SummaryMultiple phosphatidylinositol (PtdIns) 3-kinases (PI3Ks) can produce PtdIns3P to control endocytic trafficking, but whether enzyme specialization occurs in defined subcellular locations is unclear. Here, we report that PI3K-C2α is enriched in the pericentriolar recycling endocytic compartment (PRE) at the base of the primary cilium, where it regulates production of a specific pool of PtdIns3P. Loss of PI3K-C2α-derived PtdIns3P leads to mislocalization of PRE markers such as TfR and Rab11, reduces Rab11 activation, and blocks accumulation of Rab8 at the primary cilium. These changes in turn cause defects in primary cilium elongation, Smo ciliary translocation, and Sonic Hedgehog (Shh) signaling and ultimately impair embryonic development. Selective reconstitution of PtdIns3P levels in cells lacking PI3K-C2α rescues Rab11 activation, primary cilium length, and Shh pathway induction. Thus, PI3K-C2α regulates the formation of a PtdIns3P pool at the PRE required for Rab11 and Shh pathway activation.

Blockade of PI3Kγ may provide a dual therapeutic advantage in cancer therapy by simultaneously preventing anthracyclines cardiotoxicity and reducing tumor growth.

Proper organization of the mitotic spindle is key to genetic stability, but molecular components of inter-microtubule bridges that crosslink kinetochore fibers (K-fibers) are still largely unknown. Here we identify a kinase-independent function of class II phosphoinositide 3-OH kinase α (PI3K-C2α) acting as limiting scaffold protein organizing clathrin and TACC3 complex crosslinking K-fibers. Downregulation of PI3K-C2α causes spindle alterations, delayed anaphase onset, and aneuploidy, indicating that PI3K-C2α expression is required for genomic stability. Reduced abundance of PI3K-C2α in breast cancer models initially impairs tumor growth but later leads to the convergent evolution of fast-growing clones with mitotic checkpoint defects. As a consequence of altered spindle, loss of PI3K-C2α increases sensitivity to taxane-based therapy in pre-clinical models and in neoadjuvant settings.

Directional transport of recycling cargo from early endosomes (EE) to the endocytic recycling compartment (ERC) relies on phosphatidylinositol 3-phosphate (PtdIns(3)P) hydrolysis and activation of the small GTPase Rab11. However, how these events are coordinated is yet unclear. By using a novel genetically-encoded FRET biosensor for Rab11, we report that generation of endosomal PtdIns(3)P by the clathrin-binding phosphoinositide 3-kinase class 2 alpha (PI3K-C2α) controls the activation of Rab11. Active Rab11, in turn, prompts the recruitment of the phosphatidylinositol 3-phosphatase myotubularin 1 (MTM1), eventually enabling the release of recycling cargo from the EE and its delivery toward the ERC. Our findings thus define that delivery of recycling cargo toward the ERC requires spatial and sequential coupling of Rab11 activity with PtdIns(3)P turnover.

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