Phosphatidylinositol-3-kinase p110δ serves as a central integration point for signaling from cell surface receptors known to promote malignant B-cell proliferation and survival. This provides a rationale for the development of small molecule inhibitors that selectively target p110δ as a treatment approach for patients with B-cell malignancies. We thus identified 5-fluoro-3-phenyl-2-[(S)-1-(9H-purin-6-ylamino)-propyl]-3H-quinazolin-4-one (CAL-101), a highly selective and potent p110δ small molecule inhibitor (half-maximal effective concentration [EC(50)] = 8nM). Using tumor cell lines and primary patient samples representing multiple B-cell malignancies, we have demonstrated that constitutive phosphatidylinositol-3-kinase pathway activation is p110δ-dependent. CAL-101 blocked constitutive phosphatidylinositol-3-kinase signaling, resulting in decreased phosphorylation of Akt and other downstream effectors, an increase in poly(ADP-ribose) polymerase and caspase cleavage and an induction of apoptosis. These effects have been observed across a broad range of immature and mature B-cell malignancies, thereby providing a rationale for the ongoing clinical evaluation of CAL-101.

Tyrosine residues have been identified in the human platelet‐derived growth factor (PDGF) receptor beta‐subunit whose phosphorylation is stimulated by PDGF. These sites are also in vitro autophosphorylation sites. There are a total of three phosphorylation sites in the kinase insert region, tyrosines 740, 751 and 771. Mutagenesis studies show that Tyr740 and 751 are involved in the PDGF‐stimulated binding of phosphatidylinositol (PI) 3 kinase, and Tyr771 is required for efficient binding of GAP, the GTPase activator of Ras. The requirement for Tyr751 is only detected at low PDGF receptor levels, suggesting that it increases the affinity of binding of PI3 kinase but is not absolutely required. Small deletions in the kinase insert only 10 residues from Tyr740 and Tyr771 do not significantly reduce binding of PI3 kinase or GAP, indicating that distant sequences are probably unimportant for recognition. The data suggest that the receptor signals to different pathways via different phosphorylated tyrosines, and that certain proteins, such as PI3 kinase, can recognize two phosphorylated tyrosines in a single receptor.

We have identified a novel p110 isoform of phosphatidylinositol 3-kinase from human leukocytes that we have termed p110␦. In addition, we have independently isolated p110␦ from a mouse embryo library on the basis of its ability to interact with Ha-Ras V12 in the yeast two-hybrid system. This unique isoform contains all of the conserved structural features characteristic of the p110 family. Recombinant p110␦ phosphorylates phosphatidylinositol and coimmunoprecipitates with p85. However, in contrast to previously described p110 subunits, p110␦ is expressed in a tissue-restricted fashion; it is expressed at high levels in lymphocytes and lymphoid tissues and may therefore play a role in phosphatidylinositol 3-kinase-mediated signaling in the immune system. Phosphatidylinositol (PI)1 3-kinase was originally identified as an activity associated with viral oncoproteins and growth factor receptor tyrosine kinases that phosphorylates PI and its phosphorylated derivatives at the 3Ј-hydroxyl of the inositol ring (1). The purification and subsequent molecular cloning of PI 3-kinase revealed that it is a heterodimer consisting of p85 and p110 subunits (2-5).The p85 subunit acts to localize PI 3-kinase activity to the plasma membrane by virtue of the interaction of its SH2 domain with phosphorylated tyrosine residues (present in an appropriate local sequence context) in target proteins (6). Two isoforms of p85 have been identified: p85␣, which is ubiquitously expressed, and p85␤, which is primarily found in brain and lymphoid tissues (7). The p110 subunit contains the catalytic domain of PI 3-kinase, and three isoforms of p110 have thus far been reported (␣, ␤, and ␥) (3,8,9). The identification of p110␥ revealed additional complexity within this family of enzymes. p110␥ is most closely related to p110␣ and ␤ (45-48% identity in the catalytic domain) but does not make use of p85 as a targeting subunit. p110␥ contains an additional domain termed a pleckstrin homology domain near the amino terminus. The pleckstrin homology domain allows interaction with the ␤␥ subunits of heterotrimeric G proteins that appears to regulate its activity and subcellular localization (9).Additional members of this growing gene family include more distantly related lipid and protein kinases such as Vps34, TOR1, and TOR2 of Saccharomyces cerevisiae (and their mammalian homologues such as FRAP and mTOR), the human ataxia telangiectasia gene product, and the catalytic subunit of DNA-dependent protein kinase (10).The levels of phosphatidylinositol-3,4,5-triphosphate, the primary product of PI 3-kinase activation, are elevated upon treatment of cells with a wide variety of agonists (11). This observation has implicated PI 3-kinase activation in a diverse range of cellular responses including cell growth, differentiation, and apoptosis (1, 12, 13). The downstream targets of the phosphorylated lipids generated following PI 3-kinase activation have not been well characterized. However some isoforms of protein kinase C are directly activated by phosphatidyl...

In response to binding of platelet-derived growth factor (PDGF), the PDGF receptor (PDGFR) I1 subunit is phosphorylated on tyrosine residues and associates with numerous signal transduction enzymes, including the GTPase-activating protein of ras (GAP) and phosphatidylinositol 3-kinase (P13K). Previous studies have shown that association of P13K requires phosphorylation of tyrosine 751 (Y751) in the kinase insert and that this region of receptor forms at least a portion of the binding site for P13K. In this study, the in vitro binding of GAP to the PDGFR was investigated. Like PI3K, GAP associates only with receptors that have been permitted to autophosphorylate, and GAP itself does not require tyrosine phosphate in order to stably associate with the phosphorylated PDGFR. To define which tyrosine residues are required for GAP binding, a panel of PDGFR phosphorylation site mutants was tested. Mutation of Y771 reduced the amount of GAP that associates to an undetectable level. In contrast, the F771 (phenylalanine at 771) mutant bound wild-type levels of PI3K, whereas the F740 and F751 mutants bound 3 and 23%, respectively, of the wild-type levels of PI3K but wild-type levels of GAP. The F740/F751 double mutant associated with wild-type levels of GAP, but no detectable P13K activity, while the F740/F751/F771 triple mutant could not bind either GAP or P13K. The in vitro and in vivo associations of GAP and PI3K activity to these PDGFR mutants were indistinguishable. The distinct tyrosine residue requirements suggest that GAP and P13K bind different regions of the PDGFR. This possibility was also supported by the observation that the antibody to the PDGFR kinase insert Y751 region that blocks association of P13K had only a minor effect on the in vitro binding of GAP. In addition, highly purified PI3K and GAP associated in the absence of other cellular proteins and neither cooperated nor competed with each other's binding to the PDGFR. Taken together, these studies indicate that GAP and P13K bind directly to the PDGFR and have discrete binding sites that include portions of the kinase insert domain.

(27,31). Recent studies indicate that binding of p85 to the tyrosinephosphorylated insulin receptor substrate 1 activates PI-3 kinase (2).SH2 domains are structural modules composed of approximately 100 amino acids (for reviews, see references 24, 30, and 35). SH2 domains have been identified in a wide range of molecules, which can be divided into two classes. One class contains proteins with enzymatic or other known functional elements and includes cytoplasmic tyrosine kinases such as the src product, PLC-y, GAP, protein tyrosine phosphatase, the transcription factor interferon-stimulated gene factor 3 (ISGF-3), and the product of the putative GDP-GTP exchanger vav (for reviews, see references 24, 30, and 35 (5,20,23,34,39).Nck is a ubiquitously expressed protein consisting of one SH2 and three SH3 domains (19). Nck is phosphorylated on serine residues in quiescent cells and on serine, threonine, and tyrosine residues in response to a variety of extracellular stimuli, including EGF, PDGF, and nerve growth factor 6889