Identification of serine 24 as the unique site on the transferrin ...

THE JOURNAL OF BIOLOGICAL CHEMISTRY 0 1986 by The American Society of Biological Chemists, Inc

Vol. 261, No. 19, Issue of July 5, pp. 9034-9041, 1986 Printed in U.S.A.

Identification of Serine 24 as the Unique Site on the Transferrin Receptor Phosphorylated by Protein Kinase C*

(Received for publication, February 10,1986)

Roger J. Davis$, Gary L. Johnsong, Daniel J. Kelleher, Jacqueline K. Anderson, John E. Mole, and Michael P. Czech From the Department of Biochemistry, University of Massachusetts Medical Center, Worcester, Massachusetts 01605

Addition of tumor-promoting phorbol diesters to [32P]phosphate-labeled A431 human epidermoid carcinoma cells caused an increase in the phosphorylation state of the transferrin receptor. The A431 cell transferrin receptor was also found to be a substrate for protein kinase C in vitro. Tryptic phosphopeptide mapping of the transferrin receptor resolved the same two phosphopeptides (X and Y) after either protein kinase C phosphorylation in vitro or treatment of labeled A431 cells with phorbol diesters. [S2P]Phosphoserine was the only labeled phosphoamino acid detected. Phosphopeptide X was shown to be an incomplete tryptic digestion product which could be further digested with trypsin to generate the limit tryptic phosphopeptide (Y). Radiosequence analysis of [92P]phosphopeptide Y demonstrated that the [32P]phosphoserine was the second residue from amino terminus of the peptide. This receptor phosphopeptide was found to co-migrate with the synthetic peptide Phe-Ser(P)-Leu-Ala-Arg (where Ser(P) is phosphoserine) during reverse-phase high pressure liquid chromatography and two-dimensional thin layer electrophoresis and chromatography. The peptide Phe-Ser(P)-Leu-Ala-Arg is an expected tryptic fragment of the cytoplasmic domain of the transferrin receptor corresponding to residues 23-27. We conclude that the major site of protein kinase C phosphorylation of the transferrin receptor in vivo and in vitro is serine 24. This phosphorylation site is located within the intracellular domain of the transferrin receptor, 38 residues away from the predicted transmembrane domain.

The Ca2+- and phospholipid-dependent protein kinase (protein kinase C') is activated by diacylglycerol (1, 2). Addition of exogenous diacylglycerol to cultured cells causes the phos-

* This work was supported by Grants AM 30648, AM 32520, GM 30324, and NS 18779 from the National Institutes of Health and Program Project CA 39240 from the National Cancer Institute, Protein sequence analysis was performed using the National Com- puter Resource (Bionet) supported by the National Institutes of Health Grant 1 U41 RR 01685. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "aduertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

$ Recipient of a postdoctoral fellowship from the Damon Runyon- Walter Winchell Cancer Fund.

5 Established Investigator of the American Heart Assocation. The abbrevations used are: protein kinase C, Ca2+- and phospho-

lipid-dependent protein kinase; HEPES, 4-(2-hydroxyethyl)-l-piper- azineethanesulfonic acid; PMA, 4&phorbol 12&myristate 13a-ace- tab ; HPLC, high pressure liquid chromatography; EGF, epidermal growth factor; EGTA, [ethylenebis(oxyethylenenitrilo)]tetraacetic acid; NaDodSO,, sodium dodecyl sulfate; Ser(P), phosphoserine.

phorylation of specific target proteins such as the EGF receptor (3-5). The phosphorylation of these proteins by protein kinase C can also be caused by platelet-derived growth factor (6-10) which stimulates the hydrolysis of phosphatidylinositol 4,5-bisphosphate and results in an increase in the level of diacylglycerol ( l l ) , inositol 1,4,5-trisphosphate (E), and cytosolic free Ca2+ (13). Recently it has been demonstrated that protein kinase C is a major cellular receptor for tumor- promoting phorbol diesters which bind to protein kinase C at the diacylglycerol-binding site (for review see Ref. 14). The binding of phorbol diesters or diacylglycerol to protein kinase C results in an activation of the phosphotransferase activity of the enzyme (1,2,15) and the association of cytosolic protein kinase C with the inner surface of the plasma membrane (3, 16).

The association of protein kinase C with the plasma membrane suggests that membrane proteins are important substrates for protein kinase C. Furthermore, experiments dem- onstrating that phorbol diesters perturb the signaling by a number of growth factors and hormones (7, 17-20) suggest that receptors and the proteins involved in transmembrane signal transduction are physiologically relevant substrates for protein kinase C. It is, therefore, of interest that receptors such as the EGF receptor (21-26), type I insulin-like growth factor receptor (27), insulin receptor (20, 27), interleukin-I1 receptor (28), transferrin receptor (29, 30), B-adrenergic receptor (17), and a-adrenergic receptor (31) have been found to become phosphorylated after the addition of phorbol diester tumor promoters to intact cells. In addition two membrane proteins have been identified as substrates of protein kinase C that may be involved in signal transduction mechanisms: the a subunit of Ni (inhibitory guanyl nucleotide-binding regulatory protein) (32) and pp60""" (33, 34).

Several studies have been reported using synthetic peptide substrates and protein substrates in order to investigate the substrate specificity of protein kinase C (35-37). These experiments have indicated that protein kinase C has a require- ment for basic residues close to the phosphorylation site in the primary structure of the substrate. However, there is little information available concerning the structural requirements of membrane proteins for phosphorylation by protein kinase C. This is because the protein kinase C phosphorylation sites of only two membrane proteins have been identified. These are threonine 654 in the EGF receptor (22, 25) and serine 12 in pp60""" (34). Gould et al. (34) have noted that there is an intriguing similarity between threonine 654 in the EGF receptor and serine 12 in pp6OC-"". If it is assumed that the myris- tylated amino-terminal glycine residue of pp60"-"" is em- bedded in the membrane, then the distance between the membrane and the protein kinase C phosphorylation site (9 amino acids) is the same in both proteins. This suggests that the proximity of a potential phosphorylation site to the

9034

Transferrin Receptor Phosphorylation by Protein Kinase C 9035

plasma membrane surface may be an important factor in determining the specificity of protein kinase C. This hypothesis is an attractive one because of reports that indicate that protein kinase C binds strongly to membranes when activated by diacylglycerol or tumor promoters (3,14,16). Furthermore, the hypothesis suggests that the plasma membrane may be a fundamental component of the interaction of protein kinase C with membrane proteins.

The purpose of the experiments described in this report was to investigate whether the plasma membrane surface location of the protein kinase C phosphorylation sites o n the EGF receptor and pp6OC-"" are similar to the phosphorylation sites on other membrane proteins. The approach that we took was to investigate the phosphorylation of the transferrin receptor by protein kinase C. The primary structure of the human transferrin receptor has recently been deduced from the cDNA sequence (38, 39). Inspection of the predicted primary structure indicated that there is a potential phosphorylation site (serine 63) located close to the predicted cytoplasmic surface of the plasma membrane in a highly basic region of the receptor (Lys-Pro-Lys-Arg-Cys-Ser-Gly). How- ever, radiochemical sequence analysis and comparative phosphopeptide mapping demonstrated that serine 63 was not a major substrate for protein kinase C in vitro or in uiuo. The protein kinase C phosphorylation site on the transferrin receptor was localized to serine 24, a site that is 38 residues distant from the cytoplasmic surface of the plasma membrane i n the predicted primary structure of the receptor.

EXPERIMENTAL PROCEDURES

Materials-[32P]Phosphate (carrier free) and [y3'P]ATP were from New England Nuclear and Amersham Corp., respectively. PMA and leupeptin were obtained from Sigma. Goat anti-mouse Ig was from Cappel. Monoclonal antibody OKT9 was purified from ascites fluid produced by hybridoma cells (American Type Culture Collec- tion). Protein kinase C was prepared from bovine retinae as described (40). The specific activity of the protein kinase C used was about 160 nmol of phosphate transferred per min per mg of protein at 30 'C for histone H1 phosphorylation in the presence of Ca2+ and phosphatidylserine. The synthetic peptides, Lys-Arg-Thr-Leu-Arg-Arg and Lys-Pro-Lys-Arg-Cys-Ser-Gly-Ser-Ile-Cys-~r-Gly-Thr-Ile-Ala- Val-Ile-Val-Phe-Phe-Leu, were purchased from Peninsula Laborato- ries, the peptide Tyr-Thr-Arg-Phe-Ser-Leu-Ala-Arg was a gift from Applied Biosystems, and the peptide Val-Thr-Lys-Pro-Lys-Arg-Cys- Ser-Gly-Ser-Ile was prepared using an Applied Biosystems model 430A peptide synthesizer (Dr. J. Massagu6, University of Massachu- setts Medical Center).

Cell Culture and Preparation of Plasma Membranes"A431 human epidermoid carcinoma cells obtained from Dr. G. Todaro (Oncogen) were maintained in Dulbecco's modified Eagle's medium supplemented with 5% calf serum. Membranes were prepared by washing the cells with 120 mM NaCl, 6 mM KC], 1 mM MgC12, 5 mM EGTA, 25 mM HEPES (pH 7.4) and resuspending the cells in 200 mM sucrose, 5 mM EGTA, 50 mM NaF, 10 pg/ml leupeptin, 1 mM phenylmethylsulfonyl fluoride, 50 p~ N&VO6, 25 mM HEPES (pH 7.4) before homogenization. The homogenate was centrifuged at 1,000 X g for 10 min to give a supernatant which was layered onto a 35% (w/v) sucrose cushion. Membranes were collected from the interface after centrifugation for 90 min at 25,000 rpm in a Beckman SW 28.1 rotor, washed twice with 25 mM HEPES (pH 7.4), and stored at -70 "C.

Analysis of the Phosphorylation State of the Transferrin Receptor in Intact A431 Epidermoid Carcinoma CeIls"A431 cells were seeded in 35-mm dishes and grown to a density of 2 X l@ cells/well. The monolayers were then washed and incubated with 1 ml of phosphate- free Dulbecco's modified Eagle's medium supplemented with 0.1% calf serum and 3 mCi/ml [32P]phosphate. To achieve isotopic equilib- rium, the cells were incubated for 24 h. The cells were then treated with and without PMA for 30 min. The monolayers were washed once and the cells were lysed with 1.5 ml of 1.5% Triton X-100, 1% sodium deoxycholate, 0.1% NaDodS04, 0.5 M NaCl, 5 mM EDTA, 50 mM NaF, 100 pM N~VOE., 1 mM phenylmethylsulfonyl fluoride, 10 pg/ml leupeptin, and 25 mM HEPES (pH 7.8). The lysate was

centrifuged at 4'C for 30 min at 100,000 x g. The supernatant was then used for immunoprecipitation with a monoclonal anti-transferrin receptor antibody (OKT9). The lysate was incubated with 2 pg of monoclonal antibody for 30 min at 22 "C. The immune complexes were then collected by incubation with goat anti-mouse Ig antiserum (30 min, 22 "C) and subsequently with protein A-Sepharose CL-4B (60 min, 22 "C). The protein A-Sepharose beads were then extensively washed with lysis buffer and finally washed with 0.1% NaDodSO,, 0.2% Triton X-100, 25 mM HEPES (pH 7.8). The immunoprecipitated transferrin receptors were then solubilized with NaDodSO4 and electrophoresed on 7% polyacrylamide gels in the presence and absence of 50 mM dithiothreitol. The receptor was observed to electro- phorese on polyacrylamide gels in the presence of 0.1% NaDodSO4 as a protein of M, = 180,000 under nonreduced conditions, but after reduction with dithiothreitol it migrated as a protein of M, = 94,000.

Phosphorylution of the Transferrin Receptor by Protein Kinase C in Vitro-The phosphorylation of the transferrin receptor in A431 membranes by partially purified protein kinase C was investigated. A431 membranes (50 pg) were incubated in a final volume of 100 pl at 22 "C with 30 microunits of protein kinase C (unit = 1 pmol/min) 25 mM Tris/HCI (pH 7.4), 1 mM EGTA, 10 mM MgC12, 0.5 mM dithiothreitol, and 5 p~ [y3'P]ATP (50 pCi/nmol). In some incubations 1.5 mM CaC12 was also included. The reaction was terminated by the addition of 900 pl of 1.5% Triton X-100,1% sodium deoxycholate, 0.1% NaDodS04, 0.5 M NaC1,5 mM EDTA, 50 mM NaF, 100 pM Na3V06, 1 mM phenylmethylsulfonyl fluoride, 10 pg/ml leupeptin, and 25 mM HEPES (pH 7.8). Insoluble material was removed by centrifugation (4 "C) at 100,000 X g for 30 min, and the transferrin receptors in the supernatant were immunoprecipitated with an anti- transferrin receptor monoclonal antibody (OKT9). The transferrin receptors in the immunoprecipitate were solubilized with NaDodSO, and electrophoresed on a 7% polyacrylamide gel in the presence of

Phosphorylution of Synthetic Peptides by Protein Kinase C-The phosphorylation of synthetic peptides was assayed in an incubation

HEPES (pH 7.5), 0.5 mM dithiothreitol, 10 mM MgC12,0.5 mM EGTA, (100 pl, final volume) that contained 0.5 mg/ml peptide, 50 mM

50 p~ [y3'P]ATP (10 pCi/nmol), and 5 microunits of protein kinase C (1 unit = 1 pmol/min). In some conditions 1 mM CaClz and 25 pg/ ml phosphatidylserine were included in the incubations. The reaction was allowed to proceed for 10 min at 22 "C and was stopped by adding 900 pl of 30% (v/v) formic acid. The phosphorylated peptides were partially purified by passage through a column containing Dowex 1 (formate form), equilibrated with 30% (v/v) formic acid. The flow- through was lyophilized, dissolved in 0.1% (v/v) trifluoroacetic acid, and injected onto a Waters pBondapak C18 reverse-phase HPLC column. The phosphopeptides were eluted from the column with a linear gradient of acetonitrile. Fractions were collected at 1-min intervals. The phosphopeptides were detected in the eluant by mon- itoring the optical density at 214 nm and by measuring the Cerenkov radiation associated with each fraction collected.

Phosphopeptide Mapping-"hosphorylated transferrin receptors (100 pl) were reduced by heating to 60 "C for 15 min in the presence of 80 p1 of 10% NaDodS04, 14 mM dithiothreitol. After cooling, the transferrin receptors were alkylated by adding 40 pl of 0.4 M iodoacetamide, 0.25 M Tris-HC1 (pH 8.8) and incubation at room temper- ature for 15 min. Subsequently, 80 pl of 75% glycerol, 25% 2-mercap- toethanol was added, and the sample was heated to 60 "C for 15 min. After polyacrylamide gel electrophoresis, the gel slice containing the transferrin receptor was excised. The receptor was eluted with Na- DodS04 and precipitated with trichloroacetic acid as described (41). The sample was then digested with 1 pg of tosylphenylalanyl chloro- methyl ketone-treated trypsin in 100 mM N-ethylmorpholine (pH 8.0). After 5 h, a second addition of trypsin was made, and the incubation was allowed to proceed for a further 19 h.

Two methods were used to separate the complex mixture of peptides that were obtained after trypsin digestion of the transferrin receptor. The first method was two-dimensional thin layer separation on 100-pm cellulose plates (Machery-Nagel) by electrophoresis in 30% formic acid for 2 h a t 400 V and ascending chromatography using water/butan-1-ol/pyridine/acetic acid (60:755015) as solvent. The second method used to separate the peptides was reverse-phase HPLC employing a Waters pBondapak CIS column equilibrated with 0.1% trifluoroacetic acid. After injection of the sample, the column was washed for 5 min and the peptides were then eluted with a linear gradient of acetonitrile (0-60%) over 60 min. The flow rate was 1 ml/ min. Fractions eluted from the column were collected at 30-5 intervals. [32P]Phosphopeptides were detected by Cerenkov counting.

0.1% NaDodSOd.

9036 Transferrin Receptor Phosphorylation by Protein Kinase C Phosphoamino Acid Analysis-Phosphoamino acid analysis of the

transferrin receptor was performed by partial acid hydrolysis (1 h a t 100 "C in 6 M HCl) and thin layer electrophoresis by the method of Hunter and Sefton (42) as described (21).

Automated Amino-terminal Sequence Analysis-Sequence analysis of [32P]phosphate-labeled peptides was performed in the presence of 4 nmol of myoglobin using a modified Beckman 890C liquid-phase sequenator and a 0.1 M Quadrol Program (Beckman 121078). Two precycles were performed prior to the first cleavage. The anilinoth- iazolinones were converted to phenylthiohydantoins by reaction in 25% trifluoroacetic acid at 56 "C and were identified and quantitated by a modification of the reverse-phase HPLC procedure described by Zimmerman et al. (43) using acetonitrile. The radioactivity associated wtih the phenylthiohydantoins derived from the peptide that were released at each cycle was measured by liquid scintillation counting.

RESULTS

In order to compare the phosphorylation of the receptors for transferrin and EGF by protein kinase C, it is important that similar conditions are used for the experiments. Previous studies have demonstrated that the transferrin receptor of HL60 promyelocytic leukemia cells is a substrate for protein kinase C (29,30). However, as these cells do not express EGF receptors' we chose to investigate the phosphorylation of the transferrin receptor by protein kinase C in A431 epidermoid carcinoma cells, which we (22) and others (25) have used to identify the protein kinase C phosphorylation site on the EGF receptor (threonine 654). In preliminary experiments, the phosphorylation of the A431 cell transferrin receptor in vitro and in uiuo by protein kinase C was investigated.

The ability of protein kinase C to phosphorylate the transferrin receptor was investigated by incubating plasma membranes isolated from A431 cells with a highly purified preparation of protein kinase C and [Y-~~PIATP. The phosphorylation state of the transferrin receptor was then subsequently assayed by immunoprecipitation with a monoclonal antibody (OKT9), polyacrylamide gel electrophoresis, and autoradiog- raphy. The transferrin receptor was found to have an apparent molecular weight of 180,000 when electrophoresed under nonreduced conditions, but migrated as a protein with M, = 94,000 after reduction with dithiothreitol (Fig. 1). These observations are consistent with reports that have demonstrated that the transferrin receptor is a disulfide-linked dimer consisting of two polypeptide chains (44). We observed that there was a Ca'+-dependent phosphorylation of the transferrin receptor in the presence of protein kinase C (Fig. 1). We conclude that the A431 cell transferrin receptor is a substrate for phosphorylation by protein kinase C. A similar Ca'+ dependence of the phosphorylation of the EGF receptor in A431 membranes (24) at threonine 654 (22,25) has been previously reported.

In further experiments we investigated whether protein kinase C could phosphorylate the transferrin receptor in intact A431 cells that had been incubated for 24 h in medium containing [32P]phosphate. To stimulate protein kinase C in intact cells we employed the potent phorbol ester tumor promoter PMA which has been shown to stimulate protein kinase C in vitro (15) and to cause the phosphorylation of proteins by protein kinase C in intact A431 cells (21-26). We observed that the addition of PMA to A431 cells caused a large increase in the phosphorylation state of the transferrin receptor (Fig. 2). This observation suggests very strongly that protein kinase C can phosphorylate the transferrin receptor in intact A431 cells. However, to substantiate this conclusion it was necessary to demonstrate that the same site(s) on the transferrin receptor was phosphorylated in these in vitro and in uiuo experiments (Figs. 1 and 2). We, therefore, investigated

R. J. Davis, G. L. Johnson, D. J. Kelleher, J. K. Anderson, J. E. Mole, and M. P. Czech, unpublished data.

+ - nn nn

+ - Ca+ +

5 IO 5 IO 5 IO 5 IO TIME (min) - Mr B O K

- Mr = 9 4 K

" - D T T * D T T

FIG. 1. Phosphorylation of the transferrin receptor in vitro by protein kinase C. A431 membranes (50 pg) were incubated with a purified preparation of protein kinase C (40) in the presence and absence of CaCI2. Phosphorylation was initiated by the addition of [T-~'P]ATP (50 pCi/nmol) to a concentration of 5 p ~ . The reaction was allowed to proceed for 5 or 10 min at 22 "C. The transferrin receptors were then immunoprecipitated, and the phosphorylation state of the receptors was analyzed by polyacrylamide gel electrophoresis in the presence of 0.1% NaDodS04. Reduction of the transferrin receptors with 50 mM dithiothreitol (DTT) caused the [32P]phosphate-labeled receptors to migrate with an apparent M, = 94,000. Under nonreducing conditions, the transferrin receptor was observed to migrate as a protein of M, = 180,000. The figure presents an autoradiograph of a dried gel. Similar results were obtained in five separate experiments.

- + - + PMA + + DTT "

- Mr. 180K

- Mr. 94 K

FIG. 2. Effect of PMA on the phosphorylation state of the transferrin receptor in intact A431 cells. A431 cells were labeled with [32P]phosphate for 24 h and then treated with and without 10 nM PMA for 30 min. After solubilization of the cells, the transferrin receptors were isolated by immunoprecipitation. The immunoprecip- itates were then analyzed by polyacrylamide gel electrophoresis in the presence of 0.1% NaDodS04. Reduction of the transferrin receptors with 50 mM dithiothreitol (DTT) caused the [32P]phosphate- labeled receptors to migrate with an apparent M, = 94,000. Under nonreducing conditions the transferrin receptor was observed to be a protein of M , = 180,000. The figure presents an autoradiograph of a dried gel. Similar results were obtained in ten separate experiments.

the phosphorylation state of the transferrin receptor by phosphopeptide mapping.

Fig. 3 shows the results of two-dimensional phosphopeptide mapping of the transferrin receptor. It was found that phorbol esters caused the specific phosphorylation of the transferrin receptor on two tryptic phosphopeptides (designated x and y in Fig. 3). Similarly protein kinase C was found to phosphorylate two phosphopeptides on the transferrin receptor in isolated A431 cell plasma membranes. These two phosphopeptides were identified as phosphopeptides X and Y by mixing experiments. We conclude that PMA causes the phos-


A. CONTROL (IN VIVO) B. PMA ( IN VIVO) E. CONTROL (IN VIVO) F. PMA (IN VIVO)

C. C-KINASE (IN VITRO) D. MIX

X. . G. C-KINASE (IN VITRO) H. MIX

FIG. 3. Investigation of the phosphorylation state of the transferrin receptor by two-dimensional phosphopeptide mapping. Tryptic [32P]phosphopeptides derived from transferrin receptors that had been isolated from [32P]phosphate-labeled cells or phosphorylated by protein kinase C in vitro were resolved by electrophoresis (anode at left) on 100-pm cellulose thin layer plates in the first dimension followed by chromatography. The phosphopeptide maps A , B, C, and D were obtained by digesting the transferrin receptors for 24 h at 37 “C with 0.001% (w/v) trypsin as described under “Experimental Procedures.” The phosphopeptide maps E, F, G, and H were obtained by redigestion of the phosphopeptides isolated ( A , B, C, and D, respectively) with 0.1% (w/v) trypsin for 24 h at 37 “C. A and E are phosphopeptide maps of transferrin receptors isolated from control cells, and B and F a r e phosphopeptide maps of transferrin receptors isolated from cells treated for 30 min with 10 nM PMA. C and G are phosphopeptide maps of transferrin receptors phosphorylated in vitro by protein kinase C. D and H represent mixing experiments in which samples B and C were mixed together prior to phosphopeptide mapping (D) and samples F and G were mixed to give peptide map H.

phorylation of the transferrin receptor in intact cells on the same phosphopeptides that are phosphorylated by protein kinase C in vitro.

In several experiments we noticed that the relative yields of phosphopeptides X and Y were variable and that a larger amont of phosphopeptide Y and a decreased level of phosphopeptide X was observed if the trypsin digestion was performed using a higher concentration of the protease (Fig. 3). It was also found that a larger amount of phosphopeptide Y and a smaller amount of phosphopeptide X was obtained if the receptor was reduced and alkylated with iodoacetamide prior to trypsin digestion rather than oxidized with performic acid (data not shown). We conclude from these observations that phosphopeptide X is an incomplete digestion product and that phosphopeptide Y is derived from phosphopeptide X by further proteolysis. This hypothesis was confirmed directly by purifying phosphopeptide X by HPLC (Fig. 4) and incubating the phosphopeptide with trypsin. In the presence of a high concentration of trypsin (0.1% w/v) phosphopeptide Y was generated from phosphopeptide X (Fig. 3, C and G ) . Lower concentrations of trypsin did not cleave phosphopeptide X. This result suggests that either phosphopeptide X contains a poor trypsin cleavage site or that the trypsin used was contaminated with other proteases. In control experiments we found that phosphopeptide X was not sensitive to digestion with chymotrypsin or V8 protease.

In order to further characterize the phosphopeptides X and Y, the peptides were subjected to phosphoamino acid analysis. [32]Phosphoserine was the only phosphoamino acid detected after partial acid hydrolysis (Fig. 5). The phosphopeptides were also characterized by radiochemical sequence analysis (Table I). No radioactivity was released from phosphopeptide X after 15 cycles of degradation. However, if phosphopeptide X was first digested with trypsin prior to analysis, a peak of radioactivity was observed at cycle 2. Similarly, analysis of phosphopeptide Y also resulted in the observation of a peak of radioactivity in cycle 2. This is consistent with the finding that phosphopeptide Y is the proteolytic digestion product of

2000

0 0 C 3 1600 v)

73

7 1200 m

5 s eo0 3

4 00

t3*P] PHOSPHATE

Y X

P M A - CONTROL

0 IO 20 30 40 5 0

Acetonitrile Concentration, % FIG. 4. Isolation of phosphopeptides derived by trypsin

digestion of the transferrin receptor by high pressure liquid chromatography. A431 cells were labeled for 24 h with [32P]phosphate (3 mCi/ml). The cells were then treated with and without 10 nM PMA for 30 min, and the transferrin receptors were subsequently isolated by immunoprecipitation. Peptides obtained after trypsin digestion of the receptor were separated by reverse-phase HPLC with a Waters pBondapak C18 column equilibrated with 0.1% trifluoroacetic acid. The column was washed for 5 min after injection of the sample, and the phosphopeptides were eluted with a linear gradient of acetonitrile to 60% over 60 min. The flow rate was 1 ml/min, and fractions were collected at 30-s intervals; [32P]phosphopeptides were detected by measuring the Cerenkov radiation with a 8-counter.

phosphopeptide X. To account for the data, phosphopeptide X must contain at least an additional 14 amino acids at the amino terminus of phosphopeptide Y. This is consistent with the greatly increased hydrophobicity of phosphopeptide X compared with phosphopeptide Y as evidenced by the reten-

9038 Transferrin Receptor Phosphorylation by Protein Kinase C

I 2 3

' @ '

Ca" - f -

a - S e r W - Thr(P1 - Tyr(P)

- A B

FIG. 5. Phosphoamino acid analysis of the transferrin receptor. A, phosphoamino acid analysis of transferrin receptors isolated from [3ZP]phosphate-labeled A431 cells treated for 30 min at 37 "C without and with 10 nM PMA or 10 nM 4P-phorbol ( I , 2, and 3, respectively) was performed by partial acid hydrolysis and thin layer electrophoresis. The positions of phosphoamino acid standards are indicated. B, phosphoamino acid analysis of transferrin receptors phosphorylated in oitro by protein kinase C in the presence and absence of Ca2+. Thr(P), phosphothreonine; Tyr(P), phosphotyro- sine.

-

TABLE I Radiosequence analysis of phosphorylated peptides

Radiosequence analysis of [32P]phosphopeptides was performed using a Beckman 89OC liquid-phase sequenator. The radioactivity associated with the phenylthiohydantoins obtained at each cycle was measured with a scintillation counter. Phosphopeptides X and Y were purified by reverse-phase HPLC of a trypsin digest of the transferrin receptor isolated from A431 cells labeled with [32P]phosphate and treated with 10 nM PMA for 30 min. 9,756 cpm of phosphopeptide X and 5,146 cpm of phosphopeptide Y were analyzed. Another sample of peptide X (6,730 cpm) was further digested with 1% (w/v) trypsin for 24 h and analyzed. Two synthetic peptides corresponding to residues 20-27 (23,000 cpm) and 56-66 (12,614 cpm) of the transferrin receptor that had been phosphorylated by protein kinase C in the presence of [y3'P]ATP and subsequently purified by reverse-phase HPLC were also analyzed. The phosphorylated peptide corresponding to residues 20-27 (23,000 cpm) of the transferrin receptor was also analvzed after it was digested with 1% (w/v) t m s i n for 24 hat 37 "C.

Cycle

1 2 3 4 5 6 7 8 9

10 11 12 13 14 15

Radioactivity release (cpm)

Transferrin receptor Transferrin

Peptide X Peptide Y peptide receptor

(56-66; (-trypsin) (20-27) peptide

-Tryp- +Tryp- -Tryp- +TwP- "trypsin) "

sin sin sin sin

15 20 17 35 21 22 14 210 195 34 720 23 15 130 139 35 492 25 14 97 91 36 340 22 13 54 63 681 268 25 16 32 44 423 156 30 14 27 33 291 132 29 14 25 29 211 121 64 14 23 28 135 97 41 14 21 24 47 85 270 13 21 22 53 55 181 15 18 22 31 57 134 14 17 18 29 52 101 14 17 17 31 35 72 14 17 18 30 34 58

tion times of these phosphopeptides during reverse-phase HPLC (Fig. 4).

The identity of phosphopeptide Y was investigated by com- paring the results of the radiochemical sequencing of this peptide (Table I) with the predicted primary structure of the transferrin receptor deduced from the cloned cDNA (38, 39). The radiochemical sequencing data indicates that phosphopeptide Y contains a phosphoserine residue at position 2 from the amino terminus. We, therefore, searched the primary structure of the transferrin receptor for predicted tryptic peptides that contain a serine residue in the second position.

Seven peptides were identified, and these are presented in Table 11. One peptide (designated A in Table 11) is located within the predicted intracellular domain of the receptor, one peptide (B) consists of the entire putative transmembrane domain of the receptor, and five of the peptides (C-G) are located within the extracellular domain of the receptor (Table 11).

The intracellular and transmembrane locations of peptides A and B (Table 11) suggested that one of these peptides might represent the peptide that is phosphorylated by protein kinase C. The residues predicted to be the phosphorylation site are serine 24 (peptide A) and serine 63 (peptide B). To investigate these predictions we prepared synthetic peptides corresponding to the regions surrounding serine 24 and serine 63 in the primary sequence of the transferrin receptor. These peptides correspond to residues 20-27 and 58-78 of the transferrin receptor. The large peptide 58-78 was found to be very insoluble because of its extremely hydrophobic nature. A shorter peptide corresponding to residues 56-66 was, therefore, prepared and used for further analysis. The structures of these three peptides are: Tyr-Thr-Arg-Phe-Ser-Leu-Ala-Arg (residues 20-27), Lys-Pro-Lys-Arg-Cys-Ser-Gly-Ser-Ile-Cys-Tyr- Gly-Thr-Ile-Ala-Val-Ile-Val-Phe-Phe-Leu (residues 58-78), and Val-Thr-Lys-Pro-Lys-Arg-Cys-Ser-Gly-Ser-Ile (residues

The ability of two of the synthetic peptides (corresponding to transferrin receptor residues 20-27 and 56-66) to serve as substrates for protein kinase C was assayed in the presence and absence of Ca2+ and phosphatidylserine (Table 111). Both peptides were observed to be phosphorylated in a Ca2+- and phospholipid-dependent manner (Table 111). The peptide corresponding to residues 20-27 was a significantly better substrate than the peptide corresponding to residues 56-66. For comparison a peptide corresponding to the protein kinase C phosphorylation site (22, 25) on the EGF receptor (Lys-Arg- Thr-Leu-Arg-Arg) was also used (Table 111). It was found that the transferrin receptor synthetic peptide corresponding to residues 20-27 was phosphorylated to an extent that was iimilar to that observed with the synthetic peptide based on the EGF receptor phosphorylation site (threonine 654). How- ever, the synthetic peptide corresponding to residues 56-66 of the transferrin receptor was phosphorylated at a rate that was less than one-hundredth of that observed with the other peptides (Table 111).

The phosphorylated synthetic peptides were further characterized by phosphoamino acid analysis and radiochemical sequence analysis. [32P]Phosphoserine was the only phosphoamino acid detected in the peptides corresponding to residues 20-27 and 56-66 of the transferrin receptor (data not shown). Radiochemical sequence analysis indicated that the major phosphorylation sites in these peptides were serine 24 and serine 65 (Table 111). This data indicates that the peptide corresponding to residues 20-27 is phosphorylated on a site (serine 24) that is consistent with the site phosphorylated on the transferrin receptor by protein kinase C (Table 11). How- ever, the phosphorylation of the peptide corresponding to residues 56-66 is at a site (serine 65) that is not consistent with the site phosphorylated on the transferrin receptor by protein kinase C. This is because serine 65 is not located in a predicted tryptic peptide as the second residue from the NH2 terminus (Table 11).

The availability of a synthetic peptide corresponding to part of the intracellular domain of the transferrin receptor that was phosphorylated on serine 24 (Table 111) allowed us to test directly the hypothesis that the protein kinase C phosphorylation site on the transferrin receptor is serine 24.

56-66).

Transferrin Receptor Phosphorylation by Protein Kinase C TABLE I1

Predicted tryptic peptides derived from the transferrin receptor that have a serine residue in the second position The primary structure of the transferrin deduced from the cDNA sequence (20, 21) was used to predict the

peptides that would be obtained after trypsin digestion by assuming that trypsin will cleave the receptor only after lysine and arginine residues. Peptides containing a serine residue in the second position are presented. Five of these peptides are located in the extracellular domain of the transferrin receptor. One peptide is located in the intracellular domain of the receptor and one peptide corresponds to the predicted transmembrane domain of the receptor.

9039

Peptide Residues Sequence Domain

A 23-27 FSLAR Intracellular B 62-90 CSGSICYGTIAVIVFFLIGFMIGYLGYCK Transmembrane C 131-134 LSEK Extracellular D 194-205 DSAQNSVIIVDK E

Extracellular 326-339 SSGLPNIPVQTISR Extracellular

F 496-508 VSASPLLYTLIEK Extracellular G 694-698 ESPFR Extracellular

TABLE 111 Phosphorylntion of synthetic peptides by protein kinase C

synthetic peptide, 50 mM HEPES (pH 7.5), 0.5 mM dithiothreitol, 10 mM MgC12, 0.5 mM EGTA, 50 p M [y-32P] The phosphorylation of synthetic peptides was assayed in an incubation (100 pl) that contained 0.5 mg/ml

ATP (10 pCi/nmol) and 5 microunits of protein kinase C (1 unit = 1 pmol/min). In some conditions 1 mM CaC12 and 25 pg/ml phosphatidylserine were included in the incubations. The reaction was allowed to proceed for 10 min at 22 "C and was stopped by adding 900 p1 of 30% (v/v) formic acid. The phosphorylated peptides were partially purified by ion-exchange chromatography (Dowex 1; formate form) and then purified by reverse-phase HPLC. The radioactivity associated with the phosphopeptides eluted from the HPLC column was estimated by measuring the Cerenkov radiation. The results are presented as the means of triplicate determinations. Similar results were obtained in two separate experiments.

Phosphate incorporated Synthetic peptide

Control Caz+ Phosphatidylserine Phosphatidylserine + CaZ+

EGF receptor (652-657), KRTLRR P m l

0.18 0.17 0.31 16.3

Transferrin receptor (20-27), YTRFSLAR 0.15 0.16 0.28 13.2

Transferrin receptor (56-66), VTKPKRCSGSI 0.04 0.03 0.06 0.13

To do this we needed to prepare the limit tryptic phosphopeptide Phe-Ser(P)-Leu-Ala-Arg from the synthetic peptide Tyr-Thr-Arg-Phe-Ser(P)-Leu-Ala-Arg that corresponds to residues 20-27 of the transferrin receptor. Initial attempts to do this by incubating the synthetic phosphorylated peptide with trypsin failed in spite of the fact that the nonphospho- rylated peptide was very sensitive to trypsin digestion and was rapidly proteolyzed (data not shown). We conclude from this result that the phosphorylation of serine 24 inhibits the ability of trypsin to cleave the synthetic peptide at arginine 22.3 In view of these results we incubated the phosphorylated synthetic peptide in 100 mM N-ethylmorpholine containing 1% (w/v) trypsin at 37 "C for 24 h. The products of this digestion were analyzed by reverse-phase HPLC. Using this method, a proteolyzed derivative of Tyr-Thr-Arg-Phe-Ser(P)- Leu-Ala-Arg was obtained. This was identified as Phe-Ser(P)- Leu-Ala-Arg by the demonstration that the phosphorylated amino acid was the second residue from the amino terminus of the peptide by radiochemical sequence analysis (Table I). This synthetic phosphopeptide was then compared with phosphopeptide Y that had been purified by HPLC from a trypsin digestion of the transferrin receptor isolated from cells labeled with [32P]phosphate and treated with PMA. Fig. 6 shows that the synthetic peptide and phosphopeptide Y co-migrated during two-dimensional phosphopeptide mapping. Furthermore, the two phosphopeptides co-migrated during reverse-phase

It has been previously observed that phosphorylation of a protein close to a site of trypsin cleavage can render that site insensitive to trypsin digestion (55).

HPLC (data not shown). We conclude that the structure of phosphopeptide Y is Phe-Ser(P)-Leu-Ala-Arg and that the protein kinase C phosphorylation site on the transferrin receptor is serine 24.

DISCUSSION

The protein kinase C phosphorylation site on the EGF receptor has been identified as threonine 654 (22, 25) which is located in a highly basic region of the receptor close to the predicted transmembrane domain. A similar highly basic region adjacent to the transmembrane domain of many integral membrane proteins has been reported. These basic regions are thought to be involved in the "stop-transfer" signal during the biosynthesis of membrane proteins (45, 46). Potential protein kinase C phosphorylation sites that are similar to the EGF receptor threonine 654 are present in many proteins, for example: serine 247 and threonine 250 in the interleukin-I1 receptor (47,481; serine 312 and serine 313 in the class 1 HLA antigens (49, 50); serine 63 in the transferrin receptor (38, 39); and threonine 685 in the c-erbB2 gene product (51). In addition it has been reported that the protein kinase C phosphorylation site (serine 12) on pp60"-"" is in a highly basic region that may be located close to the cytoplasmic surface of the plasma membrane (34) and is homologous to the protein kinase C phosphorylation site on the EGF receptor. It is, therefore, possible that the protein kinase C phosphorylation sites on other membrane proteins may be similar to the EGF receptor threonine 654.

We have investigated the phosphorylation of the transferrin receptor by protein kinase C. This receptor contains a poten-

9040 Transferrin Receptor Phosphorylation by Protein Kinase C A. PEPTIDE Y

0

B. SYNTHETIC PEPTIDE

0

C MIX

0

FIG. 6. Comparative phosphopeptide mapping of [azP]phosphopeptide Y and a synthetic [SzP]phosphopeptide (Pl~e-['~Pl Ser(P)-Leu-Ala-Arg). A, [32P]phosphopeptide Y; E , synthetic peptide (Phe-[R2P]Ser(P)-Leu-Ala-Arg); C, mix of A and B. [32P]Phos- phopeptide Y was purified by reverse-phase HPLC of a trypsin digest of transferrin receptors isolated from A431 cells that had been labeled with ["P]phosphate and treated with 10 nM PMA for 30 min. To prepare the synthetic peptide, the peptide ?Srr-Thr-Arg-Phe-Ser-Leu- Ala-Arg was phosphorylated with [y3'P]ATP by protein kinase C. The phosphorylated peptide was purified by Dowex 1 chromatography and reverse-phase HPLC. The peptide was then lyophilized and subsequently digested with 1% (w/v)trypsin for 24 h at 37 "C in 100 mM N-ethylmorpholine, pH 8. The limit trypsin digestion product Phe-[R2P]Ser(P)-Leu-Ala-Arg was then isolated by reverse-phase HPLC.

tial phosphorylation site (serine 63) that is homologous to the EGF receptor threonine 654 because it is located in a highly basic region (Lys-Pro-Lys-Arg-Cys-Ser) that is adjacent to the transmembrane domain of the receptor. Digestion of the phosphorylated transferrin receptor with trypsin yielded two major phosphopeptides that were phosphorylated by protein kinase C in vitro. The two phosphopeptides were found to be related in structure and to be the result of incomplete trypsin digestion. Analysis of the phosphopeptides by partial acid hydrolysis, radiochemical sequencing, and comparative phosphopeptide mapping with synthetic peptides demonstrated that the major protein kinase C phosphorylation site on the transferrin receptor is serine 24. We conclude that the potential phosphorylation site (serine 63) that is homologous to the EGF receptor threonine 654 is not the major substrate for protein kinase C.

Inspection of the primary structure of the transferrin receptor indicates that the protein kinase C phosphorylation site (serine 24) is not located close to the transmembrane region of the receptor (38, 39). However, it is possible that the tertiary structure of the transferrin receptor is arranged so that serine 24 is located close to the cytoplasmic surface of the plasma membrane. We conclude that the protein kinase C phosphorylation site on many integral membrane proteins may not have a primary structure that is homologous to the protein kinase C phosphorylation site on the EGF receptor (threonine 654). However, the hypothesis that similarity may exist in the tertiary stucture of integral membrane proteins around the protein kinase C phosphorylation site remains to be tested.

May et al. (29,30) have suggested that the phosphorylation of the transferrin receptor by protein kinase C is important

for the regulation of the cycling of the transferrin receptor. It has been reported that in different cell types tumor-promoting phorbol diesters can cause the internalization (29,30,52,53) or externalization (54) of the transferrin receptor. Indentifi- cation of the protein kinase C phosphorylation site on the transferrin receptor will allow the hypothesis that receptor phosphorylation is directly involved in the regulation of the transferrin receptor to be tested directly. Site-directed muta- genesis of the transferrin receptor at serine 24 will provide evidence to indicate whether this amino acid is essential for the regulation of the transferrin receptor by phorbol diesters or by physiological agents that stimulate protein kinase C . These experiments are currently in progress.

Acknowledgments-We thank Dr. T. Hunter for useful discussions during the course of this work. John CNZ, Lori Kuck, and Mark Faucher are thanked for expert technical assistance. Dr. J. Massagui is thanked for assistance in the synthesis of synthetic peptides. The excellent secretarial work of Mary Halley, Judith Kula, and Karen Donahue is greatly appreciated.

REFERENCES 1. Kishimoto, A., Takai, Y.. Mori, T., Kikkawa, U., and Nishizuka,

2. Kuo, J. F., Andersson, R. G. G., Wise, B. C., Mackerlova, L., Salomonsson, I., Brackett, N. L., Katoh, N., Shoji, M., and Wrenn, R. W. (1980) Proc. Natl. Acad. Sci. U. S. A. 77, 7039- 7043

3. McCaffrey, P. G., Friedman, B., and Rosner, M. R. (1984) J. Bbl. Chem. 259.12502-12507

4. Davis, R. J., Ganong, B. R., Bell, R-M., and Czech, M. P. (1985) J. Bid. Chem. 260,1562-1566

5. Davis, R. J., Ganong, B. R., Bell, R-M., and Czech, M. P. (1985) J. Bwl. Chem. 260,5315-5322

6. Davis, R. J., and Czech, M. P. (1985) Proc. Natl. Acad. Sci.

7. Davis, R. J., and Czech, M. P. (1985) Cancer CeuS (Cold Spring Harbor) 3,101-108

8. Rozengurt, E., Rodriguez-Pena, A., Combs, M. D.. and Sinnett- Smith, J. (1984) Proc. NatL Acad. Sci. U. S. A. 81,5748-5752

9. Rodriguez-Pena, A., and Rozengurt, E. (1985) EMBO J. 4, 71- 76

10. Blackshear, P. J., Witters, L. A., Girard, P. R., Kuo, J. F., and Quamo, S. N. (1985) J. BWl. Chem. 260,13304-13315

11. Habenicht, A. J. R.. Glomset, J. A., King, W. C., Nist, C., Mitchell, C. D., and Ross, R. (1981) J. Bwl. Chem. 256,12329-12335

12. Berridge, M. J., Heslop, J. P., Irvine, R. F., and Brown, K. D. (1984) Biochem. J. 222,195-201

13. Moolenaar, W. H., Tertoolen, L. G., and delaat, S. W. (1984) J. Biol. Chem. 259,8066-8069

14. Nishizuka, Y. (1984) Nature 608.693-698 15. Castagna, M., Takai, Y., Kaibuchi, K., Sano, K., Kikkawa. U..

and Nishizuka, Y. (1982) J. Bwl. Chern. 257,7847-7851 16. Kraft, A. S., and Anderson, W. B. (1983) Nature 301,621-623 17. Kelleher, D. J., Pessin, J. E., Ruho, A. E., and Johnson, G. L.

18. Corvera. S.. and Garcia-Sainz, J. A. (1984) Biochem. BWphys.

Y. (1980) J. BWl. Chem 255,2273-2276

U. S. A. 82,4080-4084

(1984) Proc. Natl. Acad. Sci. U. S.A. 81.4316-4320

Res. Commun. 119,1128-1133 19. Friedman. B. A.. Frackelton. A. R.. Jr.. Ross. A.. Connors. J. M..

Fujiki, H., Suhmura, T., and Rosner, M. R. (1984) P&. Nati Acad. Sci. U. S. A. 81,3034-3038

20. Takayama, S., White, M. F., Lausis, V., and Kahn, C. R. (1984) Proc. NatL Acad. Sci. U. S. A. 81,7797-7881

21. Davis, R. J., and Czech, M. P. (1984) J. Bwl. Chern. 259,8545- 8549

22. Davis, R. J., and Czech, M. P. (1985) Proc. Natl. Acod. Sci. U. S. A. 82,1974-1978

23. Iwashita, S., and Fox, C. F. (1984) J. BWL Chem. 259. 2559-

24. Cochet, C., Gill, G. N.. Meiaenhelder, J., Cooper, J. A., and

25. Hunter, T., Ling, N., and Cooper, J. A. (1984) Nature 311,480-

26. Decker, S. (1984) MOL Cell BWL 4,1718-1723 27. Jacobs, S., Sahyoun, N. E., Saltiel, A. R., and Cuatrecasas, P.

2567

Hunter. T. (1984) J. Bid. Chem. 259,2553-2558

483

28.

29.

30.

31.

32.

33.

34.

35.

36.

37.

38.

39.

40.


(1983) Proc. Nutl. Acud. Sci. U. S.A. 80,6211-6213 Shackelford, D. A., and Trowbridge, I. S. (1984) J. Biol. Chem.

May, W. S., Jacobs, S., and Cuatrecasus, P. (1984) Prm. Nutl. Acud. Sci. U. S. A. 81,2106-2020

May, W. S., Sahyoun, N., Jacobs, S., Wolf, M., and Cuatrecasas, P. (1985) J. Biol. Chem. 260,9419-9426

Leab-Lundberg, L. M. F., Cotecchia, S., Lomasney, J. W., De- Bernardis, J. F., Lefkowitz, R. J., and Caron, M. G. (1985) Proc. Natl. Acud. Sci. U. S. A. 82,5651-5655

Katada, T., Gilman, A. G., Watanake, Y., Bauer, S., and Jakobs, K. H. (1985) Eur. J. Biochem. 151,431-437

Purchio, A. F., Shoyab, M., and Gentry, L. F. (1985) Science 229,

Gould, K. L., Woodgett, J. R., Cooper, J. A., Buss, J. E., Shallo-

Ferrari, S., Marchiori, F., Borin, G., and Pinna, L. A. (1985)

Turner, R. S., Kemp, B. E., Su, H., and Kuo, J. F. (1985) J. Biol. Chem. 260,11503-11507

Kishimoto, A., Nishiyama, K., Nakanishi, H., Uratsuji, Y., No- mura, H., Takeyama, Y., and Nishizuka, Y. (1985) J. Biol. Chem. 2 6 0 , 12492-12499

Schneider, C., Owen, M. J., Banville, D., and Williams, J. G. (1984) Nature 311,675-678

McClelland, A., Kuhn, L. C., and Ruddle, F. H. (1984) Cell 39,

Kelleher, D. J., and Johnson, G. L. (1985) J. Cyclic Nucleotide

259.11706-11712

1393-1395

way, D., and Hunter, T. (1985) CeU 42,849-857

FEBS Lett. 185,72-77

267-274

Protein Phosphorylation Res. 10,579-591

42.

43.

44.

45. 46.

47.

48.

49.

50. 51.

52.

53.

54. 55.

Hunter, T., and Sefton. B. M. (1980) Proc. Nutl. Acud. Sci. U. S.A. 77,1311-1315

Zimmerman. C. L.. Amella. E.. and Pisaro. J. J . (1976) Anal. . , Biochem. 75,77-85"

Greaves, M. (1982) Trends. Biochern. Sci. 7,397-400

. .

Newman, R., Schneider, C., Sutherland, R., Vodinelich, L., and

Blobel, G. (1980) Proc. Nutl. Acad. Sci. U. S. A. 77, 1496-1500 Sabatini, D. D., Kreibach, G., Morimoto, T., and Adesnik, M.

(1982) J. Cell BWL 92, 1-22 Leonard, W. J., Depper, J. M., Crabtree, G. R., Roudikoff, S.,

Pumphrey, J., Robb, R. J., Kronke, J., Svetlik, P. B., Peffer, N. J., Waldmann, T. A., and Greene, W. C. (1984) Nature 31 1 , 626-631

Nikaido, T., Shimizu, A., Ishida, N., Sabe, H., Teshigarawa, K., Maeda, M., Uchiyama, T., Yodoi, J., and Honjo, T. (1984) Nature 311,631-635

Guild, B. C., and Strominger, J. L. (1984) J. Biol. Chern. 2 5 9 ,

Koller, B. H., and Orr, H. T. (1985) J. Zmmunol. 134,2727-2733 Coussens, L., Yang-Fang, T. L., Liao, Y. C., Chen, E., Gray, A.,

McGrath, J., Seeburg, P. H., Libermann, T. A., Schlessinger, J., Francke, U., Levinson, A., and Ullrich, A. (1985) Science

Rovera, G., Ferreo, D., Pagliardi, G. L., Vartikar, J., Pesano, S.,

Acud. Sci. 379,211-220 Bottero, L., Abraham, J., and Lebman, D. (1982) Ann. N. Y.

Klausner, R. D., Harford, J., and Van Renswoude, J. (1984) Proc. Nutl. Acad. Sci. U. S. A. 81,3005-3009

Buys, S. S., Keogh, E. A., and Kaplan, J. (1984) Cell 38,569-576 Cohen, P., Watson, D. C., and Dixon, G. H. (1975) Eur. J.

9235-9240

230,1132-1139

41. Beemon, K., and Hunter, T. (1978) J. Virol. 28,551-556 Biochem. 61.79-92

Identification of serine 24 as the unique site on the transferrin ...

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