In an effort to identify additional regulators of HSW signaling we have examined the role of Tao-1 in growth control during development. Tao-1 depletion in either the eye or wing epithelium results in overgrowth phenotypes as well as transcriptional upregulation of HSW targets. Using a combination of genetic epistasis, experiments in cultured S2 cells, and in vitro biochemistry, we have demonstrated that Tao-1 directly phosphorylates the critical T195 regulatory residue in the activation loop of Hpo to promote HSW pathway activation. The observation that a mammalian orthologue of Tao-1, TAOK3, can phosphorylate MST kinases at the same residue further suggests that this regulatory function is conserved in mammals. Taken together, these results implicate Tao-1 as a component of HSW signaling and reveal a mechanism for regulation of Hpo activity ().
A model for Tao-1’s function in the HSW pathway
depletion results in overgrowth phenotypes that are similar to mutations in other HSW pathway genes, these phenotypes are less severe than those of core components such as hpo
. One likely explanation for this is that the RNAi transgenes we have used in these studies do not completely remove Tao-1
function.. It is also possible that there are multiple mechanisms for activating HSW signaling, including, but not limited to, Tao-1 phosphorylation of Hpo. Indeed, previous studies have demonstrated that the upstream components Mer, Ex, and Kibra act, at least in part, in parallel to activate Hpo (Baumgartner et al.; Genevet et al., 2010
; Yu et al., 2010
). Our biochemical evidence indicates that two of these proteins, Mer and Ex, function with Tao-1 to activate HSW signaling. While it is probable that Kibra functions upstream of Tao-1, we cannot rule out the possibility that Kibra functions independently of Mer and Ex to activate HSW signaling in a Tao-1-independent manner (). Further genetic analysis using a Tao-1
null allele would be helpful in defining Tao-1’s role relative to other HSW components, but unfortunately the deletions associated with the sole existing Tao-1
null allele, Tao-150
, also appear to affect an adjacent gene (CG14218; King et al., 2011
). In addition, Tao-1
maps very close to the most proximal FRT
element on the X
chromosome, making it difficult to generate recombinant chromosomes for somatic mosaic analysis.
How do Mer, Ex and Tao-1 cooperate to regulate Hpo phosphorylation? Given that Ex has been shown to interact with Hpo (Yu et al., 2010
), one possibility is that Mer and Ex function to scaffold Tao-1 together with Hpo, thereby promoting the ability of Tao-1 to phosphorylate and activate Hpo. However, despite repeated attempts we have been unable to detect Tao-1 in a complex with either Mer or Ex, and knockdown of Mer
does not diminish the ability of Tao-1 to promote Hpo phosphorylation in S2 cells. For these reasons, we favor the possibility that Mer and Ex indirectly affect Tao-1 function, perhaps by interacting with other proteins that in turn directly regulate Tao-1. For example, Tao-1 activity could be directly regulated by an unknown receptor at the cell surface whose localization or activity is controlled by interaction with Mer and Ex. This notion is consistent with the fact that both Mer and Ex have FERM domains, which are known to interact with the cytoplasmic tails of transmembrane proteins (Bretscher et al., 2002
). Previous studies have suggested that Ex interacts with the transmembrane protein Crumbs (Ling et al., 2010
), though the mechanistic significance of this interaction is unclear. It is not currently known whether Drosophila
Merlin has transmembrane binding partners.
Two additional ideas related to Tao-1 function are suggested by our data. In S2 cells, Tao-1 kinase activity is required for normal levels of Hpo phosphorylation at T195 in the kinase activation loop, suggesting that Tao-1 could function to maintain constant, low levels of pathway activation. In turn, this low level of Hpo activation might be necessary so that other, regulated inputs into HSW activity can quickly transition cells away from actively dividing and into a differentiated state following periods of growth. Alternatively, it is possible that Tao-1 activity itself is dynamically regulated during development, allowing it to rapidly alter levels of HSW pathway activity via its effect on Hpo phosphorylation. In either case, phosphorylation by Tao-1 at T195 is likely to promote Hpo’s known ability to undergo autophosphorylation (Colombani et al., 2006
), thus amplifying the effect of even a small change in Tao-1 activity. Further studies will be required to answer these questions and to determine if, and how, Tao-1 activity is regulated.
An interesting aspect of our discovery that Tao-1 regulates HSW signaling is that Tao-1, and its mammalian orthologues TAOK1–3, previously have been shown to regulate MT stability (Mitsopoulos et al., 2003
; Timm et al., 2003
; Liu et al., 2010
). Our results indicate that this effect on MT stability is not mediated through HSW signaling, since mutations in other HSW pathway components do not display similar MT phenotypes (data not shown). However, it is interesting to speculate that Tao-1’s association with MTs might affect its ability to regulate HSW pathway activation. More work will be required to determine whether the function of Drosophila
Tao-1 in HSW signaling is entirely independent of its role in microtubule dynamics, though a recent study in mammalian cultured cells found that microtubule disruption did not affect localization of Yap, a mammalian Yki orthologue (Dupont et al., 2011
), suggesting that in mammalian cells these roles might be independent.
An additional possible mechanistic link between Tao-1 and HSW signaling is suggested by studies in flies and in mammalian cells indicating that Par-1, a polarity protein, is positively regulated by Tao-1 (Timm et al., 2003
; King et al., 2011
). Par-1 has been shown to promote basolateral polarity in the Drosophila
follicular epithelium and to regulate the stability and organization of MTs in these cells (Doerflinger et al., 2003
). Recent studies have implicated components of both apical and basolateral polarity in the regulation of HSW signaling (reviewed in Halder and Johnson, 2011
). Conversely, HSW signaling also seems to feed back onto Crumbs, an apical determinant, and perhaps other components to regulate apical-basal polarity (Chen et al., 2010
; Ling et al., 2010
; Robinson et al., 2010
). Whether Tao-1 plays a role in the linkage between cell polarity and growth control remains to be established, but the ability to both directly activate Hpo function through phosphorylation and control cytoskeletal organization and cell polarity through microtubule organization potentially places Tao-1 in a unique position to coordinate these important cellular processes.