Many studies have demonstrated a role for Arf6 activity in Rac-mediated membrane ruffling and cytoskeletal reorganization at the PM. Arf6 may facilitate the trafficking of Rac1 to the PM [5
] and Arf6 activation may lead to the activation of Rac1 [7
]. However, the details of how Arf6 influences Rac functions remain to be elucidated. A recent study by Santy et al has shown that the ARNO activation of Arf6 that leads to the activation of Rac in MDCK cells may be mediated by the DOCK180/Elmo complex however, no direct interaction between ARNO or Arf6 and DOCK180/Elmo was described [13
]. In the present study, we describe a possible mechanism for how Arf6 could regulate Rac1 activity by showing the interaction between Arf6-GDP and Kalirin5, a Rac/RhoG GEF.
Kalirin5 is the smallest isoform in the Kalirin family. The amino terminal end of the protein begins at spectrin repeat 5 (SR5) and includes the Dbl homology (DH) and pleckstrin homology (PH) domains and a PDZ-binding motif in the C-terminal region (Fig. ). Most of the alternatively spliced isoforms of both Kalirin and Trio include these spectrin-like repeats [18
]. Interestingly, these spectrin repeats are also sites of interaction of Kalirin with PAM, a secretory granule enzyme required for the α-amidation of peptides [16
], with i-NOS [33
], and with Huntingtin-associated protein 1 (HAP1) [34
]. Since the larger Kalirin isoforms all contain SR5, they could interact with Arf6-GDP in a manner similar to Kalirin5. We do not know whether this binding is direct or involves other proteins.
What then could be the function of Arf6-GDP binding to Kalirin? We propose that the Kalirin interaction with Arf6-GDP is transient but brings Kalirin to membranes. The subsequent activation of Arf6 then allows Kalirin to activate Rac. Consistent with this, co-expression of wild type Arf6, which can cycle between GDP-and GTP-bound forms, increases the PM localization of Kalirin5, the steady state level of Rac1-GTP, and the ability of Kalirin5 to induce membrane ruffling. Arf6T27N, however, being trapped in the GDP-bound state, recruits Kalirin5 to endosomal membranes but fails to allow activation of Rac through Kalirin. On the other hand, EFA6 promotes activation of Arf6 but still allows inactivation, generating Arf6-GDP. When EFA6 is expressed, Kalirin does not stably associate with the PM but it may transiently be brought to the PM by Arf6-GDP to activate Rac. The ability of GEF dead Kalirin5 to block EFA6-induced cytoskeletal changes (Fig. ) is supportive of this model and suggests that a spectrin repeat-containing GEF might be responsible for Rac activation in EFA6 expressing cells.
Kalirin brought to the membrane through its interaction with Arf6-GDP could be positioned for optimal access to Rac1. Additionally, the interaction of Arf6 with SR5 of Kalirin could allosterically activate its GEF activity. In either case, the interaction of Arf6 with Kalirin provides a way to couple the Arf6 activation/inactivation cycle to that of a Rho GTPase. The sequential recruitment of Kalirin by Arf6-GDP, followed by activation of Arf6 and then activation of Rac is consistent with earlier observations of the requirement of Arf6 activation for Rac ruffling, the synergy between Arf6 and Rac [9
] and the fact that activation of Arf6 leads to activation of Rac [7
]. Furthermore, the requirement that Arf6 cycle between GDP-bound and GTP-bound forms to carry out its cellular functions is consistent with the known biology of Arf proteins [1
Intriguingly, two examples of proteins binding to Arf6-GDP that subsequently lead to Arf6 activation have been reported. TRE17, a protein involved in Rac activity, binds specifically to the GDP-bound form of Arf6, which leads to increased activation of Arf6 [24
] presumably by bringing Arf6 in proximity to an Arf6 GEF. The second example is β-arrestin, a regulator of agonist-stimulated, β2-adrenergic receptor endocytosis, which binds to both Arf6-GDP and ARNO, an Arf6 GEF, leading to activation of Arf6 [35
]. Like β-arrestin, Kalirin5 can make a complex with Arf6-GDP and perhaps lead to the recruitment of EFA6 or ARNO to promote Arf6 activation. Indeed, we have been able to capture Arf6T27N and EFA6 together bound to immobilized SR5 (unpublished observations) suggesting that Kalirin may provide a scaffold for sequential and coordinated activities.
Our studies demonstrate that Arf6 can regulate Rac1 activity through interaction with Kalirin. This novel signaling pathway may be involved in neurite outgrowth since each of these proteins are abundant in brain and have been implicated in this process. Arf6 activity is required for Rac1-mediated neurite outgrowth [36
] and the GEF1 domains of Kalirin and Trio also induce neurite outgrowth through RhoG activation [37
]. Exploring this functional relationship between Arf6 and Kalirin will give us more information about the mechanism for Rho GEF regulation and the crosstalk between these two small GTPases, Arf6 and Rac.