Activators of Protein Kinase C Up-regulate the Cell Surface ...

THEJOURNAL (c? 1989 by

OF BIOLOGICAL CHEMISTRY The American Society for Biochemistry and Molecular Biology, Inc.

Vol. 264, No. 26, Issue of September 15, pp. 15650-15655, 1989 Printed In U . S . A .

Activators of Protein Kinase C Up-regulate the Cell Surface Expression of CD2 and CD5 T Cell Glycoproteins* (Received for publication, March 3, 1989)

Ana C. CarreraS, Laura Cardenas+, Antonio TugoresS, Miguel Alonsop, Francisco Sanchez-Madrid$, and Manuel0. de LandazuriST From the SSeruicio de Inmunologia, Hospital de la Princesa, Uniuersidad Autonoma, Madrid 28006, Spain and the SCentro de Biologia Molecular, Uniuersidad Autonoma, Cantoblanco, Madrid 28049, Spain

Activation of human T cells through the CD3-T cell receptor complex caused an augmentation in the cell surface expression of CD2 and CD5 glycoproteins. Evidence that protein kinase C is involved in the upregulatory mechanism of these cell surface molecules has been obtained by three different approaches: (a) the changes in antigen expression wereobserved with activators of protein kinase C such as phorbol esters but not with activators of kinases dependent on calcium/calmodulin or CAMP; ( b ) the overexpression of CD2 and CD5is also observed in cells treated with 1,2dioctanoyl-ruc-glycerol,an analogue of the physiological activatorof protein kinase C; and ( c ) 1-(5isoquinoliny1)-2-methylpiperazine, an inhibitor of proecteinkinase C but not N-(2-guanidinoethyl)-5-isoquinolinesulfonamide dihydrochloride, an inhibitor of the CAMP-dependentkinase, impairs CD2 and CD5 upregulation.Thesechangesincellsurfaceantigen expression appear to be caused by the concomitant increase in the mRNA levels for CD2 and CD5. Phosphorylation studies of theCD2 and CD5 glycoproteins indicated that the overexpression of these molecules was not associated with a specific pattern of phosphorylationsinceitwasobservedindependently of their hyperphosphorylated or nonphosphorylated state.

H+ exchange protein that controls intracellular pH(7) or the expression of cellular receptors with either up-regulation or down-regulation of cell surfaceglycoproteins. Exposure of human neutrophils to phorbol myristate acetate induced an increased expression of CD45 and CDllb cell surface glycoproteins (8, 9). On the other hand, it has also been shown that phorbol esters could down-regulate the expression of receptors for growth factors such as transferrin, epidermal growth factor, and insulin (10-13). Similarly, down-modulation of T cell differentiation antigens CD3 and CD4 (14-16) occurs when protein kinase C is activated. In thecase of CD3 and CD4, this down-modulation isassociated with the simultaneous phosphorylation of these cell surface proteins, and it is thought that bothprocesses are interrelated (14). Theactivation of proteinkinase Cwhichoccurs after stimulation of the CD3 complex (3) might also modulate the expression of other T cell differentiation antigens. In this report, we describe the overexpression of CD2 and CD5T cell surface molecules after activation of T cells with mAb’ antiCD3. We demonstrate that protein kinase C is implicated in this up-regulatory mechanism and that these increments in cell surfaceantigen expression appearto be caused by a concomitant increase in the mRNAlevels for CD2 and CD5. The overexpression of these molecules was notassociated with a specific pattern of phosphorylation of CD2 or CD5 glycoproteins since it was observed independently of their hyperphosphorylated or nonphosphorylated state.

The T cell antigen receptor-CD3 complex plays a crucial MATERIALS ANDMETHODS role in the signal transduction system leading toT cell actiCell Cultures-Peripheral blood mononuclear cells (PBMC) were vation. In this pathway, the hydrolysis of phosphoinositides separated from heparinized venous blood by sedimentation of Ficollmediated by phospholipase C generates as second messengers Hypaque (Pharmacia LKBBiotechnology Inc.) and cultured in RPMI inositol trisphosphate, which mobilizes intracellular Ca2+, and1640 (Flow, Irvine, Scotland)supplemented with 1%antibiotics diacylglycerol, which activates protein kinase C (1-3). The (Flow) 1% L-glutamine(Flow), and 5% fetal calf serum (GIBCO). activation of proteinkinase C either by the physiological This is referred to as complete medium. T cells were purified from second messenger diacylglycerol, or by phorbol estersleads to PBMC by removal of adherent cells on plastic Petri dishes (1 h a t “C in 5% CO,) followed by passage through a nylon wool column. the phosphorylation of specific protein substrates presumed 37Activation of PBMC was carried out by culturing lo6 cells/ml for to be necessary for eliciting many of the cellular functions in 24 h in a humidified atmosphere containing 5% CO, with ( a ) a 0.2% which this enzyme is involved suchas mitogenesis, gene (v/v) final concentration of phytohemagglutinin M (Difco); ( b ) antiexpression, and cell surface membrane functions(4-6). CD3 mAb directly bound to the plastic surface; (c) Ca2+ionophore Several lines of evidence indicated that protein kinase C (A23187, Sigma) (to abrogate thetoxic effect of this compound, after modulates cell surface membrane functions such as the Na+/ 3 h of culture thecells were washed and left in culture for another 21 * This work was supported by grants from INSALUD (Fondo de Investigaciones Sanitarias Seguridad Social 88/1738) and Comision Asesora de Investigation Cientifica y Tecnica (0456-84).The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “aduertisernent” in accordance with18 U.S.C. Section 1734 solely to indicate this fact. II To whom correspondence should be sent: Servicio de Inmunologia, Hospital de la Princesa, c/Diego deLeon, 62, Madrid 28006, Spain.

h) ( d ) PDB (Sigma) a t different concentrations; or (e) prostaglandin ES(Sigma). Reagents-The following mAb were employed B9.4.2 anti-CD8 (19),and (17),W6/32 anti-HLA-A,B (18),SVP-T3banti-CD3

The abbreviationsused are:mAb, monoclonal antibody(ies); MFI, mean fluorescence intensity; H-7, 1-(5-isoquinolinyl)-2-methylpiperazine; PDB, phorbol12,13-dibutyrate; DIC8,1,2-dioctanoyl-rac-glycerol;HA-1004, N-(2-guanidinoethyl)-5-isoquinolinesulfonamide dihydrochloride; PBMC, peripheral blood mononuclear cells; PMA, phorbol 12-myristate 13-acetate; SDS,sodium dodecyl sulfate.

15650

Up-regulation of CD2 and CD5by Activators of Protein Kinase C MAR21 anti-CD7, kind1.y provided by Drs. Malissen (INSERM, Marseilles), Strominger (Dana Faber Cancer Institute Boston), De Vries (DNAX,Palo Alto, CA),andLopez-Botet(Hospitalde la Princesa, Madrid), respectively. Other mAb used were: D3/9 antiCD45 (20),TS1/18 anti-CD18 (21), TS2/18antiLCD2 type T l l , (211, FG2-12 anti-transferrin receptor (221, NA1/34 antiLCD1 (231, and TP1/21.1 anti-CD5. The TP1/21.1 was obtained in our laboratory (24) in a fusion with spleen cells of mice immunized with human T cells activated witha mixture of phorbol 12-myristate13-acetate (PMA) and anti-CD3mAb. The TP1/21.1 mAb immunoprecipitated a molecule of 67 kDa, and. both their biochemical characteristics and their cellular and tissue distribution fulfilled all the criteria of an anti-CD5 mAb (25). PMA, l-(5-isoquinolinyl)-2-methylpiperazine (H-7), phorbol 1213-dibutyrate (PDB), 1,2-dioctanoyl-rac-glycerol (DIC8), and cycloheximide were purchased from Sigma and HA-1004 N-(2-guanidinoethyl)-5-isoquinolinesulfonamidedihydrochloride from Calbiochem. All other chemicals were of the best qualityavailable from Merck. Northern Blot Analysis-Total cytoplasmic RNA was isolated by a slight modification of a method described previously and quantified by absorbance a t 260 nm(26).Samples (20 pg) were denatured, electrophoresed through 1% agarose,formaldehyde gel, and transferred to Biodyne nylon membranes (Pall Corp., Glen Cove, NY) as described (27). Filterswere hybridized overnight underhigh stringent conditions (50% formamide, 5 X Denhardt's solution, 0.1% SDS, 40 mM phosphate buffer, p l l 7, 5 X SSC,and 250 pg/ml denatured salmon sperm DNA)a t 421 "C with probes radiolabeled by the randon primermethod(28). CD2 (29)andCD5(30)cDNA probes were kindly provided by Drs. RII. J. Crumpton (Imperial Cancer Research Fund, London) and Nancy H. Jones (Dana Faber Cancer Institute), respectively. Radiolabeling, Immunoprecipitation, and Electrophoresis-Cells were radioiodinated with tetrachlorodiphenylglycoluril (Iodogen, Pierce Chemicals Co.) as described (31). For labeling with 32P, cells were washed three times in phosphate-freeEagle's minimal essential medium and resuspended((2X 106/ml) in the same medium containing dialyzed 1%fetal calf serum and carrier-free [32P]orthophosphate (Amersham Corp.) a t 40 &i/ml (1 Ci = 37 GBq). Cells were labeled with 'lP for 16 h at 37 "C and then incubated with PDB as indicated on the figures. In other experiments, thecells were treated with PDB and cultured for 24 h. In this case, the labeling was performed during the last 16 h of the assay Labeled cells (2 X lo7) were extracted for 20 min a t 0 "C with 1 ml of lysis buffer (1% Nonidet P-40 in 10 mM Tris-HCl, pH7.4, containing 0.15 M NaC1, 1% bovine serum albumin, and 1 mM phenylmethanesulfonyl fluoride plus 1 mM EDTA and 50 mM NaF). Cell debris was removed by centrifuging (195,000 X g/ rnin). Lysates were cleared once by incubating for 15 mina t 4 "C with rabbitantibodiesagainst mouseimmunoglobulin at 5pg/ml and protein A-Sepharose (10% suspension of beads; Pharmacia) a t 150 pl/ml. For immunoprecip:kation, 0.5 ml of cleared lysate (equivalent to lo7 cells) was incubated a t 4 "C with 5-10 pg of purified antibody or 5 p1 of ascetic fluid. After 20 min, 1 pg of rabbit antibodies against mouse immunoglobulin was added, and incubation was continued for 15 min a t 4 "C. Immune c'omplexes were allowed to absorb to protein A-Sepharose a t 4 "C for 45 min and then processed as described previously (14). Samples were subjected to SDS-polyacrylamide gel electrophoresis (32). I2'I- and "P-labeled proteins were detected by autoradiography using Kodak X-AR-5 x-ray film with Du Pont intensifying screens (Cronex Lightning Plus). Flow Cytometry Analysis-For the analysis of cell surface molecules, cell cultures were harvested after differentperiods of time, and cells were stained by an indirect immunofluorescence technique as described previously (33). P3X63 myeloma culture supernatant was used as a negative control. As second reagent, we used a fluorescein isothiocyanate-labeled goat anti-mouse F(ab'),. Samples were analyzed on a flow cytometer (EPICS-C, Coulter Electronics Inc., Hyaleah, FL). The percentage of positive cellsand the mean fluorescence intensity (MFI) in arbitrary units were calculated using a computer program (EPICS, Coulterl. After the incubation of the cells with the different reagents, the increments in mean fluorescence intensity for a determined antigen were calculated employing the formulaAMFI of the antigen analyzed = MFI (treated cells) - MFI (untreated cells). TOsimplify the figures; we do not represent the profiles obtained with the negative control P3X63.After thedifferenttreatments, there were no significant changes in MFI for the negative control P3X63 ( AMFI = 0 +. 2).

15651

RESULTS

Cell Surface Up-regulation of CD2 and CD5"The expression of different cell surface molecules was analyzed by flow cytometry on PBMC incubated for 24 h in the presence or absence of differentactivating reagents. Treatmentwith either solid-bound anti-CD3, phytohemagglutinin, or with a comitogenic mixture of a Ca2+ ionophore and PDB exerted differential effects on the distinct leukocyte antigens, which were clearly evidenced by variations in their mean fluorescence intensity (TableI). Some molecules were overexpressed as CD2 and CD5, whereas others such as CD45 remained unaffected. To investigate the intracellular signalsinvolved in the upregulation of CD2 and CD5, PBMC were incubatedwith phorbol esters that activate protein kinase C or with other E2 which agents such as a Ca2+ ionophore and prostaglandin mainly activate kinases dependent on calcium-calmodulin or CAMP, respectively. As shown in Table I, up-regulation of CD2 and CD5 cell surface expression was observedwith PDB (different doses indicatedthatthemaximalexpression of these molecules was achieved with 10-20 ng/ml) but not with prostaglandin E2or with a Ca'+ ionophore. To analyze further the overexpression of CD2 and CD5, PBMC were activated with 20 ng/ml PDB for different times (0.5, 2, 4, 24, and 48 h), and theexpression of cell surface molecules CD2 and CD5 wasanalyzed(Fig. 1). Forcomparison, we monitoredthe expression of CD3, which has beendescribed to bedownregulated after treatment with PMA (14) and othercell surface antigens (CD8, CD45, CD18). Treatment with PDB for a period of 30 min induced a very significantdown-regulation of CD3, but no effect on the expressionof CD2 and CD5 was observed. Interestingly, slight changes in the expression of CD2 and CD5 were appreciated at 4 h. Greater changes in the expression of these two molecules required at least 24 h of incubation and reached the plateau at 48 hours. In contrast, the expression of other cell surface antigens (CD8, CD45, CD18) didnot changesignificantly after PDB treatment (data not shown). These results indicate that the PDB-induced upregulation of CD2 and CD5 required long periods of incubation compared with the PDB-induced down-modulation of CD3, which takes place in minutes. Since the activationof protein kinase C on the CD3-Tcell receptorpathwayis a transient process (3), we studied whether the changes observed in CD2 and CD5 cell surface expression could be observed after a transient activation of protein kinase C. To this end, we have taken advantage of the fact that PDB canbe readily eliminated by washing the cells several times (34). Cells were pulsed for 4 h with PDB, washed, and left in culture for different periods of time (24 and 48 h) in the absenceof PDB (Fig. 2). These experiments demonstrate that a pulse of 4 h was sufficient to induce the overexpression of CD2 and CD5, although these changeswere not as strong ascells in maintained in the continuous presence of phorbol esters. The changes inCD2 and CD5 antigen expression were also confirmed by semiquantitative immunoprecipitation since the polypeptide bands immunoprecipitated by mAb against CD2 and CD5 from lZ5I-labeledsamples displayed stronger intensities in PMA-treated cells than those observed in the untreated controls (data not shown). T h e Up-regulation of CD2 and CD5 Cell Surface Expression Is Due to the Activation of Protein Kinase C-To determine whether the overexpression of CD2 and CD5 after treatment with phorbol esters was due to a direct activation of protein kinase C, we investigated if such up-regulation could be abrogated in the presence of agents such as H-7 which specifi-

Up-regulation of CD2 and CD5by Activators of Protein KinaseC

15652

TABLE I CD2 and CD5 expression after activation of T cells PBMC, adjusted to lo6 cell/ml, were incubated for 24 h in complete medium in the presence of the different reagents as indicated. After this incubation period, the cells were collected, washed, and the expression of CD2, CD5, and CD45 antigens was analyzed by flow cytometry. CD5

CD2

Reagents

Medium Anti-CD3 (0.5 pg/well') +13 PHA (0.2%) IONd (0.5 p (10 ~ + )PDB 84 ng/ml) ION (0.5 p M ) PGE2 M) PDB (10 ng/ml)

3'6 Positive

cells"

AMFI'

65 80 86

+11

72 60 81

% Positive

AMFI'

cells"

63 78 76 77 70 63 80

+24 +3 -1 +19

96

% Positive

AMFI'

cells"

97 96 95

+8 +10 +21 +2

-1 +2 -1 +1 0 +2

95 97 97

-1

+19

"The percentage of positivecellsfora determined antigen was calculated by subtracting the fluorescence detected from the negative control (P3X63). * After incubation of the cells with the different reagents, the changes in MFI for a determined antigen were calculated employing the formula AMFI of the antigen analyzed = MFI (treated cells) - MFI (untreated cells). In untreated cells, the MFI of the cell surface antigens was the following: the negative control P3X63, 104; CD2, 115; CD5, 121; and CD45, 170. Plastic plates were coated previously with anti-CD3 mAb by incubation for 16 h with 0.5 pg/well (16-mm diameter) purified SpVT3b, anti-CD3mAb, in PBS, pH8. ION, Ca2+ionophore; PGEz, prostaglandin Ez.

05

CD5

2

4h.PULSE WITH

PDB

CD 2

PDB ALL THE TIME

4h PULSE WITH PDB

PDB ALL THE TIME

I

CD 2

AMFI:tl6

CD 3

t I

CD 5

li LOG

F L U O R E S C EI N C TENSITY

LOG

FLUORESCENCE

INTENSITY

FIG. 1. Cell surface expression of CD2 and CD5 after treatment with PDB for different periods of time. PBMC were

FIG. 2. Overexpression of CD2 and CD5 after transient activation of protein kinase C. 4-h pulse with PDB,PBMC were

incubated in theabsence (dashed line)or in the presenceof 20 ng/ml PDB for 0.5, 2, 4, 24, and 48 h (solid line). Then cells were washed and analyzed for the expression of CD3, CD2, and CD5. The change in MFI is indicated in each case. In untreated cells, the MFI of the cell surface antigens was the following: the negative control P3X63, 101; CD2, 116; CD3, 134; CD5, 120.

incubated either in the absence (dashed line) or in the presence of PDB (20 ng/ml) for 4 h (solid line), washed, and left in culture for a total of24 or 48 h. P D B all the time, cells were incubated in the absence (dashed line)or in the continuouspresence of PDB (20 ng/ ml) during the24 or 48 h of the assay (solid line). In untreated cells, the MFI of the cell surface antigens was the following: the negative control P3X63, 102; CD2, 116; CD5, 119.

cally inhibit the activation of protein kinase C (35). In these experiments, an inhibitor of the activation of CAMP-depend- with the diacylglycerol DIC8. As observed in Fig.4, DIC8 ent kinase such as HA-1004 (36) was included as a control of induced significant changes in the expression of CD2 and specificity. Since long term treatmentwith these agentsmight CD5. Altogether theseresultsindicate that the activation of affect the cell viability, we took advantage of our previous data indicating that a short pulse with PDB was sufficient to protein kinase C is clearly implicated in the up-regulation of trigger the up-regulation of CD2 and CD5. The cells were CD2 and CD5 T cell molecules. The Overexpression of CD2 and CD5Cell Surface Molecules incubated with H-7 or HA-1004 during the PDB pulse and Levels-The analyzed 20 h later for the expression of CD2 and CD5. As Is Associated withIncreasesinTheirmRNA shown in Fig. 3, the phorbol ester-induced up-regulation of augmentation in the cell surface expression of CD2 and CD5 CD2 and CD5 was impaired in cells cultured in the presence is completely blocked by treatment with cycloheximide (data of H-7 (Fig. 3C) but was not affected by the presence of HA- not shown), indicating that intact protein synthesis is re1004 (Fig. 3 B ) . The inhibition caused by H-7 was not due to quired to observe this up-regulation. To substantiate theseobservations further, kinetic studies nonspecific side effects (toxicity, loss of cell viability, for example) since the up-regulation could be induced when the of mRNA levels for CD2 and CD5 by Northern blot analysis H-7 agent was removed by cell washing, followed by 24 h of were carried out in PMA-treated cells. As observed in Fig. 5, a substantial increase in the mRNA levels for both CD2 and further incubation with PDB (Fig. 3 0 ) . Further evidence that the overexpression of CD2 and CD5 CD5 was appreciated after 12 h of treatment, which reached was due to the activation of protein kinase C was obtained by the peak at 24 h and remained high at 48 and 72 h. Cell Surface Overexpression and Phosphorylation of CD2 studying the expression of these glycoproteins in cells treated

Up-regulation of CD2 and CD5

by Activators of Protein Kinase C

15653

HOURS

r 0

12 4 20 4

6

72

CD 5

2 8CSDb2 L.

18Sb 4

LOG

FLUORESCENCE

INTENSITY

FIG. 3. Effect of inhibitors of different protein kinases on of CD2 and CD5. PBMC were the PDB-induced overexpression incubated for 3 h with 20 ng/ml PDB in the absence or presence of the protein kinase C inhibitorH-7 (250 p ~ or) the CAMP-dependent kinase inhibitor HA-1004 (250 p ~ ) After . this period of time, the cells were washed and left in culture for an additional 21 h and then analyzed for the expression of CD2 and CD5.A, cells were incubated hy themselves (dashed line)or in the presenceof PDB (solid line).R, cells incubatedby themselves (dashed line)or in the presenceof PDB + HA-1004 (solid line).C, cells incubatedby themselves (dashed line) or in the presence of PDB + H7 (solid line). D, cells incubated in the presence of PDB and H-7 as in C were washed a t 24 h and cultured for an additional 24 h in the presence of 20 ng/ml PDB (solid line). Control cells were incubated by themselves for 48 h (dashed line). T h e changes in MFI are indicated in each case. In untreated cells, the MFI of the cell surface antigens was the following: the negative control P3X63, 102; CD2, 117; CD5, 120. CD3C D 2 AMFI= +I6

CD5 AMFI: +17

AMFI=-16

AMFI-+12

AMFI=-17

CD5

28Sb 4

FIG. 5 . Northern blot analysis of CD2 and CD5 transcripts on purified T cells treated with PMA. Purified T cells were treated with PMA (20 ng/ml) for different periods of time. Total RNA was isolated, electrophoresed through 1% agarose, formaldehyde gel, and transferred. Filters were hybridized with either the CD2 or the CD5 cDNA probes. Ethidium bromide staining revealed equal amounts of ribosomic RNA loaded per lane (data not shown).

LOG F L U O R E S C EIN NC TENSITY FIG. 4. Induction of the overexpression of CD2 and CD5 by DICS. Cells were incubated with PDB (20 ng/ml) or alternatively with DICS (100 pg/ml) for 24 h. After this incubation period, the expression of hothCD2andCD5antigenswasanalyzed by flow cytometry. In the case of CD3 antigen, samples were analyzed after 3 h of culture. The changeinMFIisindicatedineachcase.In untreated cells, the MFI of the cell surface antigens was the following: the negative control P3X6.1, 102; CD2, 118; CD3, 130; CD5, 120.

C

E

a

1 2 3 4

1 2

1 2

65,

5 6

a 45 b

and CD5"PreviousreportshavelinkedthePMA-induced 32 b down-modulation of cell surface receptors to their phosphorylation states (14-16). Therefore, it was of interest to investigate whether the overexpression of these cell surface moleFIG. 6. Phosphorylation patterns of CD2 and CD5. Purified cules wasassociated with specific a pattern of phosphorylation T cells labeled with:"PO4 were treated for 15 min with50 ng/ml PDB and then extracted with 1% Nonidet P-40 lysis buffer. Solubilized of hoth CD2 a n d CD5 glycoproteins. proteins from either untreated or PDR-treated cells were immunoFirst, "'P-labeled cells, eitheruntreated or treatedwith PDB for 10 min, were immunoprecipitated with mAb to CD2 precipitatedwiththe following mAb: A, anti-CD3fromuntreated and CD5 or with mAb (positive controls), which recognize (lane 1 ) or PDB-treated T cells (lane 2 ) ; R , anti-CD5 from untreated (lane I ) or PDB-treated T cells (lane 2 ) ; C, anti-CDP from untreated molecules that are phosphorylated either after induction with(lane 1 ) or PDB-treated T cells (lane 2 ) . anti-CDR from untreated PDF3 (CD3 and CD8) or constitutively (major histocompati- (lane 3) or PDB-treated T cells (lane 4 ) , and anti-HLA A,B from bility complex class I). The CD5 molecule, which is constitu- untreated (lane 5) or PDB-treated T cells (lane 6 ) .

15654

Up-regulation of CD2 and CD5by Activators of Protein Kinase C A

discretepattern of phosphorylation of the CD2 and CD5 glycoproteins. 1 2 3 4 5 The up-regulation of CD2 and CD5 which occurs after T 1 2 3 4 5 cell activation could be observed in the presence of reagents that activate protein kinase C but not with activators of other or CAMP.T o study theimplication kinases dependent on Ca2+ of protein kinase C, the H-7 reagent, a chemical inhibitor of this enzyme, was employed. The H-7 inhibitor (35) also interferes withCAMP-dependent kinases, and it ispossible that the blockade of the CD2 and CD5 up-regulationcould also be due to the inhibitionof a CAMP-dependent kinase. However, we have demonstrated that the up-regulation of CD2 and CD5 appears to be due to the activationof protein kinase C since an inhibitor of CAMP-dependent kinases (36) did not affect the overexpression of CD2 and CD5. Another approach to study the role of protein kinase C was the use of a synthetic diacylglycerol that mimics the physiological stimulation of the enzyme. DIC8 is a membrane-permeable diacylglycerol andcausesthetransientactivation of proteinkinase C, FIG.7. Kinetics of CD5 phosphorylation. A, purified T cells whereas phorbol esters can hardlybe metabolized, and therelabeled with “‘PO, were either incubated in complete medium (lane fore their effects on the activation of protein kinase C are 1 ) or treated for 15 min with 50 ng/ml PDR (lanes 2-5). After this virtually irreversible (4, 37). Both PMA and DIC8, although period, the cells were carefullywashed and incubated a t 37 “C in complete medium for 0 (lane 2 ) , 30 (lane 3 ) , 60 (lane 4 ) , or 180 min chemically quite distinct, are able to activate protein kinase (lane 5 ) . After each incubation period, the cells were lysed, immuno- C, and therefore it would appear that their common potent precipitated with anti-CD5 mAb, and analyzed by SDS-polyacryl- agonistic effect on protein kinase C is the most likely explaamide gel electrophoresis (12.5%). R. purified T cells were either nation for their role in the up-regulationof CD2 and CD5. incuhated in completemedium (lane I ) or in the continuous presence It hasbeen shown previously that the activationof protein of PDH for 3 (lane 2 ) or 24 h (lane 3 ) .Aliquots of the cells incubated kinase C by phorbolesterscanaugmentthe cell surface for 3 h with PDR were carefully washed and incubated in complete expression of neutrophil cell surface receptors such as CR1, medium for an additional 21 h. At the end of this incubation, the CR3, formylmethylleucylphenylalanine, decay accelerating cells were directly analyzed (lane 4 ) . To demonstrate that the previous exposure to PDR does not cause refractoriness of the CD5 to he factor, and CD45 (8, 9, 38, 39). These proteins are stored in phosphorylated, an aliquot of the cells employed in lane 4 was treated granulocyte internal pools, which, upon activation, are transwith 50 ng/ml PDF3 for 10 min (lane 5 ) . Cells labeled with ““PO, as located in a few minutes from these pools to the cell surface indicated under “Materials and Methods”were lysed, immunoprecipitated with anti-CD5 mAb, and analyzed by SDS-polyacrylamide gel membrane. The overexpression of CD2 and CD5 does not seem to be due to the mobilization to the cell surface memelectrophoresis (12..5%). brane of internal pools of these molecules because significant tively phosphorylated, strongly increased its level of phos- changes in the up-regulation of these molecules require a t process mentioned previously phorylation after treatment with PDB (Fig. 6B). However, no least 24 h of incubation, and the phosphorylation of the CD2 molecule was observed, either occurs in minutes. On the other hand, treatment with inhiboverexpression of constitutively or after treatment with PDB (Fig. 6 R ) . Addi- itors of protein synthesis abrogated the CD2 and CD5 glycoproteins, whereas this treatment does not tional experiments to study the kinetics of phosphorylation affect the translocation and expression at the cell surface of the CD5 molecule indicate that after a 10-min pulse with PDB, thepeak of phosphorylation occurred a t 10-30 min and membrane of internal pools of cell surface molecules. Our results rather indicate that the augmented cell surface expresreturned to basal levels after 1 h(Fig. 7A). These results sion of CD2 and CD5 is probably due to a mechanism by indicate that the phosphorylation of CD5 induced by PDB which activation of protein kinaseC increases the mRNA takes place very rapidly at both its induction and its return levels forCD2 and CD5. Recentdataalsoindicatethat to basal levels. treatment of T cells with phytohemagglutinin increases the Further phosphorylation analysis indicated that the over- mRNA levels for CD2(40). The mechanisms by which phorbol expression of CD5 can also take place in the absence of its esters are able to enhancegene expression and transcription hyperphosphorylation. In these experiments, after an initial remainunknown. However, itis generallyaccepted that pulse with PDB (4 h), the cells were washed and left in culture changes in the activity of protein kinase Cmay initiate a for anadditional 20h. Undertheseconditions,the CD5 cascade of events such as phosphorylation and dephosphorylmolecule is overexpressed (similar data as shown in Fig. 2), ation of various cellular components which may ultimately but thelevel of CD5 phosphorylationwas comparable to those affect the action of specific transcription factors. It has been observed in the untreated controls (Fig. 7R, lane 4 ) . demonstrated that 12-0-tetradecanoylphorbol-13-acetate-inducible genes such as c-fos, c-myc, interleukin-2, and others DISCUSSION contain an eight-base pairconserved sequence present in the In this paper, we have analyzed the up-regulation of CD2 promoter region of these genes and known as TRE (12-0and CD5 cell surface molecules which occurs after the acti- tetradecanoylphorbol-13-acetate-responsiveelements).This vation of T cells. Several conclusions canbe drawn from this and other sequences found in genes coding forinterleukin-2R study. ( a ) The activation of protein kinase C is implicated in a and type I human immunodeficiency virus may serve as the cell surfaceup-regulation of CD2 and CD5. ( b ) These binding sites for several transcription factors described rechanges in antigen expression appear to be due to a concom- centlysuchasAP-1, AP-2, or NF-KB, whose activityis itant increase in the mRNA levels for CD2 and CD5. (c) The regulated by phorbol esters (41-44). overexpression of these molecules is not associatedwitha The role of phosphorylation in the modulation of cell sur-

B

Up-regulation of CD2 and CD5 by Activators of Protein Kinase face receptors remains to be fully understood. With respect consequences of protein kinase to T cells, the best documented C phosphorylation of cell surface moleculesis the concomitant down-regulation of CD3 and CD4 (14-16). It remains to be proven that phosphorylationof these molecules leads directly or indirectly totheirdown-regulationonthe cell surface, although such a mechanism is attractive. However, there are clear examples that protein kinase C-mediated phosphorylation is not a universal signal for down-regulation. It hasbeen shown previously that after treatment with PMA,major histocompatibility comple-r class I and CD8 molecules are hyperphosphorylated, but there is not a discernible concomitant decrease in their surface expression (45). Our data demonstrated that thecell surface overexpression of CD2 and CD5 could not be associated with a specificpattern of phosphorylation of these molecules since it was observed independently of theirhyperphosphorylated or nonphosphorylatedstate. CD5 molecule can be Indeed, our dataindicatethatthe overexpressed and simultanously hyperphosphorylated. Recently, it hasbeen shown that after a short pulse with PMA, phosphorylation occurs in the CD5 but not in theCD2 molecule (46). Our resultshave defined thekinetics of phosphorylation of CD5 and additionally haveshown that the overexpression of the C:D5 can be associated either with basal or with elevated levels, of phosphorylation. All these data indicate that modulation of a cell surface antigen and phosphorylation are not necessarily associated. It is difficult to assess thebiological significance of the upregulation of CD2 and CD5 after T cell activation since the physiological role of thlese molecules, and especially in the case of CD5, is not clearly established. The up-regulationof CD2 contrasts with the differential down-regulation of the CD3-T cell receptor complex which occurs after the activation of protein kinase C (47). At the moment, there are no clear reasons for the differential regulationby protein kinase C of the cell surface glycoproteins responsible for these two actiC is stimulated vation pathways. The fact that protein kinase by triggering the CD3-1’ cell receptor complex or by the CD2 antigen and the observation that protein kinase C influences both CD3 and CD2 exp-ression presumably indicate that triggering one receptor would influence the expression and response displayed by the other. Acknowledgments-We thankDr. D. CantrellandDr. M. J. Crumpton (Imperial Cancer Research Fund, London) for teaching the phosphorylation techniques; Dr. J. M. Redondo for his helpful discussions; D. Hernandez and M. Viton for technical assistance in flow cytometry analysis; and M. A. Vallejo for manuscript elaboration. :REFERENCES 1. Nishizuka, Y. (1986) Science 233, 305-312 2. Rozengurt, E. (1986) Science 234, 161-166 3. Isakov, N., Mally, M I., Scholz,W., andAltman, A. (1987) Immunol. Reu. 95,89-111 4. Parker, P. J., Stabel, SI., and Waterfield, M. D. (1984) EMBO J . 3,953-959 5. Niedel, J. E., Kuhn, L. J., and Vandehbark, G. R. (1983) Proc. Natl. Acad. Sci. U. S . A. 80,36-40 6. Bell, R. M. (1986) Cell 45, 631-632 7. Rosoff, P. M., Stein, L. F., andCantley, L. C. (1984) J . Biol. Chem. 259, 7056-7060 8. Lacal,P.,Pulido,R.,Sanchez-Madrid,F.,and Mollinedo, F. (1988) J . Biol. Chem. 263, 9946-9951 9. Miller, L. J., Bainton, D. B., Borregaard, N., and Springer, T. A. (1987) J . Clin. Inuest. 80,535-544 10. May, W. S., Sahyoun, N., Jacobs, S., Wolf, M., and Cuatrecasas, P. (1986) J . Biol. Chtvn. 260,9419-9426 11. Takayama,. S., White, M. F., Lauris, V., and Kalhn, C. R. (1984) Proc. Natl. Acad. Sci U. S. A. 81, 7797-7801

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