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 alpha-isoform (PI3K-C2 alpha), the 5'-phosphatase synaptojanin1 (Synj1), and the 4'-phosphatase INPP4B, but not PI3K-C2 beta, Synj2, or INPP4A, were required for TGF beta-induced endocytosis of TGF beta receptor. TGF beta 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 TGF beta receptor kinase ALK5-dependent manner. TGF beta induced the recruitment of both synaptojanin1 and PI3K-C2 alpha to the PM with their substantial colocalization. Knockdown of synaptojanin1 abolished TGF beta-induced PI(4,5)P-2 decreases and PI(4)P increases. Interestingly, PI3K-C2 alpha KD abolished not only TGF beta-induced PI(3,4)P-2 increases but also TGF beta-induced synaptojanin1 recruitment to the PM, PI(4,5)P-2 decreases, and PI(4)P increases. Finally, the phosphoinositide conversions were necessary for TGF beta-induced activation of Smad2 and Smad3. These observations demonstrate that the sequential phosphoinositide conversions mediated by Synj1, PI3K-C2 alpha, and INPP4B are essential for TGF beta receptor endocytosis and its signaling.