We investigated ZAS3 as a modulator of TGFβ signaling based on its phylogenetic relationship to drosophila Shn, a known regulator of dpp, which is homologous to TGFβ in mammals. Shn directly binds to the pMAD-Medea complex, homologous to mammalian pSmad2/3-Smad4 complex, and represses transcription of Brk, a negative regulator of dpp signaling (reviewed by Affolter and Balser [13
]). Although Brk is not found outside of anthropods, the Shn-related ZAS proteins regulate Bmp signaling, a member of the TGFβ superfamily of proteins [8
]. Previously, ZAS1 and ZAS2 have been shown to modulate Bmp signaling by interacting with Smad1/4 and subsequently recruiting/interacting with additional co-activators or co-repressors to mediate transcription [8
]. Potential interactions between ZAS3 and Bmp-related Smads have not been investigated. We selected ZAS3 for study in the current work based on our prior observations that disrupted ZAS3 expression in mammalian cell lines resulted in accelerated cell proliferation, and that tumors have been observed in ZAS3 deficient mice, indicating a potential role for ZAS3 in modulating cell growth and proliferation [37
]. More recently, we reported that ZAS3−/−
mice have alterations in T-cell development and activation, and an increase in bone density [11
]. Taken together, the effects of the Zas family on growth regulation, tumor inhibition, and T-cell development, all of which are cellular processes regulated by TGFβ, we chose to investigate a potential role for ZAS3 in mammalian TGFβ signaling.
As multiple regulators of TGFβ signaling, both positive and negative, are either induced or repressed in response to TGFβ (reviewed by Massague and Miyazono [39
]), the observed induction of endogenous ZAS3 in TGFβ1-treated HEK293 cells, RIE-1 cells, and in the small intestine of TGFβ1 overexpressing transgenic mice indicated a potential regulatory role for ZAS3 in the TGFβ signaling pathway. Indeed, it was determined that ZAS3 accentuates Smad-dependent TGFβ signaling assessed by 4×-SBE-luc luciferase and 3TP-luciferase assays. In attempt to determine the structure activity relationship of ZAS3-mediated signal accentuation, several truncated constructs were synthesized. The structural insight gathered from this approach indicated the necessity for the N-term Zn-finger regions and subsequent amino acid residues extending to a.a. 1186. As the greatest signal enhancement was observed with truncated construct ZAS3(T1), the C-terminus of ZAS3 appears functionally insignificant for TGFβ signal modulation.
Previous observations that ZAS1 and ZAS2 mediate BMP signaling by binding to Smad1 and Smad4 [8
], suggest to us experiments investigating interactions between the TGFβ associated Smads and ZAS3. ZAS3(T1) increased nuclear pSmad2 and pSmad2-Smad4 levels and coimmunoprecipitated with pSmad2. It should be noted that the co-IP experiments performed herein are limited in that they cannot conclusively determine if ZAS3 interacts individually with Smad2, Smad3, or Smad4, as the Smads immunoprecipitate as a complex. A drawback of the current work is the inability to study the inverse relationship (i.e. TGFβ1 signaling in the absence of ZAS3) between TGFβ signaling and ZAS3 levels. We unsuccessfully attempted use of multiple siRNAs to inhibit ZAS1, ZAS2 and ZAS3 signaling, but were unsuccessful, presumably due to redundancy in the ZAS protein family.
As there are a multitude of Smad interacting proteins (reviewed by Brown et al.
]), we chose to examine those that negatively regulate TGFβ signaling in an attempt to unravel the mechanism by which ZAS3 accentuates TGFβ signal transduction. Although extensive negative regulation of canonical TGFβ signaling can occur at many levels [23
], we focused on the well-studied Ski family of oncoproteins as potentially intriguing candidates.
Ski was first identified based on its ability to transform chicken embryo fibroblasts and was named based on its viral homologue, v-Ski, the transforming component of the Sloan-Kettering virus (SKV) [27
]. Subsequently, SnoN (Ski related novel protein) was identified based on its homology to v- and c-Ski [27
]. The primary function of Ski and SnoN is transcriptional regulation, which occurs by interaction with numerous other transcriptional modulators [24
]. Ski and SnoN exert their TGFβ antagonistic activities primarily by disruption of the active complex by simultaneous binding to Smad2/3, via their N-term region, and Smad4, via a C2
Zn-binding module [31
]. Furthermore, the inactive Smad-Ski/SnoN complex may further attenuate TGFβ signaling by: 1) competing with the active Smad complex for DNA binding [43
]; 2) preventing binding of the transcriptional co-activator p300/CBP [31
]; and 3) recruiting a transcriptional co-repressor complex containing N-CoR and HDAC [26
]. As Ski and SnoN act in the nucleus, bind the Smad complex, and contain Zn-binding module they were identified as potential ZAS3 modulators. Indeed, the ability of ZAS3(T1) to displace Ski and SnoN from pSmad2, and presumably the entire Smad complex, indicated a potential interrelationship among these proteins.
Further examination of the relationship between Ski/SnoN and ZAS3 by site directed mutagenesis revealed a potential mechanism where ZAS3 accentuates TGFβ signaling by directly competing with Ski/SnoN for binding to the Smad complex, more specifically, Smad2 and Smad3. It is not clear at what point during TGFβ signal transduction the competition between ZAS3 and Ski/SnoN occurs, as Ski/SnoN have been shown to interact with the Smad proteins at various stages during complex formation [31
]. It is also not yet clear which Smad proteins directly interact with ZAS3. The decrease in ZAS3 activity and Smad affinity following mutation of residues that share homology to the reported Smad2/3 binding domain of Ski/SnoN [32
] indicate that ZAS3-Smad2/3 association is likely. However, the ability of Ski/SnoN to repress TGFβ signaling independent of interactions with Smad2/3 [32
] brings into question if ZAS3 competition for Smad2/3 binding alone is sufficient to antagonize Ski/SnoN-mediated signal repression. The ability to co-IP ZAS3 and Smad4 is indicative of a potential additional interaction, however, the possibility that Smad4 is precipitated due to its interactions with Smad2/3 cannot be excluded and more detailed study is needed. The N-term Zn-finger motifs in ZAS3 are of particular interest in this regard. Based on the data presented herein, two potential roles can be envisioned for the ZAS3 Zn-finger domain. ZAS3 could bind DNA to enhance affinity/transcription at the Smad binding region or compete with Ski/SnoN for binding to Smad4. The ability of ZAS3 to displace Ski/SnoN from Smad2/3 and, presumably, the Smad complex supports the latter hypothesis; that is there is competition at the Smad4 binding site, however, due to the large size of ZAS3, it is possible that interactions with Smad2/3 sterically inhibit interactions between Smad4 and Ski/SnoN.