Phosphorylation of Arl4A/D promotes their binding by the HYPK chaperone for their stable recruitment to the plasma membrane

The Arl4 small GTPases participate in a variety of cellular events, including cytoskeleton remodeling, vesicle trafficking, cell migration, and neuronal development. Whereas small GTPases are typically regulated by their GTPase cycle, Arl4 proteins have been found to act independent of this canonical regulatory mechanism. Here, we show that Arl4A and Arl4D (Arl4A/D) are unstable due to proteasomal degradation, but stimulation of cells by fibronectin (FN) inhibits this degradation to promote Arl4A/D stability. Proteomic analysis reveals that FN stimulation induces phosphorylation at S143 of Arl4A and at S144 of Arl4D. We identify Pak1 as the responsible kinase for these phosphorylations. Moreover, these phosphorylations promote the chaperone protein HYPK to bind Arl4A/D, which stabilizes their recruitment to the plasma membrane to promote cell migration. These findings not only advance a major mechanistic understanding of how Arl4 proteins act in cell migration but also achieve a fundamental understanding of how these small GTPases are modulated by revealing that protein stability, rather than the GTPase cycle, acts as a key regulatory mechanism.

The Arl4 [ADP ribosylation factor (Arf)-like 4 proteins] small GTPases, comprising Arl4A, Arl4C, and Arl4D, are developmentally regulated. In adults, they have restricted tissue expression and have been found to act in a variety of cellular events. Arl4A recruits ELMO-Dock180, a conserved Rac regulator, to membranes for actin remodeling (1) and also directs cell migration through Robo1 and Pak1 effectors (2, 3). Several studies have reported that Arl4C functions in filopodia formation (4), cancer cell invasion (5, 6), and tumorigenesis (7–9). Arl4D has been found to activate Arf6 by recruiting cytohesin-2/ARNO for actin remodeling to promote cell migration (10, 11). How the Arl4 proteins are regulated in these cellular roles has been enigmatic. Small GTPases are typically activated by guanine nucleotide exchange factors and deactivated by GTPase-activating proteins (12, 13). However, neither class of key regulators has been identified for the Arl4 proteins. Consistent with this current state of knowledge, these small GTPases have been found to undergo rapid nucleotide exchange and appear structurally unable to bind GDP (13, 14). Thus, a major goal has been to elucidate how Arl4 proteins are regulated independent of the canonical mechanism that involves the GTPase cycle.The p21-activated kinases (Paks) stand at the hub of several signaling pathways for cell proliferation, migration, and survival (15). In cell migration, Pak1 phosphorylates paxillin at the leading edge of migrating cells for the rapid turnover of focal adhesion (16). Pak1 also phosphorylates LIMK1 and myosin light chain for actin reorganization to promote cell migration (17, 18). Pak activation requires cell adhesion through the interaction between integrins and the extracellular matrix (19). A well-characterized example has been the stimulation of cells by fibronectin (FN), which recruits Pak1 to the plasma membrane, where it then interacts with other molecules to coordinate downstream events (20, 21). In this role, FN acts in a variety of physiologic and pathologic circumstances, with wound healing being an example of the former (22) and cancer cell invasion being an example of the latter (23). With respect to the latter, alternatively spliced forms of FN have been found to act in collective tumor migration and are predictive of adverse outcomes for patients with cancer (24–26).We have recently found that FN promotes the cooperative recruitment of Arl4A and Pak1 to the plasma membrane, which contributes to sustained Pak1 activation needed for cell migration (3). In this study, we find that Arl4A and Arl4D (Arl4A/D) undergo rapid proteasomal degradation. FN stimulation activates Pak1 to phosphorylate these small GTPases, which leads to their binding by the chaperone-like protein HYPK (Huntingtin-interacting protein K). This binding stabilizes the targeting of Arl4A/D to the plasma membrane to promote cell migration.