Pleckstrin homology (PH) domains may act as membrane localization modules through specific interactions with phosphoinositide phospholipids. These interactions could represent responses to second messengers, with scope for regulation by soluble inositol polyphosphates. A biosensor‐based assay was used here to probe interactions between PH domains and unilamellar liposomes containing different phospholipids and to demonstrate specificity for distinct phosphoinositides. The dynamin PH domain specifically interacted with liposomes containing phosphatidylinositol‐4,5‐bisphosphate [PI(4,5)P2] and, more weakly, with liposomes containing phosphatidylinositol‐4‐phosphate [PI(4)P]. This correlates with phosphoinositide activation of the dynamin GTPase. The functional GTPase of a dynamin mutant lacking the PH domain, however, cannot be activated by PI(4,5)P2. The phosphoinositide‐PH domain interaction can be abolished selectively by point mutations in the putative binding pocket predicted by molecular modelling and NMR spectroscopy. In contrast, the Bruton's tyrosine kinase (Btk)PH domain specifically bound liposomes containing phosphatidylinositol‐3,4,5‐trisphosphate [PI(3,4,5)P3]: an interaction requiring Arg28, a residue found to be mutated in some X‐linked agammaglobulinaemia patients. A rational explanation for these different specificities is proposed through modelling of candidate binding pockets and is supported by NMR spectroscopy.

We have reported previously that Ras interacts with the catalytic subunit of phosphoinositide 3‐kinase (PI 3‐kinase) in a GTP‐dependent manner. The affinity of the interaction of Ras‐GTP with p85alpha/p110alpha is shown here to be approximately 150 nM. The site of interaction on the p110alpha and beta isoforms of PI 3‐kinase lies between amino acid residues 133 and 314. A point mutation in this region, K227E, blocks the GTP‐dependent interaction of PI 3‐kinase p110alpha with Ras in vitro and the ability of Ras to activate PI 3‐kinase in intact cells. In addition, this mutation elevates the basal activity of PI 3‐kinase in intact cells, suggesting a direct influence of the Ras binding site on the catalytic activity of PI 3‐kinase. Using an in vitro reconstitution assay, it is shown that the interaction of Ras‐GTP, but not Ras‐GDP, with PI 3‐kinase leads to an increase in its enzymatic activity. This stimulation is synergistic with the effect of tyrosine phosphopeptide binding to p85, particularly at suboptimal peptide concentrations. These data show that PI 3‐kinase is regulated by a number of mechanisms, and that Ras contributes to the activation of this lipid kinase synergistically with tyrosine kinases.

Extensive evidence implicates activation of the lipid phosphatidylinositide 3-kinase (PI3K) pathway in the genesis and progression of various human cancers. PI3K inhibitors thus have considerable potential as molecular cancer therapeutics. Here, we detail the pharmacologic properties of a prototype of a new series of inhibitors of class I PI3K. PI103 is a potent inhibitor with low IC 50 values against recombinant PI3K isoforms p110A (2 nmol/L), p110B (3 nmol/L), p110D (3 nmol/L), and p110; (15 nmol/L). PI103 also inhibited TORC1 by 83.9% at 0.5 Mmol/L and exhibited an IC 50 of 14 nmol/L against DNA-PK. A high degree of selectivity for the PI3K family was shown by the lack of activity of PI103 in a panel of 70 protein kinases. PI103 potently inhibited proliferation and invasion of a wide variety of human cancer cells in vitro and showed biomarker modulation consistent with inhibition of PI3K signaling. PI103 was extensively metabolized, but distributed rapidly to tissues and tumors. This resulted in tumor growth delay in eight different human cancer xenograft models with various PI3K pathway abnormalities. Decreased phosphorylation of AKT was observed in U87MG gliomas, consistent with drug levels achieved. We also showed inhibition of invasion in orthotopic breast and ovarian cancer xenograft models and obtained evidence that PI103 has antiangiogenic potential. Despite its rapid in vivo metabolism, PI103 is a valuable tool compound for exploring the biological function of class I PI3K and importantly represents a lead for further optimization of this novel class of targeted molecular cancer therapeutic. [Cancer Res 2007;67(12):5840-50]

Efficient binding of active phosphatidylinositol (PI) 3′‐kinase to the autophosphorylated macrophage colony stimulating factor receptor (CSF‐1R) requires the noncatalytic kinase insert (KI) region of the receptor. To test whether this region could function independently to bind PI 3′‐kinase, the isolated CSF‐1R KI was expressed in Escherichia coli, and was inducibly phosphorylated on tyrosine. The tyrosine phosphorylated form of the CSF‐1R KI bound PI 3′‐kinase in vitro, whereas the unphosphorylated form had no binding activity. The p85 alpha subunit of PI 3′‐kinase contains two Src homology (SH)2 domains, which are implicated in the interactions of signalling proteins with activated receptors. Bacterially expressed p85 alpha SH2 domains complexed in vitro with the tyrosine phosphorylated CSF‐1R KI. Binding of the CSF‐1R KI to PI 3′‐kinase activity, and to the p85 alpha SH2 domains, required phosphorylation of Tyr721 within the KI domain, but was independent of phosphorylation at Tyr697 and Tyr706. Tyr721 was also critical for the association of activated CSF‐1R with PI 3′‐kinase in mammalian cells. Complex formation between the CSF‐1R and PI 3′‐kinase can therefore be reconstructed in vitro in a specific interaction involving the phosphorylated receptor KI and the SH2 domains of p85 alpha.