Phosphorylation of the human erythrocyte glucose transporter by protein kinase C: Localization of the site of in vivo and in vitro phosphorylation

In?. J. Biochem. Vol. 21,No. 7,pp. 807-814, 1989 Printed in Great Britain. All rights reserved

0020-711X/89 f3.00 + 0.00 Copyright 0 1989 Pergamon Press pk

PHOSPHORYLATION OF THE HUMAN ERYTHROCYTE GLUCOSE TRANSPORTER BY PROTEIN KINASE C: LOCALIZATION OF THE SITE OF IN VW0 AND IN VZTRO PHOSPHORYLATION MARK R. DEZIEL,“~ HOWARD A. LIPPES,Iv3ANRIT L. RAMPAL*~' and CHAN Y. JuNG*~’* Departments of ‘Medicine and 2Biophysical Sciences, The State University of New York at Buffalo and ‘The Veteran’s Administration Medical Center, 3495 Bailey Avenue, Buffalo, NY 14215, U.S.A. [Tel. (716) 834-92001 (Receiued 12 December 1988) Abstract-l. The human erythrocyte glucose transporter was phosphorylated in vitro by protein kinase C. 2. Tryptic cleavage of phosphorylated native transporter produced two major unphosphorylated membrane-embedded fragments weighing 23 and 19 kDa and released nmnerous water-soluble peptides. 3. Ion-exchange FPLC of the soluble tryptic peptides resolved the mixture into two phosphopeptide peaks. 4. Tryptic digestion of glucose transporter that was phosphorylated in oiuo in response to phorbol esters produced soluble phosphopeptides that eluted at identical salt concentrations. 5. Proteolytic digestion and peptide mapping of the transporter revealed that the site(s) of phosphorylation lie within the large cytoplasmic domain that bisects the molecule.

INTRODUCIlON The protein-mediated facilitated diffusion of glucose and other hexoses across the plasma membranes of mammalian cells has been the subject of intensive investigation (Klip, 1982; Lefevre, 1972). The trans-

port of these sugars is greatly accelerated by the binding of the peptide hormone insulin to a receptor on the surface of many cell types, such as those of skeletal muscle (Wardzala and Jeanrenaud, 1981) and adipose tissue (Vinten et al., 1976; Martz et al., 1986). Insulin-stimulated hexose transport is apparently accomplished through an increased maximal rate of transport, without any significant alteration in the transporter’s affinity for its substrate (Vinten et al., 1976; Martz et al., 1986). A presently favored model proposes that increased rates of sugar transport initiated by insulin are the result of a “recruitment”, or translocation of transporter molecules from an inactive intracellular pool to the plasma membrane, where they are actively engaged in transport. Such a redistribution of transporters has been demonstrated experimentally (Karnielli et al., 1981; Kono et al., 1982). The biochemical events responsible for this translocation of glucose transporters are presently unknown. Insulin triggers a diverse array of cellular processes (Davis et al., 1981; Volpe and Vagelos, 1976), including protein phosphorylation (Avruch et al., 1976; Brownsey et al., 1984). Among the phosphorylating *To whom correspondence should be addressed at the Veteran’s Administration Medical Center, 3495 Bailey Avenue, Buffalo, NY 14215, U.S.A. Abbreviations: PMSF-phenylmethylsulfonylfluoride; protein kinase C-Caz+-activated, phospholipid-dependent protein kinase; SD&odium dodecyl sulfate.

enzymes of insulin-responsive cells, protein kinase C might possibly mediate some of insulin’s effects. This enzyme requires Ca*+ and diacylglycerol, whose cytoplasmic concentrations rise rapidly following insulin binding (McDonald et al., 1976; Koepfer-Hobelsburger and Wieland, 1984). Additionally, activators of protein kinase C provoke an insulin-like response of increased sugar uptake (Martz et al., 1986; Kirsch et al., 1985; O’Brien and Saladik, 1982) by directing the translocation of transporter molecules to the cell’s surface (Kitagawa et al., 1985). The in uiuo and in vitro phosphorylation of the human erythrocyte (Witters et al., 1985) and adipocyte (Gibbs et al., 1986) hexose transporters by protein kinase C has also been demonstrated. Although recent studies have shown that insulin-directed increases in glucose transport are not accompanied by the phosphorylation of a large fraction of the cells’ transporter population (Gibbs et al., 1986; Joost et al., 1987) and that activation of protein kinase C cannot fully account for the observed increased rates of sugar transport (Klip and Ramlal, 1987; Muhlbacher er al., 1988), the possibility that protein kinase C might play a contributory role in the regulation of sugar transport has not been excluded (Christensen et al., 1987). The human erythrocyte hexose transporter has been shown to resemble that of insulin-sensitive cells in many respects (Carte&u et al., 1982; Lienhard et al., 1982; Holman and Rees, 1982), and has been widely adopted as a model for examining the physicochemical properties of the protein. Earlier work, combining specific labelling of the transporter with proteolytic dissection of the protein, has provided information concerning the location of the transporter’s carbohydrate attachment site and the site that is photoalhnity labelled by the transport inhibitor cytochalasin B (Dexiel and Rothstein, 1984;

807

MARK R. DEZ~ELet al.

808

Cairns et al., 1984; Shanahan and D’Artel-Ellis, 1984) as well as the distribution and transmembrane orientation of the polypeptide’s sulfhydryl groups (Deziel et al., 1985). The recent elucidation of the transporter’s primary structure and the resultant model of the protein’s transmembrane folding (Muekler et al., 1985) have provided a useful framework for interpreting the results of such experiments. In the present study, we have characterized the in vitro phosphorylation of the erythrocyte hexose transporter by protein kinase C in order to determine the location on the transporter molecule of the site(s) of phosphorylation and to assess whether this site might be the same as that modified by in vivo phosphorylation of the transporter. MATERIALS AND METHODS

Materials

Recently outdated human blood was obtained from the American- Red Cross. [y-‘*PI-Adenosine triphosphate (3700 Ciimmol) and 13’Pl-PO, (carrier free. 9100 Ciimmol) was obtained from I6N. -Trypi& (TPCK treated), egg whitk trypsin inhibitor, molecular weight standards (SDS-7), DEAE Sephacel, bovine brain phosphatidylserine and phenylmethylsulfonylfluoride (PMSF) were from Sigma. I,2 Diolein was purchased from PL Biochemicals. Electrophoresis reagents were BioRad products. Sephadex G-150 was obtained from Pharmacia. All other chemicals were reagent grade. Preparation of the erythrocyte glucose transporter

The human erythrocyte glucose transporter was isolated by extraction of alkali-stripped erythrocyte membranes with 1.5% n-octyl glucoside and DEAEcellulose chromatography of the extract as previously described (Baldwin et al., 1982). The transporter protein was reconstituted into lipid membranes by removal of the detergent from the transporter and co-eluting lipids by dilution and centrifugal washing (Rampal et al., 1986). Functional integrity of the transporter was assessed by measuring the glucosedisplacable binding of the transport inhibitor cytochalasin B to the protein (Jung and Rampal, 1977). Preparations used in th& study bound the inhibitor with a specific activity ranging from 15 to 17 nmol/mg protein (0.8-0.95 mol of ligand/mol of peptide, .V, = 55,000). Partial pur$cation

et a/., 1982). Protein kinase C activity of each 2 ml fraction was assayed by measuring the incdrporation j2P0, from Iv-‘*Pl-ATP into H 1 histone in the oresence and absence of b;.Srnb CaCl, and added phosphatihylserine and 1,2 diolein at 30°C (Kikkawa et al., 1982). Protein kinase C typically eluted at a salt concentration of approx. 120mM NaCl. Pooled fractions (_ 18 ml) were concentrated to _ 3 ml by placing the sample in a dialysis bag and drawing the water out of the bag with dry Sephadex beads and applied to a 100 ml column of Sephadex G-150. Two ml fractions were collected and assayed for protein kinase C activity. The pooled fractions typically contained 0.2 mg protein and 2.2nmol/min units of protein kinase C activity/ml. Initial experiments indicated that the extent of phosphorylation of the glucose transporter by this partially purified preparation in the absence of Ca*+ was less than 5% of that seen with added Cal+ (see Fig. l), and further purification steps were not employed so that a sufficient yield of kinase could be obtained to phosphorylate the quantities of transporter required for biochemical characterization. In vitro phosphorylation of the glucose transporter

The partially purified preparation of protein kinase C was used to phosphorylate the membrane-reconstituted erythrocyte hexose transporter in vitro. Transporter preparations (0.25 mg/ml) were incubated with protein kinase C (1 nmol/min U/ml) in 50 mM Tris-HCl,-pH 7.7, containing 1 mM CaCl, and 50 uM IY-‘~P~-ATP 10 mM M&l,. (10 PCijmljfo; 45 min at 30°C: Approximately 0.12 mol of 3ZP-phosphate per mol of peptide. M, 55,000, were incorporated under these conditions. Up to 0.2mol of phosphate could be incorporated by increasing the ATP concentration to 100 PM and the incubation time to 90 min. The reaction was terminated by the addition of 5 mM EGTA. Most of the radioactivity was removed from the sample by dialysis against three changes of 500 vol of 5 mM sodium phosphate, pH 7.0, containing 50 mM NaCl. Removal of final traces of radioactivity and peptides originating in the kinase preparation were removed by centrifugal washing of the transporter as described above. In vivo phosphorylation of the glucose transporter

Washed erythrocytes were suspended to 10% hematocrit in media contained 120 mM NaCl, 20 mM NaHCO,, 5 mM KCl, 1 mM MgCl,, 10 mM glucose, 1 mg/ml bovine serum albumin and 100 pg/ml each of penicillin and streptomycin (Wolfe and Lux, 1978). Intracellular ATP was labelled by

of protein kinase C

For a typical preparation of protein kinase C, 10 rats were sacrificed by decapitation, their brains removed and immediately homogenized in 60 ml of ice-cold buffer containing 20 mM Tris-HCl, pH 7.7, 10 mM dithiothreitol, 0.1 mM PMSF, 0.1 mg/ml leupeptin and 1 mM CaCI,. Under these conditions, protein kinase C remains bound to the particulate fraction of the brain homogenate, which was collected by centrifugation at 40,OOOgfor 20min (Wolf et al., 1984). The pellet was resuspended in homogenization buffer containing 0.1 mM CaCl,, rather than 1 mM, collected centrifugally and washed again in that buffer. The resulting pellet was then resuspended in homogenization buffer containing no added Ca*+ and 5 mM EGTA and 2 mM EDTA and incubated for 1 hr at 4°C with stirring to solubilize protein kinase C (Wolf et al., 1984). The suspension was then centrifuged at 200,OOOg for 40 min, and the supernatent fluid (approx. 50 ml) was collected and loaded onto a 20 ml column of DEAE-Sephacel equilibrated with homogenization buffer containing 0.1 mM EDTA and EGTA. After the sample had passed through the column, the column was washed with lOm1 of homogenization buffer containing 0.1 mM EDTA and EGTA and then eluted with a 60 ml gradient of 50-250 mM NaCl in that same buffer (Kikkawa

=P (cpm)

MIGRATION

(cm)

Fig. 1. Phosphorylation of the glucose transporter by protein kinase C. Membrane-reconstituted glucose transporter was incubated with protein kinase C (1 nmol/ min U/ml) and 50 p M [32P]-ATP in the presence (0) and absence (0) of 1 mM CaCl, and electrophoresed according to the method of Laemmli (1970) as described in “Materials and Methods”. The amount of radioactivity in 3.3 mm gel slices is represented by the symbols. The solid line represents a densitometric scan of the electrophoretogram. The migration of the molecular weight standards is shown at the top.

Phosphorylation

adding 0.1 mCi of carrier-free ‘?G, per ml and incubating for 20 hr at 37°C. The phorbol ester directed phosphorylation of the erythrocyte glucose transporter was accomplished as described by Witters et al. (1985). ‘*PO,-labelled erythrocytes were incubated with 1 PM phorbol-12-myristate-13-acetate for 30 min at 37°C. The cells were rapidly cooled, lysed and the glucose transporter was isolated from the plasma membranes as described above. Proteolytic cleavage of

809

of the glucose transporter

thephosphorylated hexose transporter

Localization of the site of phosphorylation was undertaken using limited proteolytic digestion of the transporter. Tryptic cleavage was accomplished by incubating the transporter (0.5 mg/ml) with 5 pg/ml trypsin in 10 mM sodium phosphate, pH 7.5, containmg SO-mM NaCl. Proteolysis was terminated after I hr bv the addition of 50 ueiml PMSF and 1% SDS. Limited dcgestion of the trar$orter with S. aweus V8 protease or papain, which are active in SDS, was performed using a different procedure (Cleveland et al., 1977). Aliquots of transporter (20-3Opg protein) were solubilized for electrophoresis as described below, and varying amounts of these proteases ranging from 0.05 to 2yg were added to the samples. The samples were then immediately applied to electrophoresis gels. The transporter was available to the protease while the samples were within the stacking gels (approx. 45 min), and digestion was terminated when the separating gel resolved the transporter and its fragments from the proteases. Electrophoretic analysis of the phosphorylated glucose transporter

Transporter samples were solubilized in 50 mM Tris-HCl, pH 6.8, containing 1% SDS, 5% glycerol and 5% /I-mercaptoethanol. SDS-polyacrylamide slab gel electrophoresis of the intact and proteolyzed hexose transporter was performed as described by Laemmli (1970), using stacking and separating gels of 4.5 and 12% acrylamide, respectively. Coomassie blue was employed as a general protein stain and the periodate-SchitT’s reaction was used to visualize glycopeptides (Zacharius et al., 1969). Gels stained by either of these methods were scanned using a Hoeffer GS 300 scanning densitometer. Gel lanes were cut into 0.33 cm slices and the amount of radioactivity in each slice was determined by measuring the Cerenkov radiation produced by the decay of 32P, counting in the tritium channel of a Nuclear-Chicago Mark II liquid scintillation counter. The molecular weight of the hexose transporter and its proteolytic fragments were determined by comparing the electrophoretic migration of these peptides with that of a series of peptides of known molecular weight: bovine serum albumin, 66,000; ovalbumin, 45,000; glyceraldehyde-3-phosphate dehydrogenase, 36,000; carbonic anhydrase, 29,000; trypsinogen, 24,000; soybean trypsin inhibitor, 20,100; lactalbumin, 14,200. Ion exchange FPLC of soluble tryptic phosphopeptides Soluble tryptic peptides were isolated by removing the membrane-bound portions of the transporter molecule by ultracentrifugation for 40 min at 100,000g. Aliquots of the supematant fluids containing soluble tryptic peptide (0.5 ml) were brought to pH 3.0 with phosphoric acid and applied to a 5 mm x-5 cm Mono S cation -exchange column- (Pharmacia) eauilibrated with 25 mM Na phosphate. PH 3.0. The peptides were eluted from the column with a gradient of O-200 mM NaCl in 25 mM Na phosphate pH 3.0 over 20 min at a flow rate of I ml/min. Fractions of 0.5 ml were collected, and the quantity of 32P in each fraction was determined by measuring Cerenkov radiation. Other metho&

Protein concentrations were determined according to the method of Lowry et al. (1951), or with the Bio-Radprotein assay based upon the method of Bradford (1973) if the samples contained /I-mercaptoethanol.

RESULTS

As previously described (Witters et al., 1985), the human erythrocyte hexose transporter can serve as a substrate for protein kinase C. As shown in Fig. 1, in vitro phosphorylation of the purified reconstituted transporter by partially purified preparations of the kinase depended upon the presence of calcium. The phospholipid present in the transporter preparations apparently satisfied the enzyme’s requirement for lipid (Witters et al., 1985). The electrophoretic profile of the phosphorylated protein showed a broad band of labelled polypeptides ranging from 45 to 70 kDa, characteristic of the hexose transporter in this electrophoretic system (Deziel and Rothstein, 1984; Deziel et al., 1985). The extent of in vitro phosphorylation varied between 0.12 and 0.20 mol phosphate/mol polypeptide, IV, = 55,000. As previously reported (Witters et al., 1985) the red cell hexose transporter can also be phosphorylated in vivo by endogenous protein kinase C. After exposing 32Plabelled erythrocytes to phorbol- 12-myristate- 13acetate, hexose transporter was isolated from the plasma membranes of these cells. Assuming similar yields of [32P]-P0.,incorporation into the y-phosphate of cellular ATP as those described previously (Lux et al., 1978) under these labelling conditions, approx. 0.02 mol phosphate/m01 polypeptide was found in the recovered transporter. This limited in vivo modification appears to be of similar yield as that previously observed (Witters et al., 1985), which could only be detected by immunoprecipitation of the phosphorylated transporter. Proteolytic dissection of the phosphorylated transporter was undertaken in order to determine the location of the site phosphorylated by protein kinase C. Previous studies have shown that tryptic cleavage of the transporter protein produces two membranebound segments weighing approx. 25 and 19 kDa (Deziel and Rothstein, 1984; Deziel et al., 1985; Cairns et al., 1984; Shanahan and D’Artel-Ellis, 1984) that bear the protein’s carbohydrate attachment site and cytochalasin B photolabelling site, respectively, and contain all of the protein’s cysteine residues. In contrast to these observations, the same tryptic digestion of in vitro phosphorylated transporter in the presence of 50 mM NaCl resulted in the release of most of the labelled phosphate from the transporter, with none of the label being recovered in the two major membrane-associated fragments (Fig. 2). The addition of egg white trypsin inhibitor (20pg/ml) inhibited both cleavage of the protein and release of the labelled phosphate (not shown). The labelled phosphate, therefore, is being released as a tryptic phosphopeptide, rather than as free phosphate released by a contaminating phosphatase. The membrane-bound portions of the transporter molecule were removed by ultracentrifugation and the soluble tryptic phosphopeptides were collected in the supematant fluid and characterized. The mixture of tryptic peptides was separated by cation-exchange FPLC at pH 3.0. Within the complex pattern of peptides obtained, two peaks of radioactivity eluted at 8 and 12 min (nominally 80 and 120mM NaCl, Fig. 3) raising the possibility that the transporter might be phosphorylated at two distinct sites.

MARK R. DEZIELet al.

810

r ZWd

s*P

“P (cpmi

(c.m )

Elution Volume

(ml)

Fig. 4. Comparison of phospho~ptides isolated from transporter preparations that were phosphorylated in vitro and in vim Tryptic peptides from transporter preparations phosphorylated in vitro and in vivo were isolated as described in “Materials and Methods” and analyzed by ion-exchange FPLC as described in Fig. 3. The elution of phosphopep tides from transporter modified in vivo (0, scale on left axis) and in t&o (r), scale on right axis) is shown.

Abs ,/\rteraly ““in,

Fig. 2. Tryptic cleavage of the phosphorylated glucose transporter. After phosphorylation by protein kinase C, glucose transporter preparations were cleaved with 5 &nl trypsin described in “Materials and Methods” and analyzed eiectrophoretically as in Fig. 1. The profile of radioactivity with intact (0) and digested (a) transporter is shown in the upper panel. Densitometric scans of el~trophoretogr~s of intact (solid line) and cleaved (broken line) transporter preparations are represented in the lower panel. Rechromatography of each of the radioactive peaks produced a single corresponding peak that eluted at

the same salt concentration, indicating that the two peaks are not two ionic forms of the same peptide in equilibrium. An aliquot of the soluble peptides was subjected to acid hydrolysis, and the phosphoamino acids were separated by thin layer chromatography on silica gel G, using a solvent system of n-butanol, acetic acid and HZ0 (12 : 10 : 8 ratio, respectively). 32P co-migrated with phospho~rine (rl = 0.22), but not with phosphothreonine (rf = 0.27), the other amino acid that might h phosphorylated by protein kinase C (Edelman et al., 1987), a result that agrees with that previously reported (Witters et al., 1985). Transport preparations that had been phosphorylated in vim were also studied so that in viva and in

Elutiin

Volume

vitro

phosphorylation could be compared. Tryptic digestion of these preparations released soluble tryptic phosphopeptides from the membrane-bound portions of the molecule, mirroring results seen with in vitro modified transporter. Ion-exchange chroma-

tography of this material produced two radioactive peaks that eluted at exactly the same salt concen-

tration as the phosphopeptides derived from in vitro labelled transporter (Fig. 4). Thus, it appears that the site(s) on the transporter that is phosphorylated in vitro is also modified in intact erythrocytes. The site(s) of phospho~lation was further localized by fragmenting the transporter molecule with proteases that retain activity in the presence of the denaturing detergent sodium dodecyl sulfate. Limited digestion of the denature transporter with S. uureus V8 protease (OS-1 pg) produced a broadly migrating peptide extending from 45 to 30 kDa that contained -90% of the labelled phosphate that was incorporated into the transporter (Fig. 5). Digestion with a larger quantity of protease (2 ,ug) produced a smaller labelled peptide weighing 15 kDa, with no labelled bands of intermediate size. The electrophoretic profile of protein staining with Coomassie blue and the periodateBchiff’s reagent, which stains carbohydrates, were compared. Limited proteofysis of the transporter produced a glycopeptide weighing

(ml)

Fig. 3. Ion exchange FPLC of soluble tryptic phosphopeptides. Phospho~lated glucose transporter preparations were treated with trypsin and the soluble tryptic peptides were isolated and analyzed by FPLC on a Pharmacia Mono S column at pH 3 as described in “Materials and Methods”. The elution of peptides from the column was monitored continuously at 214nm (solid line), and phosphopeptides were identified by measuring the amount of radioactivity in each 0.5 ml fraction (0).

Migration

(cm)

Fig. 5. Limited digestion of phosphorylated glucose transporter with S. ~WZUSV8 protease. Phosphorylated transporter preparations (30 pg) were denatured by the addition of 1% SDS and digested with 0 (a), 0.5 (0). 1 (m), or 2 ([I3) pug of V8 protease as described in “Materials and Methods” and electrophoresed as in Fig. 1. The amount of radioactivity measured in each gel slice is shown.

Phosphorylation of the glucose transporter

811

mediate size. were observed. Digestion conditions favoring the formation of the 17 kDa phosphopeptide (100 ng papain, and an incubation time of 1 hr) produced two peptides weighing approx. 17 and 14 kDa that stained intensely with Coomassie blue (Fig. 7). Neither of these peptides stained with the periodate-SchilT’s reagent, indicating that these peptides do not contain the protein’s carbohydrate attachment site. The 17 kDa peptide contained virtually all of the “P label seen in the electrophoretic profile. Migration

(cm)

Fig. 6. Comparison of the electrophoretic profile of phosphorylated, Coomassie blue staining and periodate-bhitl’s reactive peptides produced by V8 protease digestion of the transporter. Phosphorylated glucose transporter (30 pg) was digested with 1 pg of V8 protease as in Fig. 4 and electrophoresed as in Fig. 1. The amount of radioactivity in each 3.3 mm gel slice is shown (a), along with the densitometric scan of gels stained with Coomassie blue (solid line) and the periodate-Schitf’s reagent (broken line). Absorbance is displayed in arbitrary units.

approx. 30 kDa (assuming that the leading edge of the glycopeptide band represents the molecular weight of the polypeptide chain) and a family of carbohydrate-free peptides ranging from 20 to 24 kDa (Fig. 6). The phosphate-labelled peptide comigrated exactly with the 30 kDa glycopeptide, and no label was recovered on the non-glycosylatcd fragments. The smaller 15 kDa labelled fragment produced by more rigorous digestion overlapped with trailing glycolipids that migrate near the gel front. Thus, the presence or absence of carbohydrate on this peptide could not be established. Cleavage of the denatured transporter with papain produced similarly-sized phosphopeptides. Limited digestion of the phosphorylated protein with 50 ng of papain produced a broadly migrating labelled band of approx. 30 kDa (not shown), while more rigorous treatment produced a sharper labelled band of approx. 17 kDa, which was completely resolved from the gel front (Fig. 7). No labelled peptides of inter-

Migration

DISCUSSION

Previously, it was reported that the human erythrocyte hexose transporter can serve as an in vivo and in vitro substrate for protein kinase C (Witters et al., 1985). Although phosphoserine was identified as the product of that reaction, its location on the transporter molecule and the relevance of the in vitro reaction to the in vivo process have not been investigated. In the present study, proteolytic dissection of the phosphorylated transporter, a strategy that has been employed in the study of the location and transmembrane disposition of a number of sites on the transporter molecule (Dexiel and Rothstein, 1984; Deziel et al., 1985), was undertaken in order to address these questions. A linear representation of the overlapping fragments produced by digestion of the transporter with trypsin, S. aureus V8 protease and papain and their relationship to the site(s) phosphorylated by protein kinase C is shown in Fig. 8. Tryptic digestion of the transporter produces two membrane-bound fragments: one weighing approx. 25 kDa that contains the protein’s carbohydrate moiety and three of the protein’s cysteine residues, and a second segment weighing approx. 19 kDa that contains no carbohydrate and bears the remainder of the transporter’s sulthydryl groups and the site that is photoaffinity labelled by the transport inhibitor cytochalasin B (Dexiel and Rothstein, 1984; Deziel et al., 1985). Cairns et al. (1987) have shown that tryptic cleavage additionally releases water-soluble peptides from the membrane-bound portions of the transporter that arise from the protein’s three proposed cytoplasmic domains (Muekler et al., 1985): the N-terminal

(cm)

Fig. 7. Comparison of the electrophoretic protile of phosphorylated, Coomassie blue staining and periodate-Miff’s reactive peptides produced by papain digestion of the transporter. Phosphorylated glucose transporter (20 pg) was digested with 1OOng of papain and electrophoresed as in Fig. 1, except that a longer stacking gel was employed to increase the time of digestion to approx. 1 hr. The amount of radioactivity in each 3.3 mm gel slice is shown (e), along with the densitometric scan of gels stained with Coomassie blue (solid line) and the periodate-bhiff’s reagent (broken line). Absorbance is displayed in arbitrary units.

Fig. 8. Linear representation of the overlapping peptides produced by proteolytic digestion of the hexose transporter. The molecular weight and approximate position of the peptides within the protein’s primary structure is shown, along with the protein’s carbohydrate moiety (branched tree) and the apparent site of phosphorylation by protein kinase C (s).

812

MARK

R. DEZIELPI al.

(residues l-l 1) and C-terminal (residues 452-492) “tails” and the large intracellular loop (residues 213-269). The 25 kDa tryptic fragment bears the carbohydrate moiety that is linked to Asn 45 and contains three sul~ydryl groups at cysteines 133, 201 and 207 (Deziel and Rothstein, 1984; Deziel et (I/., 1985; Muekler et al., 1985) and so must lie between the N-terminus and the cytoplasmic loop. This peptide probably corresponds to the portion of the polypeptide chain extending from Leu I2 to Arg 2 12. The 19 kDa fragment contains the remainder of the protein’s sulfhydryl groups, including a cysteine residue that is exposed at the cell’s surface (Deziel et nl., 1985) that is proposed to reside at amino acid position 429 (Muekler ef al., 1985). Considering this peptide’s mass and the release of water-soluble peptides from the cytoplasmic loop and C-terminal tail. the 19 kDa tryptic fragment must correspond to the transporter’s membrane domain that lies between these hydrophilic regions (residues 270-45 1). Neither of these two membrane-bound peptides contain the site(s) of phosphorylation (Fig. 2). Thus, only the protein’s cytoplasmic domains (residues I-1 1, 213-269 and 452-492) can contain the site(s) phosphorylated by protein kinase C. Limited digestion with S. aurelcs VS protease, an enzyme that cleaves peptide chains specifically at aspartate and glutamate residues and retains activity in the presence of the denaturing detergent SDS (Cleveland et al., 1977; Drapeau et al., 1972). produced a glycosylated peptide of approx. 30 kDa that contained the 32P-labelled phosphate incorpo~dted by protein kinase C (Fig. 5). Non-glycosylated peptides weighing as much as 24 kDa that bore no labelled phosphate were produced concimmitantly (Fig. 5). The 30 kDa glycosylated peptide and the 24 kDa carbohydrate-free peptide appear to account for virtually all of the 55 kDa transporter’s mass. Thus, the protein’s 24 kDa C-terminal region, including the C-terminal cytoplasmic domain extending from residues 452 to 492, can be positively eliminated as a possible site of phosphorylation. As shown is Fig. 8, the 30 kDa glycosylated phosphopeptide must completely encompass the 25 kDa tryptic fragment, and contains a portion of the cytoplasmic loop (residues 213-269) and probably the N-terminal tail (residues I-1 1). Limited proteolysis of the denatured transporter with papain produced a 30 kDa broadly migrating phosphorylated peptide resembling that seen with V8 protease digestion. More extensive digestion produced a smaller phosphorylated peptide having a molecular weight of I7 kDa (Fig. 7). This peptide occupied a position sufficiently removed from the glycolipids running near the gel front that periodateSchiff’s staining of the gel could be employed to demonstrate that this phosphopeptide did not contain the transporter’s carbohydrate moiety. Because the carbohydrate attachment site lies only 5 kDa from the protein’s N-terminus (Muekler et al., 1985), this 17 kDa carbohydrate-free phosphorylated peptide cannot include the protein’s N-terminal region. Thus, as shown in Fig. 8, phosphorylation must occur only in the portion of this peptide that extends beyond the C-terminal end of the 25 kDa tryptic fragment that lies within the cytoplasmic loop that

includes residues 2 13-270. Furthermore, the phosphorylated 15 kDa peptide produced by V8 protease digestion (Fig. 5) must also be carbohydrate-free and extend beyond the C-terminal end of the 25 kDa tryptic fragment. Based upon this analysis of the overlapping proteolytic fragments. we conclude that the site of in vitro phosphorylation by protein kinase C is located within the cytoplasmic loop that bisects the molecule. Phosphorylation of the cytoplasmic loop is not unexpected for the itz 1~ko modification of the transporter in the human erythrocyte, where only the transporter’s cytoplasmic domain would be accessible to the kinase. It cannot be reasonably assumed without experimental evidence, however, that in vitro phosphorylation must occur only at a site modified in viuo. Membrane-reconstituted preparations of hexose transporter consist mainly of unsealed membranes (Gorga and Lienhard, 1982) so that both the extracellular and cytoplasmic portions of the molecule would be accessible to protein kinase C. Moreover, the extent of in viva modification appears to be quite low. Previously. this modification could be detected only by specific immunoprecipitation of the transporter, despite the large number of transporters in the erythrocyte membrane (roughly 250,000 per cell) (Witters et al., 1985) and the readily apparent phosphorylation of other membrane polypeptides, including band 4.1, which is present in numbers comparable to the glucose transporter (Bennett, 1985), and a phosphoprotein of M, = 125,000 that is not among the major erythrocyte membrane proteins and must be present in lesser amounts. The more extensive in vitro phosphorylation could, therefore, possibly modify additional sites on the transporter molecule. The results of the present study. however, indicate that in vitro phosphorylation of the glucose transporter by protein kinase C does not occur on widely scattered sites on the transporter molecule, but rather is limited specifically to one or two sites within the cytoplasmic loop. The virtually identical chromatographic profiles of phosphopeptides derived from in viva and in vitro modified transporter preparations (Fig. 4) strongly suggests that the same site(s) might be modified in each case. Sufficient info~ation exists to support some speculation concerning the possible location of the site of phosphorylation within the loop. This portion of the molecule contains only four serine residues (Muekler et al., 1985). Of these, Ser 210 can be eliminated as a possible site of phosphate incorporation [assuming that the deduced amino acid sequence (Muekler et al., 1985) is correct]: Cys 207 lies on the 25 kDa membrane-bound glycosylated tryptic fragment (see above). As no trypsin sensitive amino acids reside between this residue and Ser 210, this serine residue could not be solubilized by tryptic digestion. Although Ser 265 cannot be positively eliminated, its phosphorylation is also unlikely. This residue lies close to the non-glycosylated membrane domain that is encompassed by the 19 kDa tryptic fragment, and only one trypsin-sensitive residue (Arg 269) lies between this serine residue and the membrane domain. This residue is proposed to lie very near to the membrane’s surface, and might not be cleaved efficiently (Cairns et al., 1987) under the mild

Phosphorylation

of the glucose transporter

conditions employed in these experiments. Moreover, limited proteolytic cleavage of the transporter with V8 protease produced only glycosylated phosphopeptides during initial cleavages, along with nonglycosylated peptides as large as 24 kDa that contained none of the labelled phosphate. Thus, the site of phosphorylation appears to lie closer to the protein’s glycosylated membrane domain. We propose that the two remaining serine residues at positions 226 and 248 are possible sites of phosphorylation by protein kinase C. Both residues lie on portions of the molecule that could be readily released from the membrane-bound portions of the protein by limited tryptic digestion. Both residues are located near basic amino acid residues, which are apparently required by protein kinase C (Edelman et al., 1987; O’Brien et al., 1984). These residues lie 22 amino acid residues apart, limiting phosphorylation to this small segment of the protein weighing approx. 2500 Da. Although the location of the phosphorylation site on the transporter’s cytoplasmic domain is consistent with its in situ phosphorylation, the functional significance of this modification remains unclear. At the modest extent of phosphorylation achieved in this study (12-20%), we were unable to detect alteration to a proportionate extent in the transporter’s binding of cytochalasin B, a competitive inhibitor of hexose transport (unpublished results). The hexose transporter also appears to be a relatively poor substrate for protein kinase C, compared to other well characterized substrates such as histone Hl (Glynn et al., 1985), as well as other erythrocyte membrane proteins (Witters et al., 1985). Although extensive phosphorylation of the transporter in vivo might not occur physiologically, modification of a relatively small fraction of the transporters could play an important role in the intracellular signalling processes that occur in response to insulin and other factors that affect sugar transport. Further study of the in vivo phosphorylation of the hexose transporter is required to determine if the observed phosphorylation reflects a physiological role for this phenomenon. Acknowledgements-This work was supported by Grant AM 13376 from the National Institutes of Health and by the Veteran’s Administration Medical Center, Buffalo, New York. REFERENCES

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