TGFβ receptor endocytosis and Smad signaling require synaptojanin1, PI3K–C2α-, and INPP4B-mediated phosphoinositide conversions

Phosphoinositide conversion regulates a diverse array of dynamic membrane events including endocytosis. However, it is not well understood which enzymes are involved in phosphoinositide conversions for receptor endocytosis. We found by small interfering RNA (siRNA)-mediated knockdown (KD) that class II PI3K α-isoform (PI3K-C2α), the 5′-phosphatase synaptojanin1 (Synj1), and the 4′-phosphatase INPP4B, but not PI3K-C2β, Synj2, or INPP4A, were required for TGFβ-induced endocytosis of TGFβ receptor. TGFβ induced rapid decreases in PI(4,5)P(2) at the plasma membrane (PM) with increases in PI(4)P, followed by increases in PI(3,4)P(2), in a TGFβ receptor kinase ALK5-dependent manner. TGFβ induced the recruitment of both synaptojanin1 and PI3K-C2α to the PM with their substantial colocalization. Knockdown of synaptojanin1 abolished TGFβ-induced PI(4,5)P(2) decreases and PI(4)P increases. Interestingly, PI3K-C2α KD abolished not only TGFβ-induced PI(3,4)P(2) increases but also TGFβ-induced synaptojanin1 recruitment to the PM, PI(4,5)P(2) decreases, and PI(4)P increases. Finally, the phosphoinositide conversions were necessary for TGFβ-induced activation of Smad2 and Smad3. These observations demonstrate that the sequential phosphoinositide conversions mediated by Synj1, PI3K-C2α, and INPP4B are essential for TGFβ receptor endocytosis and its signaling.