount of MARCKS protein in the cytosolic fraction was greater compared with the amount in control heifers. This intracellular movement of MARCKS has been studied extensively [48, 52, 71]. Membrane association of MARCKS requires penetration of N-terminal myristate into the lipid bilayer as well as the electrostatic interaction of the basic phosphorylation domain with PM. The latter domain carries a total charge of +13 [48]. When first, second, and fourth (but not the third) serine residues are phosphorylated by PKC [50], the total charge is reduced to +7. Once reduced, the electrostatic charge cannot support the membrane attachment in conjunction with myristate; thus, MARCKS translocates to cytoplasm [48].

In the present study, translocation of MARCKS from the PM to the cytoplasm 5 min after PGF2{alpha} administration was found to be closely correlated with an increase in luteal OT secretion, indicating a possible role of MARCKS in exocytosis of this nanopeptide. It has been shown that phosphorylated MARCKS cannot crosslink the actin filaments and cause reorganization of the cytoskeleton in a variety of cell types [42, 72] Phosphorylated MARCKS-mediated changes in F-actin integrated cytoskeleton are thought to be responsible for exocytosis [29, 56, 73, 74]. Manipulating MARCKS phosphorylation and its intracellular location has been shown to play a role in secretion of prolactin from GH4C1 cells [75] and pepsinogen from gastric chief cells [76]. Further evidence indicates that the phosphorylation of MARCKS is enhanced by angiotensin II and inhibited by ACTH in bovine adrenal glomerulosa cells that secrete aldosterone [77]. The authors of this latter study suggested that changes in the phosphorylation state of MARCKS might regulate the cytoskeletal organization and, in turn, steroidogenesis. Based on the literature and our findings, we conclude that PGF2{alpha}-induced phosphorylation and translocation of MARCKS may cause disruption of the F-actin-based cytoskeleton and lead to fusion of OT granules with the PM, resulting in exocytosis (Fig. 7). Further studies will need to be conducted to examine the structural integrity of actin filaments following translocation of MARCKS from the PM to the cytoplasm.



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  		FIG. 7. A proposed role of MARCKS protein in PGF2{alpha}-stimulated OT exocytosis. Prostaglandin F2{alpha} binds to its receptor (R) in the plasma membrane of the large luteal cell and acts through a G-protein (G) to activate phospholipase C (PLC). Phospholipase C converts phosphatidylinositol-4,5-bisphosphate (PIP2) to diacylglycerol (DAG) and inositol trisphosphate (IP3). Inositol trisphosphate mobilizes Ca2+ from intracellular stores in the endoplasmic reticulum. The increased intracellular concentration of Ca2+ promotes translocation of cytoplasmic inactive protein kinase C (PKCi) to the plasma membrane where the enzyme interacts with DAG and another phospholipid to become fully active (PKCa). Activation of PKC results in phosphorylation of membrane-bound, actin filament-crosslinked MARCKS. Phosphorylated MARCKS protein is translocated from membrane to cytoplasm and thus contributes to disassembly of the filamentous actin cortex allowing movement and exocytosis of oxytocin granules from bovine large luteal cells


   ACKNOWLEDGMENTS
 
The authors thank Dr. Perry Blackshear, NIEHS, for the donations of monoclonal and polyclonal antibodies for MARCKS protein; Gwen Spizz, NIEHS, for technical advice and Dr. Dieter Schams, Technical University of Munich, Germany for the oxytocin antibody.


   FOOTNOTES
 
First decision: 10 December 1999.

1 This research was supported in part by USDA NRICG 9702426. Technical paper 11628 of the Oregon Agricultural Experiment Station. Back

2 Correspondence: Fredrick Stormshak, Department of Animal Sciences, Oregon State University, Corvallis, OR 97331. FAX: 541 737 4174; fred.stormshak{at}orst.edu Back

Accepted: February 24, 2000.

Received: November 16, 1999.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 ELEMENTS OF EXOCYTOSIS
 MARCKS PROTEIN: STRUCTURAL AND...
 IS MARCKS INVOLVED IN...
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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