Transferrin-induced signaling through transferrin receptor and AKT kinase mediates formation of Rab8- and MICAL-L1-positive tubules involved in receptor recycling

Transferrin and its receptor play an important role in iron homeostasis. Binding of transferrin to its receptor (TFRC, transferrin receptor protein 1) initiates endocytic trafficking and subsequent recycling of TFRC to the plasma membrane. RAB8-positive tubules emanating from the endocytic recycling compartment play an important role in receptor recycling. However, the signaling pathways or mechanisms that mediate formation of RAB8-positive tubules are not clear. Here, we have investigated the role of transferrin-induced signaling in the regulation of RAB8- and MICAL-L1-positive tubules. Addition of transferrin to the serum starved HeLa cells resulted in enhanced recruitment of RAB8 as well as MICAL-L1 to the tubules, which was mediated by TFRC. Dynasore, an inhibitor of dynamin and endocytosis, completely blocked transferrin-induced formation of RAB8/MICAL-L1-positive tubules. RAB8 showed strong colocalization with MICAL-L1 on the tubules. Blocking of SRC or AKT kinase activity by specific inhibitors abolished transferrin-induced recruitment of RAB8 and MICAL-L1 to the tubules. Recycling of transferrin receptor was inhibited by blocking of AKT activity. TBC1D17, a GTPase activating protein for RAB8, inhibited RAB8/MICAL-L1-positive tubule formation. A phospho-mimicking mutant S366D of TBC1D17 did not inhibit formation of RAB8-positive tubules. Overall, these results show that transferrin induces TFRC mediated signaling dependent on endocytosis that is essential for the formation of RAB8- and MICAL-L1-positive tubules involved in recycling of transferrin receptor. Our results also show that AKT regulates transferrin-induced formation of RAB8- and MICAL-L1-positive tubules, which might be mediated by phosphorylation of TBC1D17.