Phosphatidylinositol 3,5-bisphosphate defines a novel PI 3-kinase pathway in resting mouse fibroblasts.

PtdIns(3,5)P2 is identified as the product of an agonist-independent, wortmannin-sensitive pathway in resting mouse cells. Results are presented here to indicate that PtdIns(3,5)P2 is formed by phosphorylation of PtdIns3P at the D-5 position, and they suggest that relatively constant cellular levels of PtdIns3P and PtdIns(3, 5)P2 are maintained by the concerted action of PtdIns3P 5-kinase and PtdIns(3,5)P2 5-phosphatase. These studies imply a novel mechanism for the action of PtdIns-specific phosphoinositide 3-hydroxykinases in mammalian cells.

Platelet-derived growth factor-dependent cellular transformation requires either phospholipase Cgamma or phosphatidylinositol 3 kinase.

Although it has been well established that constitutive activation of receptor tyrosine kinases leads to cellular transformation, the signal relay pathways involved have not been systematically investigated. In this study we used a panel of platelet-derived growth factor (PDGF) beta receptor mutants (beta-PDGFR), which selectively activate various signal relay enzymes to define which signaling pathways are required for PDGF-dependent growth of cells in soft agar. The host cell line for these studies was Ph cells, a 3T3-like cell that expresses normal levels of the beta-PDGFR but no PDGF-alpha receptor (alpha-PDGFR). Hence, this cell system can be used to study signaling of mutant alphaPDGFRs or alpha/beta chimeras. We constructed chimeric receptors containing the alphaPDGFR extracellular domain and the betaPDGFR cytoplasmic domain harboring various phosphorylation site mutations. The mutants were expressed in Ph cells, and their ability to drive PDGF-dependent cellular transformation (growth in soft agar) was assayed. Cells infected with an empty expression vector failed to grow in soft agar, whereas introduction of the chimera with a wild-type beta-PDGFR cytoplasmic domain gave rise to a large number of colonies. In contrast, the N2F5 chimera, in which the binding sites for phospholipase Cgamma (PLC-gamma), RasGTPase-activating protein, phosphatidylinositol 3 kinase (PI3K), and SHP-2 were eliminated, failed to trigger proliferation. Restoring the binding sites for RasGTPase-activating protein or SHP-2 did not rescue the PDGF-dependent response. In contrast, receptors capable of associating with either PLC-gamma or PI3K relayed a growth signal that was comparable to wild-type receptors in the soft agar growth assay. These findings indicate that the PDGF receptor activates multiple signaling pathways that lead to cellular transformation, and that either PI3K or PLC-gamma are key initiators of such signal relay cascades.

Wortmannin, a phosphatidylinositol 3-kinase inhibitor, blocks the assembly of peptide-MHC class II complexes.

Peptide-class II complexes are assembled in endocytic, lysosome-like compartments where newly synthesized class II molecules are targeted from the trans-Golgi network (TGN). Recent studies have implicated phosphatidylinositol 3-kinase (PI3-kinase) as an essential component in membrane trafficking from the TGN to lysosomes. Here, using subcellular fractionation, we show PI3-kinase activity associated with subcellular fractions which contain the class II peptide-loading compartment (IIPLC) in B cells. At concentrations required for inhibition of PI3-kinase activity in vivo, wortmannin blocked the processing and presentation of antigen by B cells to T cells. Treatment of B cells with wortmannin significantly limited the proteolytic degradation of invariant chain and the formation of peptide-class II complexes. Subcellular fractionation coupled with pulse-chase analyses showed that invariant chain and class II molecules trafficked to the IIPLC in wortmannin-treated cells. However, wortmannin prevented the maturation and correct targeting to the IIPLC of cathepsin D, a protease necessary for the degradation of invariant chain and assembly of processed antigen-class II complexes. These results suggest that li-class II complexes traffic to the IIPLC via a pathway that is relatively insensitive to wortmannin, but suggest a role for PI3-kinases in the trafficking of other components necessary for the assembly of processed antigen class II complexes to the IIPLC.

D-3 phosphoinositide metabolism in cells treated with platelet-derived growth factor.

Despite extensive analysis of phosphoinositide 3-hydroxykinases (PI 3-kinases) at the molecular level, comparatively little is known about the mechanisms by which products of these enzymes exert their expected second-messenger functions. This study examines the metabolism of D-3 phosphoinositides in mouse Ph-N2 fibroblasts lacking the platelet-derived growth factor (PDGF) alpha-receptor. Treatment of these cultures with BB PDGF, but not AA PDGF, resulted in transient activation of PI 3-kinase activity measured in vitro. Treatment of myo-[3H]inositol-labelled Ph-N2 cells with BB PDGF resulted in the rapid induction of PtdIns(3,4)P2 and PtdIns(3,4,5)P3 and, to a smaller extent, PtdIns3P. The appearance of PtdIns(3,4,5)P3 preceded that of PtdIns(3,4)P2 and PtdIns3P after the addition of PDGF, suggesting that PtdIns(4,5)P2 is the preferred substrate of the agoniststimulated PI 3-kinase in intact cells. Treatment of both resting and PDGF-stimulated cells with the fungal metabolite wortmannin resulted in pronounced, selective effects on the levels of all D-3 phosphoinositides. Kinetic studies with this PI 3-kinase inhibitor revealed the presence of at least two independent routes for the biosynthesis of D-3 phosphoinositides in PDGF-treated cells.