PI3K is a heterodimer of p110 catalytic and p85 adaptor subunits that is activated by agonist-stimulated receptor tyrosine kinases. Although p85 recruits p110 to activated receptors on membranes, p85 loss, which occurs commonly in cancer, paradoxically promotes agonist-stimulated PI3K/Akt signaling. p110 localizes to microtubules via MAP4, facilitating its interaction with activated receptor kinases on endosomes to initiate PI3K/Akt signaling. Here, we demonstrate that in response to agonist stimulation and p85 knock down, the residual p110 coupled predominantly to p85 exhibits enhanced recruitment with receptor tyrosine kinases to endosomes. Moreover, the p110 C2 domain binds PI3P and this interaction is also required to recruit p110 to endosomes and for PI3K/Akt signaling. Stable knockdown of p85, which mimics the reduced p85levels observed in cancer, enhances cell growth and tumorsphere formation, and these effects are abrogated by MAP4 or p85knockdown, underscoring their role in the tumor-promoting activity of p85loss.