Identification of lymphocyte integral membrane proteins as ...

THE JOURNAL OF BIOLOGICAL CHEMISTRY Q 1986 by The American Soeiety of Biological Chemists, Inc.

Vol. 261, No. 18, Issue of June 25, pp. 8334-8341 1986 Printed in i l . ~ . ~ .

Identification of Lymphocyte Integral Membrane Proteins as Substrates for Protein Kinase C PHOSPHORYLATION OF THE INTERLEUKIN-2 RECEPTOR, CLASS I HLA ANTIGENS, AND T200 GLYCOPROTEIN*

(Received for publication, December 16,1985)

Deborah A. Shackelford and Ian S . Trowbridge From the Department of Cancer Biology, The Salk Institute for Biological Studies, San Diego, California 92138

The interleukin-2 (IL-2) receptor, the leukocyte-specific membrane glycoprotein, T200, and the class I major histocompatibility antigens (HLA) have been identified as substrates for protein kinase C in vitro. IL-2 receptors on normal human T lymphocytes and the leukemic cell line, HUT102B2, are rapidly phosphorylated in vivo in response to the tumor-promoting phorbol ester, 12-0-tetradecanoylphorbol 13-acetate (TPA). Tryptic peptide analysis showed that the in vitro and in vivo 92P-labeled IL-2 receptors were phosphorylated on the same sites. A synthetic peptide cor- responding to the carboxyl-terminal cytoplasmic tail of the IL-2 receptor was shown to be phosphorylated in vitro by protein kinase C. Tryptic digestion of the peptide generated the same 32P-labeled species as those found for the IL-2 receptor. From these studies, it was concluded that Ser-247 is the major site of phosphorylation in the IL-2 receptor and that Thr-250 is a minor site. These results also provide direct evidence that the in vivo phosphorylation of the IL-2 receptor stimulated by TPA is catalyzed by protein kinase C. The sites phosphorylated in the HLA antigens in vitro by protein kinase C or in vivo after TPA stimulation were also localized to the carboxyl-terminal cytoplasmic domain of the heavy chain by limited proteolysis.

Protein kinase C is a Ca2+- and phospholipid-dependent enzyme found in all tissues (1). The phosphotransferase activity of the enzyme is normally stimulated by diacylglycerol transiently produced from the turnover of inositol phospholipids. The breakdown of polyphosphoinositides is enhanced by a variety of extracellular signals which stimulate cell proliferation or produce numerous physiological responses (2). The tumor-promoting phorbol esters such as 12-0-tetradecanoylphorbol 13-acetate (TPA’) abrogate the requirement for phospholipid turnover and directly activate protein kinase C

* This research was supported by Grant CA 17733 from the United States Public Health Service and, in part, by Junior Fellowship J-52- 83 from the California Division of the American Cancer Society. 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.

The abbreviations used are: TPA, 12-0-tetradecanoylphorbol13- acetate; IL-2, interleukin-2; PS, phosphatidylserine; SDS, sodium dodecyl sulfate; PAGE, polyacrylamide gel electrophoresis; TPCK, L- 1-tosylamido-2-phenylethyl chloromethyl ketone; EGF, epidermal growth factor; Hepes, 4-(2-hydrosyethyl)-l-piperazineethanesulfonic acid; EGTA, [ethylenebis(oxyethylenenitrilo)]tetraacetic acid; PThr, phosphothreonine; PSer, phosphoserine.

by substituting for diacylglycerol. Phorbol esters also induce a rapid and strong association of protein kinase C with the membrane and a decrease in the cytosol-associated fraction (3). Although protein kinase C phosphorylates a wide variety of substrates found in different subcellular compartments, the phosphorylation of membrane proteins may be important to the transduction of extracellular signals which interact with membrane receptors.

In lymphocytes the growth signal delivered by mitogens is accompanied by a turnover of inositol phospholipids (2). Recently it was shown that interleukin 2 (IL-2), a growth factor for T lymphocytes, induces a transient association of protein kinase C with the membrane which parallels a loss of activity in the cytosol (4). These data suggest that protein kinase C is activated in proliferating lymphocytes, a cell type in which the enzyme is particularly abundant. However, the consequences of protein kinase C activation are ill defined, and little is known about which lymphocyte cell surface proteins are substrates for the enzyme. Previously, it was demonstrated that the membrane receptor for IL-2 is rapidly phosphorylated in vivo in response to TPA (5). Here we show directly that the IL-2 receptor is phosphorylated by protein kinase C in vitro at the same sites as observed for the in vivo TPA-stimulated phosphorylation. Studies with a synthetic peptide indicate that the primary site is serine 247 located in the cytoplasmic tail of the receptor.

Further investigation into the substrates for protein kinase C revealed that the enzyme phosphorylates other lymphocyte membrane proteins not directly involved in growth control. The class I major histocompatibility antigens (HLA) (6), which are important in immune recognition, were phosphorylated in vivo and in vitro by protein kinase C. In addition, T200, a major cell surface glycoprotein found on lymphoid and myeloid cells (71, was phosphorylated by protein kinase C in vitro. The sites of phosphorylation in the IL-2 receptor and HLA antigens, for which the primaFy sequences are known, are discussed with respect to other known sites of phosphorylation recognized by protein kinase C.

EXPERIMENTAL PROCEDURES

Radiolabeling of Cells-The cell lines HUT102B2 (8) and CCRF- CEM (9) are human T leukemic cell lines. The cells were maintained in RPMI 1640 medium supplemented with 10% fetal bovine serum. The expression of the IL-2 receptor was induced on CCRF-CEM cells by incubating cells (0.5-1 X lo6 cells/ml) with 50 ng/ml TPA (Con- solidated Midland Corp.) for 2 days.

Cell surface iodination was performed using lactoperoxidase and glucose oxidase as described (10). In uivo labeling with 32P04 was done as reported previously (5). Briefly, 0.5-1 X lo7 cells/ml were incubated at 37 “C for 4 h with 0.5 mCi/ml H;*P04 (ICN). After the 4-h incubation, 50 ng/ml TPA was added to half of the sample and the incubation continued for 15 min. The cells were washed twice

8334

Phosphorylation of IL-2 Receptor, HLA Antigens, and T200 8335 and then lysed in 1% Nonidet P-40 in PBS (0.15 M NaCI, 0.01 M Na phosphate buffer, pH 7.4) containing 5 mM Na pyrophosphate and 1 mM Na3V04.

In Vitro Phosphotmnsfeme Reactions curd Immunoprecipitation- Purified protein kinase C was generously provided by Dr. Gordon Gill and Steven Heiserman (University of California, San Diego). The enzyme was purified from rat brain by modification (11) of procedures described by Kikkawa et al. (12). The final step in the purification of the enzyme used in these experiments was chromatography on a phenyl-Sepharose column as described (11).

Immunoprecipitates of membrane proteins were prepared using unlabeled cells lysed in 1% Nonidet P-40 in PBS. The monoclonal antibody, anti-Tac (13). which recognizes the IL-2 receptor (14) was kindly provided by Dr. Thomas Waldmann (National Institutes of Health). The monoclonal antibodies, W6/32 (15) and T29/33 (7), were used to precipitate the HLA-A and -B and -C antigens and the T200 glycoprotein, respectively. Aliquots of the cell lysate (1-5 X lo' cell eq) were preincubated with 100 pl of Sepharose 4B beads followed by 50 pl of Protein A-Sepharose CL-4B (Pharmacia). The precleared lysate was then incubated with 1-3 pl of a 1:lO dilution of ascites fluid containing specific antibody and precipitated with 50 pl of Protein A-Sepharose. Alternatively, the T29/33 antibody covalently coupled to Sepharose 4B was used. The immune complexes were washed sequentially with 0.5 M NaCl, 5 mM EDTA, 50 mM Tris-HCI, pH 8, and 1% Nonidet P-40 (3 times) followed by 0.5% deoxycholate, 0.5% Nonidet P-40,0.05% SDS in PBS (3 times). The final wash was in 10 mM Tris-HCI, pH 7.5, and 5 mM MgC12.

The immunoprecipitates prepared as described above were phosphorylated by protein kinase C (0.005-0.02 units) in 100 pl of 20 mM Hepes-NaOH, pH 7.5, 10 mM MgCl2, 0.5 mM CaCI2, 5 mM dithiothreitol, 0.1 mM NasV04, 0.1-0.3 p~ [y-=P]ATP (3000 Ci/mmol, Amersham Corp.), 60 pg/ml phosphatidylserine (PS), and 6 pg/ml diolein. Reactions were incubated for 10 min at 30 "C following which the immunoprecipitates were washed again as described above. The immune complexes were eluted from the Protein A-Sepharose by boiling in Laemmli gel sample buffer (16) and analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) on 10 or 12% acrylamide gels according to Laemmli (16). The gels were exposed to film for 1-7 days with an intensifying screen.

Synthetic Peptide-The peptide Tyr-Gln-Arg-Arg-Gln-Arg-Lys- Ser-Arg-Arg-Thr-Ile was synthesized by the University of California, San Diego Peptide Facility using a Beckman 99OC synthesizer. The peptide was further purified by high pressure liquid chromatography on a C8 column using a 0-30% gradient of acetonitrile in 0.01% trifluoroacetic acid. Amino acid analyses of the major high pressure liquid chromatography fractions were kindly performed by Dr. Jean Rivier (Salk Institute). The major peak had the predicted amino acid composition.

The synthetic peptide was phosphorylated by protein kinase C as described in the previous section. The reaction contained approximately 0.5 pM peptide and was stopped by the addition of 5 mM EGTA. The phosphorylated peptide was isolated by electrophoresis a t pH 3.5 for 25 min at 1 kV on a cellulose-coated thin layer plate. The peptide was eluted with pH 1.9 buffer, lyophilized, and subjected to phosphoamino acid analysis or tryptic peptide mapping as described in the following section.

Two-dimensional Peptide Mapping-The phosphoproteins were excised from SDS-polyacrylamide gels after visualization by autora- diography, eluted, and reduced and alkylated as described (5). The samples were digested in 50 mM ammonium bicarbonate a t 37 'C by adding two 10-pg aliquots of L-1-tosylamido-2-phenylethyl chloromethyl ketone (TPCK)-treated trypsin (Worthington) added at 0 and 16 h. The reaction was stopped after 1&20 h, and the sample was lyophilized twice. The phosphopeptides were separated on thin-layer cellulose plates by electrophoresis a t pH 4.7 using n-butyl alco- ho1:pyridine:acetic acidHzO (2:1:1:36) followed by ascending chromatography in n-butyl alcoho1:pyridine:acetic acidHz0 (15:103:12) essentially as described by Gibson (17).

Phosphoamino acid analyses were performed on proteins isolated as described above or on phosphopeptides eluted from the thin-layer cellulose plates. The samples were hydrolyzed in 5.7 N HCI at 110 "C for 2-3 h. The phosphoamino acids were separated by electrophoresis a t pH 1.9 followed by electrophoresis a t pH 3.5 (18).

Limited Proteolysis of HLA Antigens-The HLA antigens from iodinated cells or cells labeled with 32P04 in vivo were immunoprecipitated with the monoclonal antibody W6/32 as described above for the unlabeled cell lysates. In some experiments, heat-killed and formalin-fixed Staphylococcus wreus Cowan I strain bacteria (Pan-

sorbin, Calbiochem-Behring) were used instead of Protein A-Sepha- rose. The HLA antigens phosphorylated in vitro by protein kinase C were isolated and labeled as described earlier.

In a typical experiment the immunoprecipitate was washed and resuspended in 60 pl of 10 mM Tris, pH 7.5, and 5 mM MgC12. The sample was divided into 4 aliquots and incubated at 37 "C for 1 h with 0-1.0 pg of TPCK/trypsin or papain (Sigma). The papain was preactivated in 2 mM dithiothreitol. The reaction was stopped by adding 30 pl of Laemmli gel sample buffer and boiling. The samples were analyzed immediately by SDS-polyacrylamide gel electrophoresis.

RESULTS

Phosphorylation of the ZL-2 Receptor by Protein K i m e C- It has been shown previously that incubation of HUT102B2 cells or phytohemagglutinin-stimulated peripheral blood lymphocytes with the tumor-promoting phorbol ester, TPA, causes a rapid phosphorylation of the receptor on serine and threonine residues (5). To demonstrate directly that the IL-2 receptor is a substrate for protein kinase C, the receptor was immunoprecipitated from HUT102B2 cells and incubated with purified protein kinase C in vitro. Under nonreducing conditions, phosphorylated proteins of M, = 49,000 and 105,000 were observed (Fig. 1, lane 2). The IL-2 receptor immunoprecipitated by anti-Tac from cell surface-iodinated HUT102B2 cells co-migrated with the major phosphorylated protein (lane 1 ).

Previously, it was found that after prolonged incubation with TPA, the IL-2 receptor was induced on some T leukemic cell lines, including CCRF-CEM (5, 19). However, phospho-

1 2 3 4 5 6 7 8 9 '00 - M~ X

-200

-116

- 93

- 66

IL-2 R-

-45

FIG. 1. Phosphorylation of the IL-2 receptor by protein kinase C in vitro. The IL-2 receptor was immunoprecipitated with anti-Tac antibody from CEM cells induced with 50 ng/ml TPA for 2 days or from HUT102B2. The immunoprecipitates were phosphorylated in vitro by protein kinase C as described under "Experimental Procedures" and analyzed by SDS-PAGE. Lane 1 is an anti-Tac immunoprecipitate from '261-labeled HUT102B2 cells. Lanes 2 and 3 are anti-Tac immunoprecipitates from HUT102B2 and TPA-induced CEM cells, respectively, phosphorylated in vitro by protein kinase C. Lanes 4-6 and 8-9 are anti-Tac immunoprecipitates from HUT102B2 cells phosphorylated in vitro with the following modifications to the phosphotransferase reaction buffer: lanes 4 and 8, without phosphatidylserine and diolein; lanes 5 and 9, without Ca2+ and with 2.5 mM EGTA; and lanes 6 and 10, standard conditions. The Protein A- Sepharose CL-4B beads used to preclear the cell lysate were also incubated with protein kinase C (lanes 7 and 11). The samples in lanes 1-7 were subjected to electrophoresis in the absence of reducing agents, whereas those in lanes 8-11 contained 2-mercaptoethanol. M, X of the standards are indicated at right.

8336 Phosphorylation of IL-2 Receptor, HLA Antigens, and T200

rylation of the receptor in vivo could not be detected with TPA-induced CEM cells under the same conditions used to demonstrate phosphorylation of the IL-2 receptor from HUT102B2 cells. To investigate whether in vitro phosphorylation of the receptors from the two cell lines also differed, the IL-2 receptor from TPA-induced CEM cells was immunoprecipitated and incubated with protein kinase C. Fig. 1 (lane 3) shows that the IL-2 receptor from TPA-induced CEM cells was also phosphorylated by protein kinase C. Under nonreducing conditions, a major phosphorylated protein of M, = 53,000 and an additional protein of MI = 113,000 were observed. As shown previously, the MI of the IL-2 receptor from TPA-induced CEM cells was greater than that of the receptor from HUT102B2 cells, probably due to differences in glycosylation or other post-translational modifications which also result in the diffuse migration of the receptor on SDS-polyacrylamide gels (5 , 20). The IL-2 receptors from both HUT102B2 and TPA-induced CEM cells were phosphorylated by protein kinase C primarily on serine residues and to a lesser degree on threonine as observed for the in vivo phosphorylated receptor from HUT102B2 cells ( 5 ) (data not shown). The 105-113,000-dalton 32P-labeled species is probably a disulfide-bonded dimer of the IL-2 receptor. Under reducing conditions, the amount of the 105,000-dalton phosphoprotein was decreased (Fig. 1, lane 6 verszu 10). Further- more, the tryptic phosphopeptide maps of this high MI protein contained the same peptides as obtained with the 49,000- dalton phosphoprotein (data not shown). In the presence of reducing agents, the phosphorylated IL-2 receptor migrated more slowly at approximately M, 54,000 (Fig. 1, lane 10). This was also reported for the lZ5I-labeled 50,000-dalton receptor and is consistent with the presence of intramolecular disulfide bonds (20).

Phosphorylation of the IL-2 receptor by protein kinase C in vitro was dependent upon the addition of Ca". The elimi- nation of Ca2+ and the addition of EGTA prevented the phosphorylation of the IL-2 receptor (Fig. 1, lanes 5 and 9). However, omission of PS and diolein only minimally reduced the level of phosphorylation observed (lanes 4 and 8). It is possible that residual membrane lipid associated with the immunoprecipitated IL-2 receptor was sufficient to activate protein kinase C. Previous studies have shown that the addition of PS and diolein was not required for the protein kinase C-catalyzed phosphorylation of the epidermal growth factor (EGF) receptor in A431 membranes (11) or of the glucose transporter solubilized in octyl glucoside and erythrocyte membrane lipids (21). However, phosphorylation of the ex- tensively purified EGF receptor required phospholipid and diacylglycerol(l1). The phosphorylation of the synthetic peptide described in the next section was increased about 10-fold by PS and diolein and to a much lesser extent by the addition of Nonidet P-40 (data not shown). This is consistent with previous work showing that PS and diolein stimulated phosphorylation of the water-soluble substrate, histone H1, 6-10- fold by a preparation of protein kinase C similar to that used in the present studies (11).

The in vivo and in vitro 32P-labeled IL-2 receptor molecules were compared by two-dimensional tryptic peptide mapping. An identical set of four major phosphopeptides was obtained from the IL-2 receptor isolated from TPA-stimulated HUT102B2 cells and from the receptor phosphorylated in vitro by protein kinase C (Fig. 2, A and B). This indicates that the sites on the IL-2 receptor modified by protein kinase C-catalyzed phosphorylation in vitro are the same as those phosphorylated in vivo. In addition, the peptide map of the in vitro phosphorylated IL-2 receptor from TPA-induced CEM

cells was indistinguishable from that of the receptor from HUT102B2 cells (Fig. 2C). Thus, failure to detect phosphorylation of the IL-2 receptor on TPA-induced CEM cells in vivo cannot be accounted for on the basis of detectable struc- tural differences between the receptors of the two cell lines.

Identification of the Sites of Phosphorylation on the IL-2 Receptor-The complete amino acid sequence of the IL-2 receptor has been deduced from the nucleotide sequence of cDNA clones (22-24). The carboxyl-terminal portion of the molecule contains a stretch of 19 hydrophobic amino acids which probably comprise the transmembrane domain followed by a cytoplasmic domain of 13 amino acids (Fig. 3). The likely sites of phosphorylation in the IL-2 receptor are serine-247 and either threonine-239 or -250. To conclusively demonstrate that the intracellular carboxyl terminus contains the sites of phosphorylation by protein kinase C, the peptide Tyr-Gln- Arg-Arg-Gln-Arg-Lys-Ser-Arg-Arg-Thr-Ile was synthesized. A tyrosine residue was added at the amino terminus to allow iodination of the peptide. The synthetic peptide was phosphorylated by kinase C in vitro, and phosphoamino acid analysis demonstrated that serine was the major phosphorylated residue (Fig. 4). A small amount of phosphothreonine was also observed, but no phosphotyrosine was detected despite the added residue.

The tryptic peptide map of the phosphorylated synthetic peptide was identical to that of the native IL-2 receptor phosphorylated in vitro or in vivo (Fig. 20). Thus, the carboxyl-terminal 11 amino acids of the receptor, which includes Ser-247 and Thr-250, contain all the sites of protein kinase C-catalyzed phosphorylation. Four major spots, each repre- senting one or more tryptic phosphopeptides, were detected in each of the peptide maps shown in Fig. 2. The sequence predicts only two limit phosphopeptides, PSer-Arg and PThr- Ile. Therefore, some of the peptides identified probably rep- resent partial tryptic products. To further characterize the tryptic peptides derived from the synthetic peptide, each of the four major species were subjected to partial acid hydrolysis and then analyzed by thin-layer electrophoresis. Tryptic peptide 1 contained only phosphothreonine whereas tryptic peptides 2, 3, and 4 contained only phosphoserine. On the basis of its negative charge and hydrophobicity relative to the other phosphopeptides, tryptic peptide 1 is probably PThr-Ile. Tryptic peptide 2, the most acidic peptide containing phosphoserine, is likely to be the limit product PSer-Arg. This latter assignment is supported by the observation that more extensive digestion of the 12-mer synthetic peptide with trypsin (50 fig for 3 days) increased the yield of peptide 2 relative to peptide 3 and the minor component peptide 4 was unde- tectable. Furthermore, upon elution and redigestion with trypsin, peptide 3 was partially converted to peptide 2 (data not shown). Phosphopeptide(s) 3 probably contains more than one component, although the broad spot has not been resolved clearly into multiple peptides. Peptide(s) 4 can be resolved into 3 minor spots. The structures of the tryptic peptides migrating as spots 3 and 4 cannot be definitively assigned. Seven partial tryptic peptides containing phosphoserine and 2,3,4, or 5 positively charged residues are predicted from the sequence. These results are consistent with those of Hunter et al. (25) who found that trypsin digestion of the EGF receptor phosphorylated by protein kinase C generated a series of three phosphopeptides which concomitantly decreased in hydrophobicity and increased in positive charge as observed with the IL-2 receptor peptides. The most acidic and hydrophobic of the three peptides was shown to be the limit product (25). The other partial peptides contained one or two additional basic residues.

Phosphorylation of IL-2 Receptor, HLA Antigens, and T200 8337

FIG. 2. Tryptic phosphopeptide maps of IL-2 receptor and synthetic peptide. The samples are: A, IL-2 receptor from HUT102B2 cells labeled in uiuo with 32P0, and pulsed for 15 min with 50 ng/ml TPA; B, IL-2 receptor from HUT102B2 cells phosphorylated by protein kinase C in uitro; C, IL-2 receptor from TPA-induced CEM cells phosphorylated by protein kinase C in vitro; and D, synthetic peptide phosphorylated in uitro by protein kinase C. The IL-2 receptor, isolated by immunoprecipitation, and the synthetic peptide were phosphorylated and isolated as described under "Experimental Procedures." For samples A-C, the phosphoprotein of M, = 50- 55,000 in the anti-Tac immunoprecipitate was isolated by SDS-PAGE in the absence of reducing agents. The samples were digested with 20 pg of trypsin for 18 h at 37 "C. The phosphopeptides were separated by electrophoresis a t pH 4.7 toward the cathode (right side) followed by ascending chromatography. The origins are indicated by arrows. The major phosphopeptides are designated l , 2, 3, and 4 in p a n e l D. Maps A-C were exposed to film for 1-3 weeks with an intensifying screen whereas map D was exposed for 1 day.

A. HUT 102 B2 W o

4 c. CCRF-CEM invitro

a

3. HUT 102 82 in vitro

+ 1. Synthetic peptide

n

4

Electrophoresisl",

# WR (645662)

R R R H I V R K R T L R R L L Q E R

FIG. 3. Amino acid sequences of intracellular regions of cell membrane proteins containing sites of protein kinase C-catalyzed phosphorylation. The transmembrane regions are immediately amino-terminal to the sequences listed. The numbers in paren- theses indicate the location of the residues in the proteins. The sequences of the IL-2 receptor and HLA-.-\2 heavy chain extend to the carboxyl terminus. The sequences are from the following sources which site the original references: IL-2 R (22-24), HLA-A2 (35), and EGF R (25). The underlined residues in the HLA-A2 sequence denote positions a t which substitutions are observed in other HLA specificities. The residues phosphorylated by protein kinase C are indicated by arrows. The broken arrows indicate the proposed sites of phosphorylation. The tfyptic cleavage site(s) in the HLA heavy chain which generate p39b are designated by the filled arrowheads.

7'200 Glycoprotein and HLA-A and -B Antigens Are Also Substrates for Protein Kinuse C-Tumor-promoting phorbol esters are known to have both immediate and long-term pleiotropic effects on the cell membranes of leukemic cell lines and normal lymphocytes. In addition to induction of the IL-2 receptor, prolonged exposure to TPA alters the expression of other cell surface antigens on a variety of T leukemic cell lines. Generally, it has been found that there is an increased expression of T3, T8, and T11 glycoproteins, consistent with the differentiation of the cells to a more mature T cell phenotype (26, 27). Concomitantly, surface expression of the transferrin receptor decreases (5, 26). Similarly, TPA induces the differentiation of the promyelocytic cell lines,

FIG. 4. Phosphoamino acid analysis of synthetic peptide phosphorylated in vitro by protein kinase C. The phosphorylated peptide was hydrolyzed in 5.7 N HCI for 2 h a t 110 "C. The acid hydrolysate was resolved in two dimensions by electrophoresis toward the anode (left side) a t pH 1.9 followed by electrophoresis toward the anode (top) a t pH 3.5. The origin is designated by the arrow. Phos- phoserine (S), phosphothreonine (T), and phosphotyrosine (Y) standards were co-analyzed with the labeled sample and detected by staining with ninhydrin.

HL-60, to more mature myeloid cells with accompanying changes in cell surface antigen expression (28). A rapid change induced in HL-60 or lymphoid cells by TPA is an increase in the phosphorylation of the transferrin receptor (29, 30) and the class I major histocompatibility antigens (HLA) (31).

To investigate the role of protein kinase C in the TPA- stimulated phosphorylation and to identify other substrates among the cell surface glycoproteins of HUT102B2 and CCRF-CEM cells, immunoprecipitates of various cell membrane proteins were prepared and incubated in vitro with


protein kinase C. Using this approach, we have shown that the transferrin receptor from both cell lines is phosphorylated in vitro by protein kinase C (32). As shown in Fig. 5A, the T200 glycoprotein, a major leukocyte-specific cell surface molecule (7), was also found to be a substrate for protein kinase C in vitro. Limited acid hydrolysis of the in vitro "P- labeled T200 glycoprotein and analysis by thin-layer electrophoresis revealed serine to be the major phosphorylated residue (Fig. 5B). Although the T200 glycoprotein in mouse lymphoma cell lines is heavily phosphorylated (33), it was only weakly labeled in either of the human T cell lines, and the phosphorylation was not detectably increased by treat- ment of cells with TPA (data not shown).

The HLA-A, -B, and -C antigens were immunoprecipitated with the monoclonal antibody W6/32 which recognizes all HLA specificities and incubated in vitro with protein kinase C. The HLA antigens are composed of a 44,000-dalton glycoprotein heavy chain and a 12,000-dalton noncovalently associated subunit, &microglobulin (6). As shown in Fig. 6A, the HLA heavy chain was phosphorylated by kinase C. Phos- phoamino acid analysis of the gel-purified heavy chain revealed predominantly phosphoserine (data not shown). In some experiments, an additional 32P-labeled species was obtained which was identified as the heavy chain of the W6/32 antibody by demonstrating that protein kinase C phosphorylated the W6/32 antibody bound to Protein A-Sepharose in

200 -

116-

93-

66 -

45 -

30 -

FIG. 5. I n vitro phtmphorylation of T200 glycoprotein by protein kinase C. A, T200 was immunoprecipitated from HUT102B2 cells using T29/33 antibody covalently coupled to Seph- arose 4B. The immunoprecipitate was phosphorylated by protein kinase C ae described in the text ( l u n e 7'200). The T29/33-Sepharose beads which had not been incubated with cell lysate were also incubated with protein kinase C in vitro as a control ( l o n e 0. B, phosphoamino acid analysis of T200. The T200 phosphorylated by protein kinase C was g e l purified and subjected to partial acid hydrolysis for 3 h as described. The phosphoamino acids were separated by two- dimensional electrophoresis as described for Fig. 4 and in the text.

the absence of cell lysate. In vivo labeling with 32P04 showed that the HLA heavy chain was constitutively phosphorylated in both HUT102B2 and CCRF-CEM cells and that TPA increased the level of phosphorylation less than 2-fold (Fig. 6B).

To localize the site(s) phosphorylated by protein kinase C and to determine if this differed from the constitutive site of phosphorylation, the in vitro and in vivo labeled HLA antigens were subjected to limited proteolysis. The carboxyl-terminal cytoplasmic and transmembrane domains of the HLA heavy chain can be cleaved stepwise by limited proteolysis of the HLA-&microglobulin heterodimer with papain or trypsin (6, 34). The first cleavage during papainolysis converts the HLA heavy chain from M, = 44,000 (p44) to 39,000 (p39a) as part of the 30-amino acid cytoplasmic region is removed (Fig. 6, A and B) . The second cleavage, which occurs after Leu-270 or Thr-271, removes the hydrophobic membrane-spanning region converting the heavy chain to M, = 34,000 (p34). Trypsin also converts the HLA heavy chain to a species of approximately M, = 39,000 (p39b) by a series of cleavages in the cytoplasmic domain (Fig. 6A). The major site of in vivo phosphorylation of the HLA-B7 heavy chain was localized to Ser-335 which is conserved in all specificities (35) (see Fig. 3). Conversion of p44 to p39a by papain removes the phosphate label from the in vivo phosphorylated HLA heavy chain as reported previously (34) (Fig. 6B, -TPA). However, the first papain cleavage of the HLA antigens phosphorylated in vivo in the presence of TPA did not remove all the phosphate label. Fig. 6B ( + P A , 50-250 ng of papain) shows that 32P- labeled p39a was observed. Further cleavage to p34 removed all the label. Thus, TPA stimulated the phosphorylation of a new site in the cytoplasmic domain. However, 32P-labeled p39b was not observed after limited trypsinolysis of the HLA antigens phosphorylated in vivo in the presence or absence of TPA (data not shown). In vitro, protein kinase C also phosphorylates sites in the intracellular domain of the HLA heavy chain as demonstrated by limited proteolysis. A fraction of the phosphate label was retained after the first papain cleavage of the in vitro-labeled p44 heavy chain to p39a and subsequently lost after the second cleavage by papain which generates p34 (Fig. 6A). In contrast to the results with the in vivo 32P-labeled HLA antigen, limited tryptic cleavage of the in vitro-labeled heavy chain generated a 32P-labeled p39b species. Thus, the HLA heavy chain is phosphorylated in vivo after TPA stimulation or in vitro by protein kinase C at new site(s) in the intracellular segment of the molecule which are amino-terminal to the site phosphorylated constitutively in vivo. An additional site appears to be phosphorylated in vitro by protein kinase C which may not be phosphorylated in vivo.

DISCUSSION

Protein kinase C is a ubiquitous enzyme which is activated in response to many different extracellular signals (for review see Ref. 1). A wide variety of substrates for protein kinase C have been identified recently, including the nuclear proteins, histones (36), and high mobility group proteins (37), enzymes such as guanylate cyclase (38) and tyrosine hydroxylase (391, ribosomal S6 protein (40), and various cell membrane proteins important for cell growth such as the EGF receptor (11, 25, 411, transferrin receptor (30,321, and the glucose transporter (21). In this paper it was demonstrated that the receptor for IL-2, the T200 glycoprotein, and the class I major histocompatibility antigens are also substrates.

A consensus on the constraints of primary and secondary structure for protein kinase C-catalyzed phosphorylation has not been established. Protein kinase C and the cyclic AMP-

A. PAPAIN (ng):

TRYPSIN (ng):

044 - 039 - 034 <

Phosphorylation of IL-2 Receptor, HLA Antigens, and T200 8339

B. PAPAIN Inn). n 10 50 250 0 10 50 250 0 10 50 250

0 10 50 250 - - - - 0 10 5 0 250 - - - - 0 - - - 10 5 0 2 5 0 1000 0 - - - 10 50 250 1000

p44- p39-

P34-E

I

I 1 1 I 125 I 32 P

FIG. 6. Limited proteolysis of class I HLA antigens phosphorylated in vitro and in vivo. A. in uitro 9-labeled HLA antigens. The HLA antigens were immunoprecipitated with W6/32 and Protein A-Sepharose and phosphorylated in vitro with protein kinase C. For comparison, the HLA antigens were immunoprecipitated from '251-labeled HUT102B2 cells. The 32P- or '261-labeled immunoprecipitates were aliquoted and incubated at 37 "C for 1 h with no additions, 10.50, or 250 ng of papain, or 10, 50, 250, or lo00 ng of TPCK/trypsin as indicated in the figure. No phosphorylation was detected in the absence of protein kinase C. p34 is doublet due to separation of the HLA-A and -B specificities (34). B, in uiuo 32P-labeled HLA antigens. The HLA antigens were immunoprecipitated with W6/32 and Pansorbin from HUT102B2 cells labeled in vivo for 4 h with "PO, followed by no addition (-TPA) or a 15-min pulse with 50 ng/ml TPA (+TPA). The HLA antigens were also immunoprecipitated from HUT102B2 cells labeled by lactoperoxidase-catalyzed cell surface iodination (lZ51). The immunoprecipitates bound to Pansorbin were aliquoted and incubated with no enzyme or 10, 50, or 250 ng of papain as indicated in the figure. The samples were analyzed by SDS-PAGE in the presence of 2-mercaptoethanol. &.-microglobulin ( D m ) co-migrated with the dye front.

dependent protein kinase (kinase A) phosphorylate many of the same substrates although at different relative rates (36- 40,42). Both enzymes require multiple basic residues close to the site of phosphorylation. However, the basic residues can be amino- or carboxyl-terminal to the site recognized by protein kinase C , whereas kinase A requires basic residues amino-terminal to the site of phosphorylation (42, 43). The EGF receptor is the only integral membrane protein in which the site of phosphorylation by protein kinase C had previously been firmly established. The phosphorylated residue is threonine 654 which is in the intracellular domain of the receptor 9 residues from the cytoplasmic face of the membrane and situated amidst multiple basic residues (25, 41) (see Fig. 3). As shown in this report, serine 247 and threonine 250 in the IL-2 receptor are phosphorylated by protein kinase C . These residues are 8 and 11 amino acids, respectively, from the membrane, and each site is immediately adjacent to 2 arginine or lysine residues.

The heavy chain of the class I major histocompatibility antigens is phosphorylated constitutively in vivo at Ser-335 in the intracellular domain. It was shown by limited proteolysis of the HLA antigens that stimulation with TPA in vivo or incubation with protein kinase C in vitro causes phosphorylation of additional serine residues in the intracellular domain. The proteolytic cleavage data are consistent with

residues, being new sites of protein kinase C-catalyzed phosphorylation. However, Ser-312 or -313 may only be phosphorylated by protein kinase C in vitro as the phosphate label was lost after tryptic cleavage of in vivo-labeled HLA antigens. These sites may be too close to the membrane to be accessible to the enzyme in vivo. Thus, Ser-319 which is probably amino- terminal to the first papain cleavage site is a likely new site phosphorylated in vivo after TPA stimulation. Serines 312, 313, and 319 are also possible phosphorylation sites for CAMP-dependent protein kinase A in vitro (44). The HLA heavy chains are highly polymorphic proteins. However, most of the variability is clustered in regions of the extracellular domain, whereas the intracellular domain is highly conserved at both the amino acid and nucleotide levels (35,45). Only 9 out of 33 residues show limited variability in the intracellular domain of the HLA heavy chain (see Fig. 3). The sequence Arg-Arg-Lys-Ser-Ser (residues 309-313), which abuts the transmembrane region, and Lys-Gly-Gly-Ser (residues 316- 319) contain the most likely sites of phosphorylation by protein kinase C and are totally conserved in the 6 HLA-A, -B, and -C heavy chains sequenced thus far (35,451. The in uitro site of phosphorylation of the integral membrane glycoprotein T200 by protein kinase C was not determined. The complete amino acid sequence of T200 from rat thymocytes has been determined from the nucleotide sequence of cDNA

serines 312, 313, and 319, which are all situated near basic clones, and numerous potential sites for phosphorylation exist


in the 705-amino acid intracellular domain (46). Little is known about how phosphorylation might affect the

function of cell membrane proteins. Integral membrane proteins are characterized by a cluster of basic residues near the intracellular face of the membrane. These residues may interact with the negatively charged phospholipid head groups, thus anchoring the molecule in the membrane. Phosphoryla- tion of a serine or threonine residue near the membrane could affect the interaction of the phosphorylated protein with membrane or cytoskeletal components. Phosphorylation of EGF receptor at Thr-654 decreases the EGF-dependent tyrosine phosphorylation of the receptor (11, 47). In vivo, this is induced by either phorbol esters (11,47) or platelet-derived growth factor (48), each of which acts synergistically with EGF to induce cell proliferation and a concomitant loss of high affinity EGF-binding sites. In lymphocytes, phorbol esters induce phosphorylation of the insulin receptor (49) and decrease ligand binding (50, 51). In hepatoma cells, however, phosphorylation of the receptor is not accompanied by a decrease in insulin binding (52). The transferrin receptor is also a substrate for protein kinase C in vitro and in vivo, and stimulation by TPA decreases transferrin binding due to internalization of the receptor (29, 30, 32,53). However, it is not clear if phosphorylation of the receptor is a signal for internalization.

Physiological stimuli which can induce phosphorylation of the IL-2 receptor by protein kinase C or other kinases have not been identified. Phosphorylation of the IL-2 receptor in response to IL-2 binding has not been observed (20) as it has been upon ligand binding to other growth factor receptors. However, it was recently reported that IL-2 (4), like TPA (3), induces an increase in the membrane-associated protein kinase C activity and a concomitant decrease in phosphotransferase activity in the cytosoI fraction. The increase in membrane activity observed with TPA is approximately 2-fold greater than with IL-2, and the protein kinase C activity remains preferentially membrane associated for a prolonged period compared to the transient redistribution observed with IL-2. Thus, it is possible that the failure to detect IL-2- stimulated phosphorylation of the IL-2 receptor may be due to the transient nature of the protein kinase C stimulation compared to activation by TPA.

The binding of IL-2 to its receptor distinguishes two classes of receptors with different affinities as noted with the receptors for EGF and insulin. The high affinity IL-2-binding sites comprise only 10% of the total number of sites (54). Incuba- tion of cells bearing IL-2 receptors with IL-2 has been shown to induce a decrease in the number of the high affinity-binding sites and an increase in the total number of receptors meas- ured by anti-Tac binding (55). The effects of TPA on IL-2 binding have not been investigated. However, preliminary data indicates that TPA caused a 50% increase in anti-Tac binding to HUT102B2 cells after 1 h.2 Further work is required to evaluate whether phosphorylation of the IL-2 receptor by protein kinase C regulates either internalization of receptors or IL-2 binding under physiological conditions.

Acknowledgments-We thank Dr. Gordon Gill and Steve Heiser-

thank Kathy Gould, Jim Woodgett, and Jean Rivier for their help, man for providing the purified protein kinase C. We also wish to

Clare Isacke and Matt Thomas for their comments, and Ami Koide for typing the manuscript.

REFERENCES

1. Nishizuka, Y. (1984) Nature 308,693-698

D. A. Shackelford and I. S. Trowbridge, unpublished observation.

2. Michell, R. H. (1979) Trends Bwchem. Sci. 4 , 128-131 3. Kraft, A. S., and Anderson, W. B. (1983) Nature 301.621-623 4. Farrar, W. L., and Anderson, W. B.-(1985) Nature 315,233-235 5. Shackelford, D. A., and Trowbridge, I. S. (1984) J. Biol. Chem.

259,11706-11712 6. Ploegh, H. L., Orr, H. T., and Strominger, J. L. (1981) Cell 2 4 ,

7. Omary, M. B., Trowbridge, I. S., and Battifora, H. (1980) J, Exp. Med. 152,842-852

8. Gazdar, A., Carney, D. N., Bunn, P. A., Russell, E. K., Jaffe, E. S., Schechter, G. P., and Guccion, J. G. (1980) Blood 55, 409- 417

9. Foley, G. E., Lazarus, H., Farber, S., Uzman, B. G., Boone, B. A., and McCarthy, R. E. (1965) Cancer 18,522-529

10. Trowbridge, I. S., Ralph, P., and Bevan, M. J (1975) Proc. Natl. Acud. Sei. U. S. A. 72, 157-161

11. Cochet, C., Gill, G. N., Mersenhelder, J., Cooper, J. A., and Hunter, T. (1984) J. Biol. Chem. 259,2553-2558

12. Kikkawa, U., Takai, Y., Minakuchi, R., Inohara, S., and Nishi- zuka, Y. (1982) J. Bwl. Chem. 2 5 7 , 13341-13348

13. Uchiyama, T., Broder, S., and Waldmann, T. A. (1981) J. Im- munol. 126,1393-1397

14. Leonard, W. J., Depper, J. M., Uchiyama, T., Smith, K. A., Waldmann, T. A., and Greene, W. C. (1982) Nature 300,267- 269

15. Barnstable, C. J., Bodmer, W. F., Brown, G., Galfre;G., Milstein, C., Williams, A. F., and Ziegler, A. (1978) Cell 14, 9-20

16. Laemmli, U. K. (1970) Nature 227 , 680-685 17. Gibson, W. (1974) Virology 62,319-336 18. Hunter, T., and Sefton, B. M. (1980) Proc. Natl. Acad. Sci. U. S.

19. Greene, W. C., Robb, R. J., Depper, J. M., Leonard, W. J., Drogula, C., Svetlik, P. B., Wong-Staal, F., Gallo, R. C., and Waldmann, T. A. (1984) J. Immunol. 133,1042-1047

20. Leonard, W. J., Depper, J. M., Kronke, M., Robb, R. J., Wald- mann, T. A., and Green, W. C. (1985) J. Biol. Chem. 260,

21. Witters, L. A., Vater, C. A., and Lienhard, G. E. (1985) Nature

22. Leonard, W. J., Depper, J. M., Crabtree, G. R., Rudikoff, S., Pumphrey, J., Robb, R. J., Kronke, M., Svetlik, P. B., Peffer, N. J., Waldmann, T. A., and Greene, W. C. (1984) Nature 3 1 1 ,

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

24. Cosman, D., Cerretti, D. P., Larsen, A., Park, L., March, C., Dower, S., Gillis, S., and Urdal, D. (1984) Nature 312 , 768- 771

25. Hunter, T., Ling, N., and Cooper, J. A. (1984) Nature 31 1,480- 483

26. Delia, D., Greaves, M. F., Newman, R. A., Sutherland, D. R., Minowada, J., Kung, P., and Goldstein, G. (1982) Int. J. Cancer

27. Ryffel, B., Henning, C. B., and Huberman, E. (1982) Proc. Natl.

28. Omary, M. B., Trowbridge, I. S., and Minowada, J. (1980) Nature

29. May, W. S., Jacobs, S., and Cuatrecasas, P. (1984) €'roc. Natl.

30. May, W. S., Sahyoun, M., Jacobs, S., Wolf, M., and Cuatrecasas,

31. Feuerstein, N., Monos, D. S., and Cooper, H. L. (19%) Biochem.

32. Trowbridge, I. S., and Shackelford, D. A. (1986) Bwchem. sot.

33. Omary, M. B., and Trowbridge, I. S. (1980) J. Bid. Chem. 2 5 5 ,

34. Pober, J. S., Guild, B. C., and Strominger, J. L. (1978) Proc. Natl.

35. Guild, B. C., and Strominger, J. L. (1984) J. Biol. Chem. 259,

36. Nishizuka, Y., Takai, Y., Kishimoto, A., Hashimoto, E., Inoue, M., Yamamoto, M., Criss, W. E., and Kuroda, Y. (1978) A&. Cyclic Nucleotide Res. 9,209-220

37. Ramachandran, C., Yau, P., Bradbury, E. M., Shyamala, G., Yasuda, H., and Walsh, D. A. (1984) J. Bioi. Chem. 259,13495- 13503

287-299

A. 77,1311-1315

1872-1880

315,777-778

626-631

29,23-31

Acud. Sci. U. S. A. 79,7336-7340

286,888-891

Acad. Sci. U. S. A. 81,2016-2020

P. (1985) J. Biol. Chem. 260,9419-9426

Bwphys. Res. Commun. 126,206-213

Symp. 51,117-129

1662-1669

Acad. Sei. U. S. A. 75,6002-6006

9235-9240

Phosphorylation of IL-2 Receptor, HLA Antigens, and TZOO 8341 38. Zwiller, J., Revel, M-O., and Malviya, A. N. (1985) J. Biol. Chem.

39. Albert, K. A., Helmer-Matyjek, E., Nairn, A. C., Muller, T. H., Haycock, J. W., Greene, L. A., Goldstein, M., and Greengard, P. (1984) Proc. Nutl. Acad. Sei. U. S. A. 81,7713-7717

40. Parker, P. J., Katan, M., Waterfield, M. D., and Leader, D. P. (1985) Eur. J. Bwchem. 148,579-586

41. Davis, R. J., and Czech, M. P. (1985) Proc. Nutl. Acad. Sei. U. S. A. 82,1974-1978

42. Kemp, B. E., Graves, D. J., Benjamini, E., and Krebs, E. G. (1977) J. Bwl. Chem. 252,4888-4894

43. Ferrari S.. Marchiori. F.. Borin. G.. and Pinna. L. A. 11985)

260,1350-1353

FEBS Gtt. 184,72171' I . . I

44. Guild. B. C.. and Stromineer. J. L. (1984) J. Biol. Chem. 259. I , . ,

13504-13510

Biol. Chem. 260,13414-13423

(1985) Cell 41,83-93

45. Davidson, W. F., Kress, M., Khoury, G., and Jay, G. (1985) J.

46. Thomas, M. L., Barclay, A. N., Gagnon, J., and Williams, A. F.

47. McCaffrey, P. G., Freidman, B., and Rossner, M. R. (1984) J.

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

49. Jacobs, S., Sahyoun, N. E., Saltiel, A. R., and Cuatrecasas, P.

Biol. C k m . 259,12502-12507

A. 82,4080-4084

(1983) Proc. Nutl. Acud. Sei. U. S. A. 80,6211-6213 50. Grunberger, G., and Gorden, P. (1982) Am. J. Physiol. 243,

E319-E324 51. Thomopoulos, P., Testa, U., Gourdin, M. F., Hervy, C., Titeux,

M., and Vainchexiker, W. (1982) Eur. J. Biochem. 29,389-393 52. Takayama, S., White, M. F., Lauris, V., and Kahn, C. R. (1984)

Proc. Natl. Acad. Sei. U. S. A. 81,7791-7801 53. Klausner, R. D., Harford, J., and Van Renswoude, J. (1984) Proc.

Natl. Acad. Sci. U. S. A. 81,3005-3009 54. Robb, R. J., Greene, W. C., and Rusk, C. M. (1984) J. Exp. Med.

55. Smith, K. A., and Cantrell, D. A. (1985) Proc. Natl. Acad. Sci. U. 160,1126-1146

S. A. 82,864-868

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