We provide evidence that the RabGAP AS160 plays both negative and positive regulatory roles in vesicle transport. This supports the existence of Rab regulatory networks whereby individual components play active roles both in promoting and repressing flux through the pathway. This is consistent with the notion of Rab cascades where the function of multiple Rabs that act in series in a pathway can be coupled by a countercurrent mechanism encoded both by GEFs and GAPs (10
). The current studies extend this model by showing that phosphorylation and/or 14-3-3 binding switches AS160 from a negative to a positive regulator of vesicle fusion. In view of the vast number of different RabGAPs found in the human genome, these studies have broad implications for the role of this family of proteins in eukaryotic vesicle transport.
In addition to the TBC domain, RabGAPs possess a range of modular domains, the function of which in many cases has not been ascertained. A key observation in the present study was the identification of a lipid-binding domain encoded within the second PTB domain in the N terminus of AS160 that conferred its localization to the PM. This was a striking observation because previous studies had suggested that AS160 acted principally as a negative regulator of GLUT4 trafficking by binding to intracellular GLUT4 vesicles and inhibiting GTP loading of a Rab that was required to facilitate docking of the vesicles at the PM. The identification of a PM binding domain was also intriguing in light of previous observations identifying a pool of highly phosphorylated AS160 at the PM in adipocytes (23
PTB domains bind to phosphopeptides and phospholipids, although the locations of the phospholipid binding sites differ drastically between PTB domains (6
). For example, the PI(4,5)P2
binding site in Dab1 is not likely to be a canonical binding site for phospholipids, as indicated by the lack of conservation of the relevant basic residues in paralogs. Indeed, the phospholipid binding site in the Shc PTB domain is located on a completely different surface compared to Dab1 (30
). Using the Dab1 PTB domain as a homology model, it is predicted that the phospholipid binding site in the second PTB domain of AS160 is located on a surface proximal to the peptide binding groove and not where the PI(4,5)P2
site is located in the Dab1 () or Shc PTB domains. Intriguingly, the lysine and arginine residues conserved in AS160 orthologs are not broadly conserved in other PTB domain paralogs. Further, the second PTB domain of AS160 carries an insertion of 107 amino acids not found in other PTB domains. Therefore, it is likely that the second PTB domain of AS160 is unique. Also noteworthy is that the second PTB domain in AS160 regulates binding to GSV cargo proteins, although this function is distinct from phospholipid binding (). Nevertheless, the fact that both of these functions are encoded within the same domain suggests that the inhibitory and facilitative roles that are encoded by these distinct interactions coevolved. By examining a series of AS160 mutants with various degrees of IRAP binding, we mapped the negative regulatory function of AS160 to the IRAP binding domain ( and ). However, given that this domain also interacts with other proteins in GSVs, such as LRP1 (), this suggests that the interaction with GSVs is mediated via interactions with multiple cargo components. This is consistent with the observation that the intracellular distribution of AS160 is unaltered in adipocytes from IRAP−/−
mice and 3T3-L1 adipocytes that have reduced IRAP expression (13
Thus, the interaction of AS160 with GSVs likely confers an inhibitory effect on GLUT4 translocation by inhibiting a Rab associated with GSVs. Evidence indicates that the inhibition of AS160 RabGAP activity is mediated by AS160 phosphorylation and 14-3-3 binding (28
), although this has not been formally proven. The present study extends this model. We propose that AS160 associated with GSVs might become phosphorylated at the PM when it encounters active Akt at this location. Consistent with this, it has been shown that Akt functions principally at the PM (1
) and that insulin stimulates GSV trafficking to the PM in an Akt-independent manner (43
). Moreover, AS160 is highly phosphorylated at the PM and not at other locations (23
). Notably, constitutive targeting of AS160 to the PM enhanced its phosphorylation in the absence of insulin, and this was accompanied by increased GLUT4 translocation. This surprising result suggests that under basal conditions there must be a small amount of active Akt at the PM that under normal circumstances is insufficient to phosphorylate endogenous AS160. By targeting AS160 to the PM, we have likely shifted the equilibrium in favor of AS160 phosphorylation. These findings indicate that phosphorylation of AS160 at the PM positively regulates GLUT4 trafficking. AS160 phosphorylation at the Thr642 site encodes 14-3-3 binding (28
), and here we show that constitutive binding of 14-3-3 to AS160 was sufficient to replicate its facilitative role in GLUT4 trafficking. Collectively, these findings suggest that phosphorylation and 14-3-3 binding not only suppress the GAP activity of AS160 but that this also confers an additional facilitative regulatory function. We have yet to resolve the nature of this role, but we speculate that phosphorylated/14-3-3-bound AS160 at the PM plays an active role in the docking of GSVs at the PM. It will be intriguing to determine whether other RabGAPs display a similar dual role. Hence, we conclude that AS160 possesses two separate and mutually exclusive functions that can be interchangeably regulated by Akt-dependent phosphorylation and 14-3-3 binding. An elegant feature of the use of its PTB domain for targeting AS160 to the PM is that by strategically localizing a very small pool of AS160 to the precise site of vesicle docking at the PM, this makes it relatively easy for Akt to disarm the inhibitory function of AS160 only at this location to promote the facilitative role of AS160 on the docking and fusion reaction ().
Fig 11 Model for the positive role of AS160 at the PM. In the absence of insulin, the AS160 PTB domain interacts with the PM, depicted by red lines. This interaction is transient and with no further GSV-PM interaction the vesicle dissociates from the PM. With (more ...)
The fact that we detected a stimulatory effect of PM targeted AS160 on GLUT4 trafficking in the absence of any other perturbation was noteworthy. First, insulin-dependent GLUT4 translocation requires many steps other than phosphorylation of AS160, including trafficking of GLUT4 vesicles to the adipocyte cortex, actin rearrangement (21
), recruitment of the exocyst complex to the PM (11
), and posttranslational modification of SNARE proteins (4
) or their regulators such as Munc18c (37
). Second, in addition to disarming the GAP, one imagines that in the absence of an active effect on the Rab GEF this might have a relatively minor effect. Hence, the impact of constitutive targeting of AS160 to the PM, in the absence of any of these other changes, on GLUT4 trafficking probably points to the highly significant dual role of AS160 in this process.
In summary, we have mapped out the critical residues for the association of AS160 with phospholipids at the PM and unravel an additional role for AS160 in GLUT4 translocation. These data implicate AS160 as a major fork in the pathway that determines the probability that GSVs will either fuse with the PM or recycle back to the cell interior. This is a highly efficient way of coupling the activity of the Rab on the vesicle to the correct location in the cell, to which the vesicle is destined to fuse, and the nutrient status of the cell, which is encoded by the activity state of Akt also found at the same location.