Human ARL4A blocking peptide

Cell migration requires the coordination of multiple signaling pathways involved in membrane dynamics and cytoskeletal rearrangement. The Arf-like small GTPase Arl4A has been shown to modulate actin cytoskeleton remodeling. However, evidence of the function of Arl4A in cell migration is insufficient. Here, we report that Arl4A acts with the serine/threonine protein kinase Pak1 to modulate cell migration through their cooperative recruitment to the plasma membrane. We first observed that Arl4A and its isoform Arl4D interact with Pak1 and Pak2 and showed that Arl4A recruits Pak1 and Pak2 to the plasma membrane. The fibronectin-induced Pak1 localization at the plasma membrane is reduced in Arl4A-depleted cells. Unexpectedly, we found that Pak1, but not Arl4A-binding-defective Pak1, can recruit a cytoplasmic myristoylation-deficient Arl4A-G2A mutant to the plasma membrane. Furthermore, we found that the Arl4A-Pak1 interaction, which is independent of Rac1 binding to Pak1, is required for Arl4A-induced cell migration. Thus, we infer that there is feedback regulation between Arl4A and Pak1, in which they mutually recruit each other to the plasma membrane for Pak1 activation, thereby modulating cell migration through direct interaction.

The prototypical DOCK protein, DOCK180, is an evolutionarily conserved Rac regulator and is indispensable during processes such as cell migration and myoblast fusion. The biological activity of DOCK180 is tightly linked to its binding partner ELMO. We previously reported that autoinhibited ELMO proteins regulate signaling from this pathway. One mechanism to activate the ELMO-DOCK180 complex appears to be the recruitment of this complex to the membrane via the Ras-binding domain (RBD) of ELMO. In the present study, we aimed to identify novel ELMO-interacting proteins to further define the molecular events capable of controlling ELMO recruitment to the membrane. To do so, we performed two independent interaction screens: one specifically interrogated an active GTPase library while the other probed a brain cDNA library. Both methods converged on Arl4A, an Arf-related GTPase, as a specific ELMO interactor. Biochemically, Arl4A is constitutively GTP-loaded, and our binding assays confirm that both wild-type and constitutively active forms of the GTPase associate with ELMO. Mechanistically, we report that Arl4A binds the ELMO RBD and acts as a membrane localization signal for ELMO. In addition, we report that membrane targeting of ELMO via Arl4A promotes cytoskeletal reorganization including membrane ruffling and stress fiber disassembly via an ELMO-DOCK1800-Rac signaling pathway. We conclude that ELMO is capable of interacting with GTPases from Rho and Arf families, leading to the conclusion that ELMO contains a versatile RBD. Furthermore, via binding of an Arf family GTPase, the ELMO-DOCK180 is uniquely positioned at the membrane to activate Rac signaling and remodel the actin cytoskeleton.