Aug 5, 2016 - marked âadvertisementâ in accordance with 18 U.S.C. Section 1734 solely to ... signals and transformation (25, 26, 28-30). ..... 9, 1651-1658. 31.
THEJOURNAL OF BIOLOGICAL CHEMISTRY Vol. 266, No. 22, Issue of August 5 , pp. 14167-14170,1991 Printed in U.S.A.
Communication
The interleukin 2 (IL-2)’ receptor is a multimeric complex of transmembrane proteins (1-6). The p55 subunit (a chain or Tac antigen) is thelow affinity receptor for IL-2 (& lo-’ M ) and has a 13-amino acid intracytoplasmic domain which has no apparent role in signal transduction (7, 8). The p75 (Received for publication, January 18,1991) chain (@ chain) possesses an intermediate affinity for IL-2 (Kd lo-’ M ) and has a 286-amino acid intracytoplasmic doBrian Remillard$, Ray Petrillog, main. A restricted 46-amino acid segment of this domain is Wlodzimierz Maslinskig, Mitsuru Tsudon, essential for IL-2-mediated signal transduction (9). Mutant Terry B. Stromg, Lewis CantleyII, and receptors lacking this region, although able to bind and interLyuba Varticovski**$$ nalize IL-2, fail to proliferate in response to IL-2. The nonFrom the $Deaconess/Joslin Division of Nephrology, New covalent association of these two proteins generates the high England Deaconess Hospital, Boston, Massachusetts 02215, affinityIL-2receptor ( K d lo-” M) (5, 6, 10).Despitethe the §Department of Medicine, Beth IsraelHospital and wealth of structural information, the intracellular eventsfolHaruard Medical School, Boston, Massachusetts 02215, the lowing binding of IL-2 to its receptor areunknown.The VDepartment of Medicine, Unitika Central Hospital, Kyoto, binding of IL-2totheIL-2receptor does notappearto Japan, the 11 Department of Physiology, Tufts U. School of stimulate canonical phosphatidylinositol turnover, elevate inMedicine, Boston, Massachusetts 02111, and the tracellular calcium, or require activation of protein kinase C **Departments of Medicine and Biomedical Research, St. (11-13). Although the intracytoplasmic domains of the 01 or Elizabeth’s Hospital, Tufts University School of Medicine, the @ subunits of the IL-2 receptorlack a consensus sequence Boston, Massachusetts 02135 for a protein kinase, phosphorylationof several intracellular proteins on serinelthreonine and tyrosine residues is observed Interleukin-2 (IL-2) stimulates proliferation of T in response to IL-2 stimulation(14-16). Furthermore, the ILlymphocytes and is involved in the activation of both 2 receptorformsanimmunoprecipitable complex with an natural killer and lymphokine-activated killer precur- unidentified protein-tyrosine kinase in response to IL-2 (17). sor cells. The intracellular messengers which mediate Recently, several transmembrane growth factor receptors, IL-2-dependent events have not yet been identified. which act as ligand-activated protein-tyrosine kinases, have IL-2 receptor is not a protein-tyrosine kinase. Activa- been shown tophysically associate with and activate a novel tion of a cellular protein-tyrosine kinase and direct phosphoinositide kinase, termed type I phosphatidylinositol association of a protein-tyrosine kinase activity with kinase or phosphatidylinositol 3-kinase (PI 3-kinase) (18).P I the IL-2 receptor occurs within minutes of IL-2 stim- 3-kinase has beenpurified to homogeneity and will phosulation. We investigated the activation of phosphati- phorylate phosphatidylinositol (PI), phosphatidylinositol 44,5-bisphosdylinositol 3-kinase(PI3-kinase) in IL-2-mediated phosphate(PI-4-P),andphosphatidylinositol position of the inositol ring (19). signal transduction using the IL-2-dependent murine phate (PI-4,5-P2) at the D-3 T-cell line, CTLL-2, and human phytohemagglutinin- P I 3-kinase has been shown to form a complex with PDGF, stimulated peripheral blood lymphocytes (phytohe- epidermal growth factor, CSF-1, and insulin receptors (20magglutinin blasts). Within a minute following stimu- 23) and with oncogene products of polyoma middle T , v-src, v-fms, v-abl, and v-yes (20, 24-27). The exact role and fate of lation of these cells with IL-2, PI 3-kinase activity could be detected in antiphosphotyrosine (anti-P-Tyr) the novel polyphosphoinositides produced by PI 3-kinase are antibody immunoprecipitates. IL-2 triggered a direct unknown; however, mutants of receptors and oncoproteins that fail to associate with P I 3-kinase fail to mediate growth association of PI 3-kinase with the IL-2 receptor as signals and transformation (25, 26, 28-30). We investigated detected in immunoprecipitates using anti-IL-2recepthe activation of P I 3-kinase in response to IL-2 in human tor @ chain antibody. In vivo labeled CTLL-2 cells have PHAblastsand cloned IL-2-dependentmurineT-cells, a time-dependent increase in D-3-phosphorylated CTLL-2. polyphosphoinositides following stimulation with IL2. This is the first group of second messengers identiMATERIALS AND METHODS fied in IL-2-mediated signal transduction. Preparation of Cell Lysates-Peripheral blood mononuclear cells
Interleukin-2 Receptor Regulates Activation of Phosphatidylinositol 3-Kinase”
were isolated from random donors using Ficoll-Hypaque. Cells were
* This work was supported by National Institutes of Health Grant A122882 and National Cancer Institute Grant A18512 (to T. B. S.), National Institutes of Health Grants GM36624 and GM41890 (to L. C. C.), and National Cancer Institute GrantCA53094 (to L. V.) The costs of publication of thisarticle were defrayed inpart by the payment of pagecharges. Thisarticlemusttherefore hehereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate thisfact. $$ To whom correspondence should headdressed.
’ Theabbreviations used are:IL-2,interleukin-2; PI 3-kinase, phosphatidylinositol 3-kinase; PI-4-P, phosphatidylinositol 4-phosphate; PI-4,5-P2, phosphatidylinositol 4,5-bisphosphate; PI-3-P, phosphatidylinositol 3-phosphate; PI-3,4-P2, phosphatidylinositol 3,4-bisphosphate; PIP:,, phosphatidylinositol 3,4,5-trisphosphate; gPI-3-P, glycerophosphoinositol 3-phosphate; gPI-4-P, glycerophosphoinositol 4-phosphate; gPI-4,5-P2, glycerophosphoinositol 4,5-bisphosphate; gPI-3,4-P2,glycerophosphoinositol3,4-bisphosphate;gPI3,4,5-P:1, glycerophosphoinositol 3,4,5-trisphosphate; Ins-1,4,5-P:{, inositol1,4,5-trisphosphate;In~-1,3,4,5-P:~,inositol1,3,4,5-tetrakisphosphate; BSA, bovine serumalbumin;PDGF,platelet-derived growth factor; HPLC,high performance liquid chromatography; CSF1, colony stimulating factor-1.
14167
IL-2 Receptor Regulates Activation
14168
of PI 3-Kinase
stimulated with PHA and maintained in culture for 72-96 h a t which time they express 3.5-7 X loLhighaffinity 11,-2 receptorhinding sites/cell hut do not secrete ahundant amounts of 11,-2 (10,31). These IL-2 and are very I'HA hlasts proliferate in response to exogenous sensitive to a chimeric 11,-2 diphtheria toxin fusion protein (32, 33). I'HA hlasts were suspended in 1% RSA, RPMI for 4 h prior to 11,-2 stimulation to induce quiescence (14). Cells were exposed to human recomhinant 11,-2 (100 units/:! X 10" cells/ml) (Riogen) for indicated t imes. Samples were washed with ice-cold media and lysed in buffer containing 1 % Nonidet P-40. 20 mM Hepes, pH 7.5. 5 mM sodium orthovanadate. 137 mM NaCI. 1 mM PMSF, a n d 10 pg/ml leupeptin. C T I L - 2 cells were maintained in culture with 10% rat T-cell growth l'artor (Hiocell) in supplemented RPMI media and passaged everv 48 h. Cells were grown to a density of 1-2 X 10"/ml, placed in media with 1"; RSA, RPMI for 12-14 h to induce quiescence (32). Irnrnunopr~cipilnlitnand PI Kinare Acfiuify-Appropriate mono2 hfollowedhygoatanti-mouse clonalantihodvwasaddedfor iantihody linked to Sepharose heads (Cappell). The heads were washed twice with each of t h e following: I ) phosphate-huffered saline; 2) 0.5 M I X I , 10 mM Tris-HCI, pH 7.4; 3 ) 0.1 M Nacl, 1 mM EDTA in 0.1 M Tris. pH 7.4. Lipid kinase assays were performed on the heads in the presence of 25 mM Hepes, pH 7.4, and 10 mM MgCI,. A mixture o f either PI-4.5-P, and phosphatidylserine (1:l)or PI, P1-4..5-P2, and
A 0
B 1'
2'
C 0
5' PIP
2'
5'
.
phosphatidylserine (l:l:2) at a final concentratinn of 0 . 2 mg/ml was dispersed hv sonication in 2.5 mM Hepes. pH 7.4, 1 mY F:I)TA and then added to the reaction mixture. The reactinn was initiated hy addition of 10-20 pCi of [y-"I']ATP (6,000 Ci/mmol: I)u I'ont-New England Nuclear) and I 0 0 p~ ATP and terminated t ~ yaddition of I N NCI. The products were extracted into chloro!r~rm:n~ethanol ( ? : I ) and separated hy TIX. Thin /,oyer ('hrornnlo~roph\.-'rheproducts o f 1'1 kinase were separated hy T1.C in acidic conditions on oxalate-treated silica gel plates (Merck). The mohile phase was composed of 1-propanol, 2 Y acetic acid (13.75; v:v). LJnlaheled lipid standards (1'1-4-1' and 1'1-4P,) were ohtained from Sigma. antlI 'I']l'lI', was prepared frnm antiP-Tyr immunoprecipitates o f 1'IX;F-stimulateti cells (22. 3.1 ). T h e standards were co-chromatographed with the samples a n d viwalized hv exposure to iodine vapor antl autoradiography. prodIlc~ncylutionnnd HP/,(' AnnlTsis-The"l'-laheledreaction ucts were deacylated with methylamine reagent (18) antl identified as tlrsrritwd by H P L C o n a I'artisphere SAX column (\Vhatman) (34). gl'I-:l-P, gP1-:3,4-P2, and gI'l-:l,4,5-1', standards were prepared from 1't)C.F-stimulated NIH :YI':j fihrohlasts (22. 341. In Viuo /Ahding n f Infort C'dl.v-CTI,IA-2 cells were deprivedo f Tcell growth factor for 12 h. The cells w'ere Iaheled with 0.1 mCi/lf)' lfK)-200 cells/ml of '"Poi- for t h e last h prior to stirnulation with 11,-2. Cellwerewashedhrieflyin RI'MI, the lipids units/mlof extracted into 1 N HCI:chloroform:methanol f 1 2 1 ), and dearylated as ahove. The products were separated hy HI'1,C and identified hy comparison with deacylated commercially nvailahle'H standards antl with authentic D-3-phosphorylated pol~~hopshoin[)sititlrs prepared from PDGF-stimulated N I H 3T3 fihrohlasts (22, 3 4 ) .
b
RESULTS
FIG. 1. Association of PI 3-kinasewith IL-2 receptor fl chain and withphosphotyrosine phosphorylated proteins. I'HA hlasts were stimulnted with human recomhinant IL-2 for the indicated times. IL-2 receptor was immunoprecipitated using anti11,-2 receptor /j chainmAh (panels A a n d H ) . Phosphotyrosinecontaining proteins were immunoprecipitated using anti-1'-Tyr mAh ( p n n d ('). T h e PI %kinase assay wasperformedontheheads; as descrihed extraction and separation of the products was performed of the standunder "Materials and Methods." The migration position ards was determined as descrihed under "Materials and Methods." IGve separate harvests of I'HA hlasts from normal volunteers were analyzed. The TIL represents one of those experiments. The standards were co-chromatographed with the samples and visualized hy Pond A, anti-11,-2 exposure to iodine vapor and autoradiography. receptorimmunoprecipitahle PI kinaseproductsfromPHAhlasts stimulated with II,-2 for 0, I. 2. a n d 5 min. PI-4,5-P2was used as suhstrate. /'and H. PI kinase products ohtained in anti-lL-2 receptor PI and PI-4.5-P, as suhstrates. immunoprecipitates usinga mixture of (:ells were exposed to IL-2 for 2.5 min prior to lysis.Panrl C , a n t i - P T y r (IJHI) immunoprecipitahle PI kinase products from PHA hlasts stimulatedwith 11,-2 for 0. 2. a n d 5 min. PI-4,5-P2 wasused as suhstrate.
Flc. 2. HPLC identification of deacylated polyphosphoinositides generated using anti-l1,-2 receptor antihody immunoprecipitahle P I 3-kinase from IL-2-stimulnted PHA hlnsts. ('losrd syrnhols represent the migrat ion of "1'-laheled deacylated products. Solid line indicates deacylated "H standards (Du I'ont-New England Nuclear) coinjected with the samples. The of gl'I-3-1' and migration positions gI'll', generated from I'DGI~'-stimulated N I H 3'1'3 cells is indicated hy thenrrows. I'nnrl A , separation of glycerophosphoinositol 3-phosphate (g/'I-.'l-I') from glycerophosphoinositol4-phosphate (g/'/4 - / ' ) . I'nnel R , separation ofglvceroI)hosphoinositoltrisphosphate (g/'IP,) from Ins-1,4,5-P,, and Ins-l,3,4,5-P4.
The IL-2 receptor was immunoprecipitatedfromhuman PHAblastsusing a murinemonoclonalantibodvdirected against a non-IL-2 hinding epitope of the I L - 2 receptor (f chain (35)before and after stimulationof intact cells withIL2. P I .?-kinase activity was measured either usingPI-4,.5-P2or a mixture of P I a n d PIP, as substrates (Fig. 1, panP1.s A and E?).Conversion of PI-4,5-P2to PIP., was detected within a minute after addition of IL-2, was maximal hy 2 min, and declined at 5 min (Fig. 1,p a n d A ). A dose-dependent increase PI .?-kinaseactivitvoccurs in inIL-2receptor-associated response to IL-2 stimulation in the physiological range: activity plateaus at 200 units/ml of11,-2 (not shown). PI :]-kinase was also immunoprecipitated using anti-P-Tyr antihodv in response to IL-2 (Fig. 1, p a n d C). HPLC analysisof the lipids produced byanti-11,-2 receptor or anti-P-Tyr antibody immunoprecipitates (Fig. 2) demonstrated that these lipids comigrate with authentic D-3-phosphorylated polyphosphoinosititles. IL-2-dependent association of PI 3-kinase with phosphotyrosine phosphorylated proteins was also demonstrated in
A
B el-3-P
el-4-P
J.
J.
Ins- 1.4.5 PJ
h,-P4 *lPJ
3000
2000
I
I
IL-2 Receptor Regulates Activation
PIP
0
1’
5’
10’
30’
1
2
3
4
5
-
P I P3-
FIG. 3. Anti-P-TyrimmunoprecipitablePI3-kinasefrom 11,-2-stimulated CTLL-2 cells.Quiescent CTLL-2 cells were stimulated with 11,-2 for the indicated times,washed, and lysed in Nonitlet P-40-contnining huffer. Immunoprecipitation, in uitro kinase assav and T I L analvsis of the products were performed as described in Fig. 1. A mixture of 1’1 and PI-4,5-P2 was used as suhstrate.
B
A
5001
400
400
300
300
200
200 -
1
PI 3-Kinase
14169
murine IL-2-dependent T-cell line CTI,I,-2. These cells express approximately 3 X 10.’ high affinity 11,-2 hinding sites/ cell. CTLL-2 cells were induced to quiescence in 1 7 HSA, RPMI for 12-14 h prior to 11,-2 stimulation. Previous studies suggest that under these conditions, the cells are quiescent metaholically but respond rapidly to exogenous 11,-2 f%). A time-dependent increase in anti-1’-Tyr immunoprecipitahle PI 3-kinase activity was seenin CTI,L“L cells stimulated with IL-2(Fig. 3). HPLCanalysis of thedeacylatedproducts confirmed the identity of the pol.~hopshoinositidesas P I - 3 P and PIP.,. We also investigated accumulationof P I 3-kinase products inintactCTLL-2cells.CTLI,-2cellsweremetaholically
C PPI-3.4 P2
500
of
II’J
+
*PIPS
f
IP4
+
100
0 26
2tI
30
32
34
56
D
TIME (rnln)
FIG. 4. HPLC analysis of polyphosphoinositides from intact CTI,1,-2 cells. rTI,I,-2 rells werr metabolically laheledwith .“l’O.~ for 3 hpriortostimulation with ll,-2. C‘ells were washedhrielly in RI’VI, the phospholipids extracted into 1 N HC1:chloroform:methanol (1:2:1), and dearvlated as tlesrrihed (18). T h r produrts were separated by HPLC and identified hy comparison with the migration properties of gl’l-:bI’, gPl-3,4-l’2,and gPI-3,4,5-P, standards ohtained from anti-P-‘l’yr immunoprecipital)le 1’1 :I-kinase from P I X F stimulated SI11 :{‘I3cells (indicated by the arrows). Open triangles represent phospholipids from unst imulated rells. ( ’ l o s r d tricrnglr,s represent phospholipids from cells stimulated with 11,-2 for 12 min. Solid line represents ‘ H stnndards o f gi’l.4-I’ and gI’I-4,5-P2,and arrows indicateelution positionsof Ins-1,4,5-l’, and Ins-l,:~.4,T,-l’,ro-injerted with the samples. I’anel A , the elution pattern of gl’l-.‘bl’ and ~1’1-4-1’; panrl H , the elution pattern of gI’l-3,4-1)~and g l ’ I - 4 , ~ 1 ~ l ’ : : panel C, the elution pattern of gl’1-3,4,5-P.l.The counts/min in each one o f the lipids at zero time from onr o f the experiments was: PI-3-1’ = 2,498; PI-4-P = 69,935; I’I-3,4-1’? = 535; PI-4,5-I’, = 24,078; I’ll’, = 238. The trnrings arerepresentative of one of theexperiments. I’and 11, time course of thecontent of pol.~hos’ph~~inositi~lc~s normalized to a total ”1’ incorporated into phospholipids andexpressed as percent ront rol. Hnrs represent standnrd deviations from four experiments a t times 0,1, and 12 min and three experiments at 2, 5 , and 25 min. ’I’hc. rrsrrlts are expressed using the following symhols: closed syunres. PI-3-P; npen syunres. P I - 4 - P : closed rirrlrs, l’I.:L4.1’.,: open circles, PI-4,5-P,;closed triangles, PIP.
