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
