Interleukin-2 Receptor Regulates Activation of Phosphatidylinositol 3-Kinase”

Interleukin-2 (IL-2) stimulates proliferation of T lymphocytes and is involved in the activation of both natural killer and lymphokine-activated killer precur-sor cells. The intracellular messengers which mediate IL-2-dependent events have not yet been identified. IL-2 receptor is not a protein-tyrosine kinase. Activation of a cellular protein-tyrosine kinase and direct association of a protein-tyrosine kinase activity with the IL-2 receptor occurs within minutes of IL-2 stimulation. We investigated the activation of phosphatidylinositol 3-kinase (PI 3-kinase) in IL-2-mediated signal transduction using the IL-2-dependent murine T-cell line, CTLL-2, and human phytohemagglutinin-stimulated peripheral blood lymphocytes (phytohe-magglutinin blasts). Within a minute following stimulation of these cells with IL-2, PI 3-kinase activity could be detected in antiphosphotyrosine

The interleukin 2 (IL-2)' receptor is a multimeric complex of transmembrane proteins (1)(2)(3)(4)(5)(6). The p55 subunit (a chain or Tac antigen) is the low 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 chain (@ chain) possesses an intermediate affinity for IL-2 (Kd lo-' M ) and has a 286-amino acid intracytoplasmic domain. A restricted 46-amino acid segment of this domain is essential for IL-2-mediated signal transduction (9). Mutant receptors lacking this region, although able to bind and internalize IL-2, fail to proliferate in response to IL-2. The noncovalent association of these two proteins generates the high affinity IL-2 receptor ( K d lo-" M) (5,6,10). Despite the wealth of structural information, the intracellular events following binding of IL-2 to its receptor are unknown. The binding of IL-2 to the IL-2 receptor does not appear to stimulate canonical phosphatidylinositol turnover, elevate intracellular calcium, or require activation of protein kinase C (11)(12)(13). Although the intracytoplasmic domains of the 01 or the @ subunits of the IL-2 receptor lack a consensus sequence for a protein kinase, phosphorylation of several intracellular proteins on serinelthreonine and tyrosine residues is observed in response to IL-2 stimulation (14- 16). Furthermore, the IL-2 receptor forms an immunoprecipitable complex with an unidentified protein-tyrosine kinase in response to IL-2 (17).
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).

RESULTS
The IL-2 receptor was immunoprecipitated from human PHA blasts using a murine monoclonal antibodv directed against a non-IL-2 hinding epitope of the IL-2 receptor (f chain (35) before and after stimulation of intact cells with IL-2. P I .?-kinase activity was measured either using PI-4,.5-P2 or a mixture of PI and PIP, as substrates (Fig. 1, panP1.s A and E?). Conversion of PI-4,5-P2 to 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 in IL-2 receptor-associated PI .?-kinase activitv occurs in response to IL-2 stimulation in the physiological range: activity plateaus at 200 units/ml of 11,-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).
IL-2-dependent association of PI 3-kinase with phosphotyrosine phosphorylated proteins was also demonstrated in 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 seen in CTI,L"L cells stimulated with IL-2 (Fig. 3). HPLC analysis of the deacylated products confirmed the identity of the pol.~hopshoinositides as P I -3 -P and PIP.,.

IL-2 Receptor Regulates Activation of PI 3-Kinase
labeled with "PO!-and the content of individual phospholipids was analyzed by HPLC. No significant changes in the D-3-phosphorylated polyphosphoinositides were observed at one minute following addition of IL-2. Within 2 min, there was a significant increase in the levels of PI-3,4-Pz and PI-3,4,5-P3 with a maximum increase detected at 12 min (269% increase in the level of PI-3,4-P2 and a 417% increase in PI-3,4,5-P3) without significant changes in the content of PI-3-P (Fig. 4, panel D). The levels of these lipids subsequently declined but remained above control values at 25 min. No decline in the levels of PI-4-P or PI-4,5-P2 were seen, confirming previous observations that IL-2 does not stimulate PI turnover. These results are similar to accumulation of PI 3kinase products observed in response to PDGF or CSF-1, except that the initial response is delayed for 1-2 min (22,34).

DISCUSSION
The data presented here suggests that IL-2-dependent signal transduction involves activation of PI 3-kinase. PI 3kinase activity co-immunoprecipitates with the anti-P-Tyr antibody, suggesting that PI 3-kinase is either phosphorylated on tyrosine, as has been shown for the PDGF receptorassociated enzyme (20) or physically associates with a proteintyrosine kinase in response to IL-2.
A direct association of PI 3-kinase with the IL-2 receptor was observed following ligand binding. This association could be a result of receptor association with a protein-tyrosine kinase (17, 37). Alternatively, PI 3-kinase might associate directly with the IL-2 receptor which is phosphorylated on tyrosine in response to IL-2 (38). Several protein-tyrosine kinases are expressed in lymphocytes, and previous studies suggest that signals from the T-cell and the IL-2 receptors may involve activation of distinct protein-tyrosine kinases (39). Recent information suggesting that IL-2 receptor complex may contain additional subunits (4, 40) may provide the crucial information for our understanding of the assembly of the IL-2 receptor. The role of the 46-amino acid signal transducing domain of the IL-2 receptor /? chain in the assembly of this complex is currently under investigation.
The increase in the content of PI-3,4-P2 and PIP, in intact CTLL-2 cells begins at 1-2 min. This delay in not observed in PDGF-stimulated smooth muscle cells (34) or CSF-1 stimulated murine macrophages (22) and could be explained by less enzyme being activated in CTLL-2 cells or the need of additional molecular assembly in order to activate PI 3kinase.
The PI 3-kinase pathway represents a new signaling pathway whose physiological responses are not yet understood. Since PI-3,4-P2 and PIPB are not produced until cells are stimulated and since they are not substrates for the known PI-specific phospholipases C (41), it is likely that they act as messengers rather than being precursors for messengers. The nearly ubiquitous association of PI 3-kinase with mitogenically active protein-tyrosine kinases indicates that this pathway constitutes a critical component of the growth signaling mechanism of those enzymes (42).
The IL-2 receptor is a member of the cytokine receptor family all of which have relatively small cytoplasmic domains. These receptors do not contain intrinsic protein-tyrosine kinase activity or any other known enzymatic activity. The results of this study are the first report that a member of this family of receptors, like the protein-tyrosine kinase family, can associate with PI 3-kinase.