CD45 phosphotyrosine phosphatase and p56lck protein tyrosine kinase: a functional complex crucial in T cell signal transduction.

Tyrosine phosphorylation of several intracellular proteins is observed very early during T cell activation. p56lck, a non-receptor src-like protein tyrosine kinase (PTK) which is associated with the intracellular domains of CD4 and CD8 co-receptors, has been implicated in these early signal transduction events. Furthermore, recent experiments indicate that the receptor phosphotyrosine phosphatase, CD45, might be important in the regulation of p56lck PTK activity and that its expression is required for the generation of second messenger molecules following TCR triggering. Here, using co-capping experiments and double indirect immunofluorescence microscopy in functional human T lymphocytes, a specific co-distribution of a significant fraction of p56lck with CD45, but not with several other cell surface proteins, has been revealed. This is the first demonstration of a physical interaction between a receptor phosphotyrosine phosphatase and a PTK under physiologically relevant conditions. In addition, after antibody-induced capping of CD4, both a co-localization of p56lck and CD4, and concomitantly a significant increase in intracellular phosphotyrosine at the sites of CD4 caps were observed. In strong contrast to these results, co-clustering of CD4 with CD45 did not result in any detectable intracellular phosphotyrosine at the cap sites. These data indicate that CD45 can act on CD4-associated phosphoproteins in viable human T lymphocytes. Further, this provides evidence that p56lck PTK is a substrate of CD45 phosphotyrosine phosphatase in vivo and thereby supports the idea that CD45 is an early regulator of T cell activation involved in the modulation of the coupling of receptor-triggered events to intracellular signalling pathways.     

The lymphocyte-specific protein tyrosine kinase p56lck is hyperphosphorylated on serine and tyrosine residues within minutes after activation via T cell receptor or CD2

Human T cells can be activated and induced to proliferate through either the antigen-specific receptor complex (TcR-CD3) or the CD2 surface molecule. Following stimulation, both serine and tyrosine phosphorylation of cellular protein have been demonstrated to occur. p56lck, a protein tyrosine kinase associated to the inner face of the plasma membrane, is almost exclusively expressed in lymphoid cells, especially T cells. Within minutes after activation of a human T cell-derived line (Jurkat) via stimulation of either the TcR-CD3 complex or the CD2 glycoprotein, we observed a hyperphorphosylation of p56lck. A concomitant shift to a higher molecular weight in sodium dodecyl sulfate-polyacrylamide gel was also observed. Similar changes were obtained with phorbol 12-myristate 13-acetate. Tryptic phosphopeptide analysis of the hyperphosphorylated form of p56lck yielded new phosphorylated sites in serine residues and an increased tyrosine phosphorylation. These results suggest that p56lck may be intimately connected to the signaling pathway in T cell activation.     

The tyrosine kinase activity of p56lck is increased in human T cells activated via CD2.

An early biochemical event associated with T cell activation is tyrosine phosphorylation. We have previously shown that p56lck, a lymphocyte-specific protein tyrosine kinase, is hyperphosphorylated on serine and tyrosine residues 15 minutes after activation via CD2 with a concomitant shift to a higher molecular mass. We now demonstrate that the tyrosine kinase activity of p56lck is increased within seconds following CD2 triggering. This activity decreases thereafter correlating with the appearance of changes in phosphorylation previously described. These results suggest that p56lck may play an important role in the CD2 activation pathway.     

T cell receptor and CD8-dependent tyrosine phosphorylation events in cytotoxic T lymphocytes: activation of p56lck by CD8 binding to class I protein

Tyrosine phosphorylation of proteins plays a central role in T cell activation. Mitogens or anti-receptor antibodies have been employed to study these signaling events, but the extent to which these mimic receptor interactions with native ligands is unclear. Cytotoxic T lymphocytes can be activated for functional responses using purified, native class I ligands presented on a surface. Previous work showed that stimulation with fluid-phase anti-T cell receptor (TCR) monoclonal antibody (mAb) activates CD8 to mediate adhesion to class I proteins and that activated CD8 generates a co-stimulatory signal upon binding to class I. Changes in tyrosine phosphorylation of substrates and activity of the p56^lck kinase have now been examined in this two-step process. The observed changes are small in comparison to those found using more potent nonphysiological stimuli, but may more accurately reflect the events required for activation of functional responses. Fluid-phase anti-TCR mAb caused increased tyrosine phosphorylation of a discrete subset of cellular substrates. Increased phosphorylation of additional substrates occurred upon CD8 binding to class I, resulting in a phosphorylation pattern comparable to that found in cells stimulated with class I alloantigen. Anti-TCR mAb alone caused increased tyrosine phosphorylation of p56^lck. When CD8 bound to class I, phosphorylation of p56^lck decreased to below the basal level found in unstimulated cells, accompanied by a substantial increase in kinase activity. These results are consistent with the two-step model for TCR activation of CD8/class I interactions and directly demonstrate that CD8 binding to class I leads to up-regulation of p56^lck activity.     

The p56lck SH2 domain mediates recruitment of CD8/p56lck to the activated T cell receptor/CD3/ζ complex

The CD4 or CD8 co-receptors and the T cell receptor (TCR) are thought to interact with the same antigen-presenting major histocompatibility complex molecule in a stable ternary complex. Therefore, the TCR and its co-receptor need to come into close proximity on the surface of the T cell. We have previously shown that the interaction of the p56^lck SH2 domain with ζ-associated, tyrosine phosphorylated ZAP-70 and Syk kinases leads to an enhanced association of CD4 with TCR/CD3/ζ complex after CD3 stimulation of Jurkat cells. In this report, we analyzed whether a similar mechanism can mediate recruitment of the CD8αβ and CD8αβ isoforms to the TCR. We demonstrate in vivo in association of CD8αα/p56^lck with the tyrosine kinase ZAP-70 after CD3 stimulation of Jurkat cells. A phosphopeptide competing in vitro for the binding of tyrosine phosphorylated proteins to the SH2 domain of p56^lck specifically impedes the association of ZAP-70 with CD8αα/p56^lck without affecting the ζ/ZAP-70 interaction. The same peptide is able to compete for the activation-dependent association of the CD8αα or CD8αβ isoform with the TCR/CD3/ζ complex. Moreover, co-precipitation of the TCR with both CD8 isoforms was observed after CD3 stimulation. These findings strongly suggest that the p56^lck SH2 domain mediates recruitment of CD8/p56^lck to the activated TCR/CD3/ζ complex.     

CD4-dependent and -independent association of protein tyrosine kinases to the T cell receptor/CD3 complex of CD4+ mouse T lymphocytes

Tyrosine phosphorylation of different substrates is the earliest intracellular signal detected after T cell receptor (TcR) ligation. Several tyrosine kinases have been detected associated to the CD3-TcR complex in stimulated or unstimulated cells, including p56^lck, p59^fyn and ZAP-70. We have observed, in one mouse T helper CD4 T cell line, that most TcR- or CD3-associated tyrosine kinase activity comes from CD4:p56^lck (Diez-Orejas, R., Ballester, S., Feito, M. J., Ronda, M., Ojeda, G., Criado, G., Portolées, P. and Rojo, J. M., EMBO J. 1994. 13: 90). To analyze whether this is a major way of tyrosine kinase association to the TcR in normal CD4⁺ T cells, we examined the nature and mode of association of tyrosine kinases to the TcR complex in normal spleen CD4⁺ T lymphocytes. Our results show that, in normal CD4⁺ T lymphocytes, as in CD4⁺ T cell lines, there is a stable and readily detectable association between CD4: p56^lck and the TcR/CD3 complex, as determined by in vitro kinase activity in immunoprecipitates from cell lysates. However, TcR/CD3 complexes from nature CD4⁺ lymphocytes have detectable amounts of p56^lck associated in a CD4-independent manner, as shown by immunodepletion of the lysates with anti-CD4 antibodies. In addition, TcR/CD3 also bind p59^fyn regardless of the presence of CD4. Conversely, we have observed that CD4 co-precipitates small quantities of p56^fyn in a TcR/CD3-independent manner. Overall, our data suggest the existence of different possible molecular complexes between TcR/CD3, CD4 and their attending kinases, as well as some quantitative and qualitative differences between CD4⁺ T cells and CD4⁺ T cell lines in kinase association to the TcR/CD3 complex.     

Interaction of CD4:lck with the T cell receptor/CD3 complex induces early signaling events in the absence of CD45 tyrosine phosphatase.

Antibody-mediated ligation of the CD3/T cell antigen receptor (TcR) activates phospholipase C (PLC) via a tyrosine kinase signaling pathway that requires expression of the transmembrane tyrosine phosphatase CD45. In normal T cells, CD3-mediated PLC activation is significantly augmented by co-ligation of CD3 with the CD4 co-receptor; however, unlike CD3-associated tyrosine kinases, antibody-induced activation of the CD4-associated tyrosine kinase p56lck does not require CD45 expression. To explore the role of CD45 in the CD3 and CD4 activation pathways further, we examined the effect of CD3/CD4 cross-linking on tyrosine phosphorylation and activation of phospholipase C in CD45- mutant cells of the T cell leukemia line HPB.ALL. In accord with previous observations, anti-CD3 stimulation of the CD45-deficient cells failed to activate tyrosine kinases, or PLC as measured by mobilization of intracellular calcium. However, we show here that ligation of CD3 with CD4 leads to tyrosine phosphorylation of PLC gamma 1 and elevation in the intracellular free Ca2+ concentration in CD45- cells that is in excess of that seen in CD45+ cells. Since CD4 stimulation alone did not activate PLC, a component of the CD3 signaling pathway must be independent of CD45. Anti-CD4-induced tyrosine phosphorylation and activation of CD4-associated lck was also enhanced in CD45- cells, suggesting that increased lck activation compensates for the defect in CD3/TcR signaling, such that interaction of the CD3 signaling pathway with the CD4-associated pathway activates PLC even in the absence of CD45. The data demonstrate that the requirement for CD45 in coupling CD3/TcR to the PI-PLC activation cascade is not absolute, but rather substantiates a role for CD45 in modifying molecular interactions that control T cell activation.     

A functional complex is formed in human T lymphocytes between the protein tyrosine phosphatase CD45, the protein tyrosine kinase p56lck and pp32, a possible common substrate.

In vitro protein kinase assays of CD45 immunoprecipitates prepared from digitonin lysates of resting human T lymphocytes resulted in exclusive tyrosine phosphorylation of a 32-kDa protein (pp32). Reprecipitation of the in vitro phosphorylated proteins and Western blot analysis of whole CD45 immunoprecipitates employing antisera specifically directed at different protein tyrosine kinases demonstrated that the p56lck protein tyrosine kinase was responsible for in vitro phosphorylation of pp32. Since in vitro kinase assays of p56lck immunoprecipitates also resulted in phosphorylation of pp32, the present data strongly suggest that a functional complex is formed between CD45, p56lck and pp32. Such a notion is supported by the findings that phosphorylation of pp32 by p56lck correlated with expression of the CD45 molecules and that in vitro phosphorylated pp32 was completely dephosphorylated by purified CD45.     

CD45 modulates T cell receptor/CD3-induced activation of human thymocytes via regulation of tyrosine phosphorylation.

Stimulation of thymocytes or mature T cells via the T cell receptor (TcR)/CD3 complex activates a cascade of processes inducing cells to enter the cell cycle. A key step is the activation of phosphatidylinositol-specific phospholipase C (PI-PLC) within seconds following TcR/CD3 stimulation, an event which is strongly enhanced by co-ligation of the CD4 (or CD8) accessory molecule with TcR/CD3. In contrast, co-ligation of CD45 inhibits the same TcR/CD3 responses. The machinery which couples the TcR/CD3 complex, CD4, and CD45 to PI-PLC appears to involve regulation of tyrosine phosphorylation, as the TcR/CD3 and CD4 receptors are associated with the tyrosine kinases p59fyn and p56lck, respectively, and CD45 has intrinsic tyrosine phosphatase activity. Here, we have examined the ability of CD45 to regulate signal transduction via TcR/CD3 in human thymocytes. Co-cross-linking CD45 to the TcR/CD3 complex strongly suppressed the tyrosine phosphorylation of several intracellular substrates normally seen following TcR/CD3 stimulation. This effect of CD45 was associated with inhibition of a rise in intracellular calcium following TcR/CD3 ligation. Since TcR/CD3 stimulation of mature T cells induces tyrosine phosphorylation of PLC gamma 1, we investigated this phenomenon in thymocytes, and asked whether ligation of CD45 might regulate this process. By immunoprecipitation we found that TcR/CD3 stimulation induced tyrosine phosphorylation of PLC gamma 1, an effect which was enhanced by co-cross-linking CD4 to TcR/CD3. In contrast, co-ligation of CD45 strongly blocked PLC gamma 1 phosphorylation induced by either stimulus. Consistent with previous findings in mature T cells, CD45 cross-linking was able to partially inhibit TcR/CD3-induced thymocyte proliferation when interleukin 2 was used as a second signal, but almost completely (80%-90%) blocked proliferation when anti-CD28 mAb was used as the second signal, suggesting that CD45 cross-linking may be able to block interleukin 2 production via the CD28 pathway. These effects of CD45 on TcR/CD3 signaling and proliferation in thymocytes point towards a potential role for this pathway in thymic selection.     

The CD45 tyrosine phosphatase regulates CD3-induced signal transduction and T cell development in recombinase-deficient mice: restoration of pre-TCR function by active p56(lck).

The pre-TCR complex regulates the transition from CD4(-)CD8(-) double-negative (DN) to CD4(+)CD8(+) double-positive (DP) thymocytes during T cell development. In CD45(-/-) mice there is an accumulation of DN cells, suggesting a possible role for CD45 in pre-TCR signaling. We therefore crossed CD45(-/-) with Rag-1(-/-) mice to investigate the signaling functions of the CD3 complex in DN thymocytes. Remarkably, treatment of Rag-1(-/-)/CD45(-/-) mice with a CD3 mAb caused maturation to the DP stage at only 3% of the level measured in Rag-1(-/-) mice. Furthermore, ligation of the CD3 complex on Rag-1(-/-) /CD45(-/-) thymocytes in vitro induced less tyrosine phosphorylation in specific proteins when compared to Rag-1(-/-) thymocytes. CD45(-/-) mice were also crossed with pLGFA mice expressing a constitutively active form of the lck tyrosine kinase which restored the DN to DP transition to near normal levels. Our results are consistent with a model in which CD45-activated p56(lck) is critical for pre-TCR signal transduction.     

Interleukin-2 signal transduction in human NK cells: multisite phosphorylation and activation of the tyrosine kinase p56lck.

Interleukin-2 (IL-2) potently stimulates natural killer (NK) cell proliferation and cytotoxic function. However, the molecular mechanisms by which IL-2 delivers activation signals from the IL-2 receptor to the NK cell interior are incompletely understood. Previous studies demonstrated that IL-2 stimulation induced the tyrosine phosphorylation of multiple proteins in NK cells, together with a prominent reduction in the electrophoretic mobility of p56lck. The present studies indicate that IL-2 induces a rapid (< or = 1 min) increase in the catalytic activity of p56lck, as measured by increases in protein tyrosine kinase activity in vitro. Furthermore, in response to IL-2, p56lck itself undergoes complex alterations in serine and tyrosine phosphorylation. Cyanogen bromide cleavage maps indicate that IL-2 stimulates a pronounced increase in the phosphorylation of the NH2-terminal region of p56lck containing multiple known sites of serine phosphorylation. In addition, IL-2 induced a marked increase in the phosphorylation of a COOH-terminal peptide containing the regulatory Tyr-505 residue of p56lck. These results suggest that p56lck serves as a substrate for both protein serine and tyrosine kinases activated during stimulation of this cell type with IL-2. Furthermore, these results indicate that the pleiotropic effects of IL-2 on NK cell physiology are initiated and regulated by a complex and multitiered interaction of different protein kinases including p56lck.     

Biochemical association of CD45 with the T cell receptor complex: regulation by CD45 isoform and during T cell activation.

CD45 is the predominant transmembrane tyrosine phosphatase in lymphocytes and is required for the efficient induction of T cell receptor signaling and activation. However, the regulation of CD45 activity and substrate specificity are poorly understood. In the present study, we demonstrate a basal biochemical association of CD45 with the T cell receptor complex that is regulated in part by CD45 isoform expression. Further, maintenance of CD45/TCR association is differentially regulated following TCR ligation with peptide: a partial agonist peptide induces CD45/TCR dissociation while an agonist peptide promotes sustained association in a CD4-dependent manner. These data suggest that T cell receptor signaling pathways may be modulated by altering access of CD45 to TCR-associated substrates involved in T cell activation.     

Genetic reconstitution of the T cell receptor (TcR) alpha/beta heterodimer restores the association of CD3 zeta 2 with the TcR/CD3 complex.

The cell surface expression of the T cell receptor (TcR)/CD3 complex and, consequently, the functional competence of the cell is partly dependent on CD3 zeta. In its absence, a pentameric complex (TcR alpha/beta/CD3 gamma delta epsilon) is formed which is inefficiently transported to the cell surface. Reconstitution of CD3 zeta by transfection, in turn, restores the cell surface expression and function of the complex. Through the use of transfection experiments, we here provide direct evidence that the association of CD3 zeta 2 with the TcR/CD3 complex is dependent on the presence of both the TcR alpha and beta polypeptide chains. Despite wild-type levels of the CD3 zeta protein in a TcR alpha-negative mutant human T cell line, a complex was formed intracellularly which lacked CD3 zeta 2 and consisted of beta gamma delta epsilon and beta 2 gamma delta epsilon. Upon transfection of the mutant with a TcR alpha cDNA, a TcR/CD3 complex which contained CD3 zeta 2 was observed intracellularly. In contrast to the partial subcomplex on the cell surface of the untransfected cell line, the TcR/CD3 complex on the transfectant was functional as demonstrated by its ability to mobilize intracellular calcium after stimulation with a mitogenic CD3 epsilon-specific monoclonal antibody. Transient transfection studies performed in COS cell fibroblasts indicated that CD3 zeta 2 was not interacting with the TcR alpha protein alone, implying that a conformation provided by either the TcR alpha/beta heterodimer or the TcR alpha/beta/CD3 gamma delta epsilon complex was necessary for the association of CD3 zeta 2. Transfection studies performed in a TcR alpha/beta-negative murine T-T hybridoma confirmed the requirement of both the TcR alpha and beta proteins in CD3 zeta 2 binding. We conclude that the TcR alpha and beta chains harbor polypeptide sequences essential for the association of CD3 zeta 2 with the TcR/CD3 complex.     

Association of the p56lck protein tyrosine kinase with the FCγRIIIA/CD16 complex in human natural killer cells

The multimeric FcγRIIIA (CD16) complex is expressed on the surface of natural killer (NK) cells and is composed of a 50–70-kDa transmembrane glycoprotein Fcγ receptor (CD16), the T cell receptor (TCR)-ζ chain, and the FcεRIγ chain. Cross-linking FcγRIIIA initiates the rapid tyrosine phosphorylation of multiple substrates including the ζ, subunit and causes subsequent cell activation and antibody-dependent cellular cytotoxicity (ADCC). The subunits of the FcγRIIIA complex lack intrinsic protein tyrosine kinase (PTK) activity, suggesting that receptor-induced tyrosine phosphorylation events are mediated by a nonreceptor PTK. We report here that the human FcγRIIIA is complexed with p56^lck, a src-family PTK previously found associated with the CD4 and CD8 receptors on T cells. Upon engagement of the CD16 receptor, p56^lck is rapidly (within 30 s) and transiently phosphorylated on tyrosine residues. Several FcγRIIIA-associated proteins are identified in immune complex kinase assays including the TCR-ζ, subunit, a p70–90 ζ-associated protein (ZAP), p50a (acidic) and p50b (basic), and p56^lck. We demonstrate that the src-family protein tyrosine kinase inhibitor, herbimycin A, blocks increased intracellular calcium levels and ADCC caused by CD16 cross-linking on NK3.3 cells. Likewise cross-linking CD16 with the protein tyrosine phosphatase CD45, abrogates CD16-induced calcium mobilization. These data suggest that p56^lck is physically associated with FcγRIIIA(CD16) and functions to mediate signaling events related to the control of NK cellular cytotoxicity.     

Activation of MAP-2 kinase activity by the CD2 receptor in Jurkat T cells can be reversed by CD45 phosphatase.

We have recently characterized a serine kinase in T lymphocytes which phosphorylates microtubule-associated protein-2 (MAP-2) in vitro. This kinase is activated in a rapidly reversible fashion during ligation of CD3/Ti by a process which involves tyrosine phosphorylation of the enzyme itself. We show that the stimulatory anti-CD2 mAb combination, anti-(T11(2) + T11(3), stimulates MAP-2K activity in Jurkat cells with kinetics that are more prolonged than during anti-CD3 treatment. The principal difference is not in the rate of response induction, but in the decline of the response beyond the peak, to which end anti-CD2 stimulation resembles the sustained phytohaemagglutin (PHA) response. Parallel immunoblotting, utilizing anti-phosphotyrosine antibodies, also revealed differences in the rate at which tyrosine phosphorylation of pp43 (MAP-2K) disappears after induction. In spite of these differences, CD2 was absolutely dependent on the presence of CD3 for inducing a MAP-2K response in Jurkat cells. These results indicate that, even though CD2 and CD3 are using a common signalling pathway in Jurkat cells, additional differences such as the involvement of a tyrosine phosphatase may have to be considered in response generation. We also demonstrate that the common CD45 isoform, when cross-linked to CD2 by mAb, could inhibit the MAP-2K response during both induction as well as the disappearing phase of the response.     

Biochemical identification of a direct physical interaction between the CD4:p56lck and Ti(TcR)/CD3 complexes.

The CD4 and CD8 antigens function in synergy with the TcR/CD3 complex in the generation of intracellular signals leading to T cell proliferation. The association of the protein-tyrosine kinase p56lck with CD4 and CD8 provides a potential mechanism in the generation of intracellular signals. Several studies have shown that CD4 can co-modulate with TcR/CD3 suggesting that these receptor complexes may associated on the surface of the T cell. Nevertheless, it has proven difficult to formally demonstrate a direct physical interaction between the CD4 and TcR/CD3 complexes using biochemical techniques. In this study, we have used the sensitivity of the in vitro kinase assay to show a direct physical linkage between the CD4:p56lck complex and various CD3 subunits. Immunoprecipitation of CD4 from cell lysates derived from the T lymphoblastoid line HPB-ALL results in the co-purification of p56lck with an additional polypeptide at 20 kDa. Re-precipitation analysis and isoelectric focusing demonstrated that this band corresponds to the CD3 epsilon chain. An alternative approach which involves the labeling of microsomal membranes with [gamma-32P]ATP revealed the presence of CD3 epsilon and zeta chains in anti-CD4 immunoprecipitates. By contrast, we were unable to demonstrate the association of the CD4:p56lck and TcR/CD3 complex in resting peripheral blood lymphocytes. These data indicate that the CD4:p56lck and TcR/CD3 complexes have the ability to form stable complexes on the surface of certain T cell lines.     

Decreased TcR-CD3+ T cell numbers in healthy aged humans. Evidence that T cell defects are masked by a reciprocal increase of TcR-CD3- CD2+ natural killer cells

While there is accumulating evidence to indicate the presence of functional abnormalities in T cells from aged healthy humans, their cellular basis remains unclear. By using two-color immunofluorescence and multiparameter flow cytometry we show that (a) the number of peripheral blood antigen receptor-positive (TcR-CD3+) T cells is significantly lower in aged than in young adults; (b) the numbers of E-rosette-forming (CD2+) cells are maintained in the elderly due to a reciprocal increase in the frequency of TcR-CD3- cells, which constitute only a minor lymphocyte subpopulation in young adults, and (c) TcR-CD3-CD2+5- lymphocytes exhibit the phenotypic features of natural killer (NK) cells. By using functional assays we show the TcR-CD3-CD2+16+ lymphocytes are indeed NK cells because they are activated by and lyse NK targets. In contrast, they are unresponsive to either phytohemagglutinin or mitogenic CD2 monoclonal antibody stimulation, which in turn activates TcR-CD3+CD2+16- T cells. We conclude that the increase in TcR-CD3-CD2+ NK cells masks the T cell reduction in aged humans by normalizing CD2+ cell frequencies. However, NK cells cannot functionally substitute the thymus-derived lymphocytes they replace.     

Activation of phosphatidylinositol-3-kinase in Jurkat T cells depends on the presence of the p56lck tyrosine kinase

Activation of resting T lymphocytes by ligands to the T cell receptor (TcR)/CD3 complex is initiated by phosphorylation of a number of key regulatory proteins on specific tyrosine residues. One such protein is the heterodimeric enzyme phosphatidylinositol-3-kinase (PI3K). We recently found that this enzyme is also rapidly activated following TcR/CD3 triggering and that immunoprecipitated PI3K was activated in vitro by direct tyrosine phosphorylation. Here we show that TcR/CD3-induced tyrosine phosphorylation and activation of PI3K in Jurkat T leukemia cells depend on the presence of the p56^lck tyrosine kinase: in a variant of the Jurkat T cell line lacking p56^lck, JCaM1, these responses were absent. We also show that p56^lck directly activates PI3K purified from transfected COS-1 cells, indicating that other T cell-specific proteins are not required for the process. Finally, tryptic peptide maps show that p56^lck phosphorylates three tyrosine residues in the p85α subunit of PI3K and two in p110 of PI3K. Our results suggest that p56^lck is required for activation of PI3K in Jurkat T cells and can itself directly activate it by phosphorylating one or several stimulatory sites.     

Regulation of D-3 phosphoinositides during T cell activation via the T cell antigen receptor/CD3 complex and CD2 antigens.

An immediate consequence of T cell activation via the T cell receptor (TcR)/CD3 complex and CD2 antigen is the hydrolysis of phosphatidylinositol-(4,5)-bisphosphate and the generation of inositol-(1,4,5)-trisphosphate and diacylglycerol which then regulate intracellular calcium and protein kinase C. Changes in cellular levels of phosphoinositides phosphorylated on the D-4 and D-5 position during T cell activation have been well documented. Recently it has been proposed that phosphoinositides phosphorylated on the D-3 position of the inositol ring by a novel phosphoinositide (PI) 3 kinase may also be important in cell activation. In the present study we have examined the levels and regulation of D-3 phosphoinositides in T cells activated by the TcR/CD3 complex and CD2 antigens. The data show the existence of phosphatidylinositol-(3)-monophosphate [PtdIns(3)P], phosphatidylinositol-(3,4)-bisphosphate [PtdIns(3,4)P2] and phosphatidylinositol-(3,4,5)-trisphosphate [PtdIns(3,4,5)P3] in T cells. Activation of the TcR/CD3 complex or CD2 antigen results in modulation of PtdIns(3,4)P2 and a putative PtdIns(3,4,5)P3 in T cells but does not change levels of PtdIns(3)P. These data provide the first evidence that lipid products of a PI3 kinase exist in T cells.