In vivo binding properties of SH2 domains from GTPase-activating protein and phosphatidylinositol 3-kinase.

We have used a transient expression system and mutant platelet-derived growth factor (PDGF) receptors to study the binding specificities of the Src homology 2 (SH2) regions of the Ras GTPase-activator protein (GAP) and the p85 alpha subunit of phosphatidylinositol 3-kinase (PI3 kinase). A number of fusion proteins, each tagged with an epitope allowing recognition by a monoclonal antibody, were expressed at levels comparable to those of endogenous GAP. Fusion proteins containing the central SH2-SH3-SH2 region of GAP or the C-terminal region of p85 alpha, which includes two SH2 domains, bound to PDGF receptors in response to PDGF stimulation. Both fusion proteins showed the same requirements for tyrosine phosphorylation sites in the PDGF receptor as the full-length proteins from which they were derived, i.e., binding of the GAP fusion protein was reduced by mutation of Tyr-771, and binding of the p85 fusion protein was reduced by mutation of Tyr-740, Tyr-751, or both residues. Fusion proteins containing single SH2 domains from either GAP or p85 alpha did not bind detectably to PDGF receptors in this system, suggesting that two SH2 domains in a single polypeptide cooperate to raise the affinity of binding. The sequence specificities of individual SH2 domains were deduced from the binding properties of fusion proteins containing one SH2 domain from GAP and another from p85. The results suggest that the C-terminal GAP SH2 domain specifies binding to Tyr-771, the C-terminal p85 alpha SH2 domain binds to either Tyr-740 or Tyr-751, and each protein's N-terminal SH2 domain binds to unidentified phosphorylation sites.(ABSTRACT TRUNCATED AT 250 WORDS)</jats:p

Phosphorylation sites at the C-terminus of the platelet-derived growth factor receptor bind phospholipase C gamma 1.

We have identified two tyrosine phosphorylation sites, Tyr 1009 and Tyr 1021, in the C-terminal noncatalytic region of the human platelet-derived growth factor (PDGF) receptor beta subunit. Mutant receptors with phenylalanine substitutions at either or both of these tyrosines were expressed in dog epithelial cells. Mutation of Tyr 1021 markedly reduced the PDGF-stimulated binding of phospholipase C (PLC) gamma 1 but had no effect on binding of the GTPase activator protein of Ras or of phosphatidylinositol 3 kinase. Mutation of Tyr 1009 reduced binding of PLC gamma 1 less severely. Mutation of Tyr 1021, or both Tyr 1009 and Tyr 1021, also reduced the PDGF-dependent binding of a transiently expressed fusion protein containing the two Src-homology 2 domains from PLC gamma 1. Mutation of Tyr 1021, or both Tyr 1009 and Tyr 1021, greatly reduced PDGF-stimulated tyrosine phosphorylation of PLC gamma 1 but did not prevent the tyrosine phosphorylation of other cell proteins, including mitogen-activated protein kinase. We conclude that Tyr 1021, and possibly Tyr 1009, is a binding site for PLC gamma 1

GTPase-activating protein and phosphatidylinositol 3-kinase bind to distinct regions of the platelet-derived growth factor receptor beta subunit.

In response to binding of platelet-derived growth factor (PDGF), the PDGF receptor (PDGFR) beta 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 (PI3K). Previous studies have shown that association of PI3K 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 PI3K. 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 PI3K activity, while the F740/F751/F771 triple mutant could not bind either GAP or PI3K. 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 PI3K 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 PI3K 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 PI3K bind directly to the PDGFR and have discrete binding sites that include portions of the kinase insert domain

CAL-101, a Potent Selective Inhibitor of the p110δ Isoform of Phosphatidylinositol 3-kinase, Attenuates Pathway Signaling, Induces Apoptosis, and Overcomes Signals From the Microenvironment In Cellular Models of Hodgkin Lymphoma

Abstract Abstract 3926 Phosphatidylinositide 3-kinases (PI3Ks) are a family of lipid kinases that are involved in signaling events which control a diverse number of cellular processes. The class I kinases contain 4 isoforms designated p110α, β, δ, γ, and are activated by cell surface receptors. Aberrant regulation of the PI3K signaling pathway is frequently observed in human malignancies including those of hematological origin. CAL-101 is an oral p110δ-specific inhibitor which has shown preclinical and clinical activity in non-Hodgkin lymphoma (NHL) and chronic lymphocytic leukemia (CLL). This compound is a potent p110δ inhibitor (EC50 of 65 nM in a whole-blood assay) with &gt;200-fold selectivity over the other class I PI3K isoforms and no activity against Class II and III PI3K family members or other PI3K-related proteins, including mTOR and DNA-PK. Prior in vitro NHL studies revealed that CAL-101 induces caspase-dependent apoptosis, and inhibits CD40L-, BAFF-, CXCL12- and CXCL13-derived survival signals in cellular models (Lannutti BJ, et al., Blood 2010). To investigate the potential role of p110δ in Hodgkin lymphoma (HL) we screened a number of HL cell lines for p110δ isoform expression and constitutive PI3K pathway activation. We report high levels of p110δ protein and activated Akt in 5 of 5 HL cell lines evaluated (L428, L540, L591, L1236, KM-H2). Inhibition of p110δ with CAL-101 treatment of cell lines resulted in a reduction of Akt phosphorylation and a decrease in cellular viability. Because previous studies have established the importance of signals from the microenvironment for the survival and proliferation of malignant cells as well as for their resistance to standard therapies, we investigated the effect of p110δ inhibition by CAL-101 in HL cell line-stroma cocultures. In this setting, CAL-101 overcame tumor cell growth induced by coculture of HL cells with bone marrow stromal cells. In addition, CAL-101 induced dose-dependent apoptosis of HL cells at 48 hours. Furthermore, stromal cell coculture resulted in increased CCL5, CCL17, and CCL22 levels; productions of these chemokines by HL cells cultured in the presence of stromal cells were reduced by CAL-101 in a dose-dependent manner. These results indicate that specific inhibition of p110δ may disrupt signals between HL cells and their microenvironment, thereby providing the preclinical rationale for clinical evaluation of CAL-101 as a novel therapeutic approach in patients with Hodgkin lymphoma. Disclosures: Meadows: Calistoga Pharmaceuticals: Employment. Kashishian:Calistoga Pharmaceuticals: Employment. Johnson:Calistoga Pharmaceuticals: Employment. Lannutti:Calistoga Pharmaceutical Inc.: Employment. </jats:sec