The generation of phosphatidylinositide 3-phosphates has been observed in a variety of cellular responses. The enzymes that mediate synthesis are the phosphoinositide 3-kinases (PI3-Ks) that form a family of structurally diverse enzymes with distinct substrate specificities. In this paper, we describe the cloning of a novel human PI3-K, namely PI3-K-C2 alpha, which contains a C-terminal C2 domain. This enzyme can be assigned to the class II PI3-Ks, which was defined by characterization of the Drosophila 68D enzyme and includes the recently described murine enzymes m-cpk and p170. Despite the overall similarity in the amino acid sequence of the murine and human enzymes, which suggests that they are encoded by closely related genes, these molecules show marked sequence heterogeneity at their N-termini. Biochemical analysis of recombinant PI3-K-C2 alpha demonstrates a restricted lipid substrate specificity. As reported for other members of this class, the enzyme only phosphorylates PtdIns and PtdIns4P when the lipids are presented alone. However, when lipids were presented together with phosphatidylserine acting as a carrier, phosphorylation of PtdIns(4,5)P2 was also observed. The catalytic activity of PI3-K-C2 alpha is refractory to concentrations of wortmannin and LY294002 which inhibit the PI3-K activity of other family members. The comparative insensitivity of PI3-K-C2 alpha to these inhibitors suggests that their use should be reevaluated in the study of PI3-Ks.

not shown). Consistent with previous results I I ,Jak-3 is expressed in breast-tis sue-derived ceIl lines (K.S.L. and E.T.L., unpublished observations). To assess Jak-3 in sign aIling, cytokine-induced tyrosine phosphorylation was examined. Phosphorylation was not seen with erythropoietin, IL-3, granulocyte macrophage colony-stimulating factor (GM-CSF), G-CSF, interferon-a (IFN-a), IFN-y or IL-6 (data not shown), but was seen in IL-2-or IL-4-stimulated cells. In CTLL ceIls, IL-2 and IL-4 induced tyrosine phosphorylation of several proteins (Fig. 4), including proteins of 120K and 130K, as recently shown 16. IL-2 and IL-4 induced tyrosine phosphorylation of Jak-3 (aJak-3), which co migrated with the major l20K substrate, and Jak-l (aJak-l), which comigrated with the 130K substrate. No Jak-2 or Tyk-2 phosphorylation was detected (data not shown). Last, the Jak-3/Jak-l-crossreactive Tyk-2 antiserum immunoprecipitated proteins that comigrated with Jak-l and Jak-3 but not Tyk-2. Phosphorylation was detectable within 1 min, peaked at 20-30 min and subsequently declined (data not shown). Cytokine-induced tyrosine phosphorylation of Jaks activates their in vitro kinase activityl.2,4,7,9. Therefore, we examined the effects of IL-2 or IL-4 on kinase activity. No kinase activity was detected in immunoprecipitates of Jak-l or immunoprecipitates of Jak-3 obtained with the Jak-3-specific antiserum (data not shown). However, the Jak-3 antiserum is against a peptide containing the autophosphorylation site (KDYY; single-letter amino-acid code) and could interfere with activity. Therefore immunoprecipitates obtained with the Jak-l/Jak-3 crossreactive antiserum were examined. With these, IL-2-and IL-4-induced in vitro kinase activity was readily detected (Fig. 4b) and the single phosphorylated protein comigrated with Jak-3. No detectable Jak-l phoshorylation was seen. Amino-acid analysis of phosphorylated Jak-3 detected exclusively phosphotyrosine (data not shown). To assess the specificity of Jak-3 activation, we examined CTLL cells expressing the erythropoietin receptor, which shares homology in the cytoplasmic domain with the IL-2 receptor [3-chain17. Transfected CTLL ceIls express levels of high-affinity erythropoietin receptors comparable to transfected myeloid ceIls (O.M. and J.N.J, unpublished data). Erythropoietin, but neither IL-2 nor IL-4, induced tyrosine phosphorylation of Jak-2 (Fig. 4c). Conversely, IL-2, but not erythropoietin, induced phosphorylation of Jak-3. The IL-2 receptor consists of a-, [3-and y-chains IR • The ychain is also used in the IL-4 receptor l9 ,20 and is essential for function. The [3-chain is also essential and contains two functional domains. An acidic domain is required for association and activation of p56 lck , p59" y n or p53/561yn18 but is dispensable for mitogenesis, In contrast, the membrane-proximal, serine-rich domain is required for mitogenesis. To assess their role in Jak-3 activation, the wild-type and receptors lacking the acidic (Amutant) or serine-rich (S-mutant) domains were intro...

The engagement of CD28 with its ligand B7.1/CD80 results in potent costimulation of T-cell activation initiated through the CD3/T-cell receptor complex. The biochemical basis of CD28 costimulatory function is poorly understood. The signalling pathways used by CD28 are unlike those used by the CD3/T-cell receptor in that they are resistant to cyclosporin A and independent of changes in cyclic AMP concentrations. These differences suggest that each pathway provides unique biochemical information which is required for T-cell activation. We report here that CD28 becomes tyrosine-phosphorylated following interaction with B7.1/CD80, which induces formation of a complex with phosphatidylinositol-3-OH kinase, mediated by the SH2 domains of the p85 subunit of the kinase. Phosphatidylinositol-3-OH kinase is a heterodimer of this 85K regulatory subunit and a 110K catalytic subunit, and is a common substrate for most receptor tyrosine kinases and some cytokine receptors, binding through its SH2 domain to phosphotyrosine in the motif Tyr-X-X-Met in the CD28 sequence, which is highly conserved between human, mouse and rat and lies in the intracellular domain. We show that CD28 mutants that have their kinase-binding site deleted or the tyrosine at position 173 substituted by phenylalanine do not associate with the kinase after CD28 stimulation and cannot stimulate production of interleukin-2. Our results suggest that phosphatidylinositol-3-OH kinase is critical for signalling by CD28.

Through the interaction with its ligands, CD80/B7-1 and CD86/B7-2 or B70, the human CD28 molecule plays a major functional role as a costimulator of T cells along with the CD3-TcR complex. We and others have previously reported that phosphatidylinositol 3-kinase inducibly associates with CD28. This association is mediated by the SH2 domains of the p85 adaptor subunit interacting with a cytoplasmic YMNM consensus motif present in CD28 at position 173-176. Disruption of this binding site by site-directed mutagenesis abolishes CD28-induced activation events in a murine T-cell hybridoma transfected with human CD28 gene. Here we show that the last 10 residues of the intracytoplasmic domain of CD28 (residues 193-202) are required for its costimulatory function. These residues are involved in interleukin-2 secretion, p85 binding, and CD28-associated phosphatidylinositol 3-kinase activity. In contrast, the CD28/CD8O interaction is unaffected by this deletion, as is the induction of other second messengers such as the rise in intracellular calcium and tyrosine phosphorylation of CD28-specific substrates. Furthermore, we also demonstrate that, within these residues, the tyrosine at position 200 is involved in p85 binding, probably together with the short proline-rich motif present between residues 190 and 194 (PYAPP).

Phosphoinositide 3-kinases (PI3-kinases) have been shown to be recruited to cell surface receptor signal complexes whose formation is triggered by growth factors, cytokines and other ligands. PI3-kinases are also involved in protein sorting phenomena. A number of PI3-kinase isotypes have been characterised in several laboratories. Here the relations between the PI3-kinases, PI4-kinases and PI5-kinases and other potential phosphoinositide kinases are analysed. A study of the relation of structure to function for sequence motifs defined through the use of homology searches and protein modelling techniques is described and used to assign the family of phosphoinositide kinases to subgroups.