Ubiquitin-like protein 4A (Ubl4A) was identified as a housekeeping gene at X chromosome. It involves in the guided entry of tail-anchored (GET... Show moreUbiquitin-like protein 4A (Ubl4A) was identified as a housekeeping gene at X chromosome. It involves in the guided entry of tail-anchored (GET) protein pathway in which tail-anchored (TA) proteins are transported to endoplasmic reticulum (ER). However, Ubl4A also involves other functions not related to GET pathway, such as tumor suppression and DNA damage-mediated apoptosis. Up to date, the function of Ubl4A in mammals is still largely unknown. We found that either overexpression or knockdown of Ubl4A promoted cell death in cell culture system. Using an in vivo genetic knockout system, we found that Ubl4A knockout mice displayed a high neonatal mortality and had a defect in glycogen synthesis, which is mainly controlled by a key protein kinase Akt. Loss of Ubl4A resulted in the impairment of insulin-induced Akt translocation to the plasma membrane, an essential step for Akt activation. We demonstrated that Ubl4A directly interacted with actin-related protein 2/3 (Arp2/3) complex, further accelerated Arp2/3 complex-dependent actin branching, thereby bringing Akt to proximity into the plasma membrane for activation. Furthermore, we showed that Ubl4A-mediated actin branching also played important roles in other cellular activities, such as formations oflamellipodia and filopodia, macrophage phagocytosis, wound healing, and neutrophil chemotaxis. These findings provide us a new insight into understanding the roles of Ubl4A in cellular function and a molecular basis for treatment of related human diseases. Ph.D. in Biology, December 2015 Show less
Ubl4A is a member of the ubiquitin-like protein family with multiple functions, such as pro-survival and anti-tumor. Our group previously... Show moreUbl4A is a member of the ubiquitin-like protein family with multiple functions, such as pro-survival and anti-tumor. Our group previously found that Ubl4A directly interacts with actin-related protein Arp2/3 complex to promote Arp2/3-dependent actin “Y shape” branching formation. However, the binding region of Ubl4A for Arp2/3 still remains unknown. To address this question, we generated several Ubl4A mutant and truncated constructs, and cloned them into a GST vector in which GST was fused in-frame with Ubl4A at the N-terminus. We used Glutathione-beads to purify GST fusion proteins and performed pull-down assay with purified Arp2/3 complex. We show that C-terminus of Ubl4A is the region where the Arp2/3 complex interacts with. A single point mutation (D122A) in Ubl4A C-terminal “DYD” motif can abolish Ubl4A ability to bind Arp2/3. These results indicate that C-terminus, especially D122, is critical for Ubl4A association with Arp2/3. Show less
