The present study shows that zyxin is a novel interacting partner for SIRT1. SIRT1 and zyxin are co-localized in the nucleus after LMB treatment, suggesting that nuclear-accumulated zyxin interacts with SIRT1. SIRT1 and zyxin transcript are both preferentially expressed in developing mouse brain and various adult tissues, including lungs, spleen, and testis. Furthermore, SIRT1 deacetylates zyxin, especially after LMB treatment, raising the possibility that SIRT1 could modulate zyxin's functions in the nucleus via deacetylation. Zyxin-null mice exhibit almost no abnormal phenotype, probably due to genetic redundancy [31
]. On the other hand, SIRT1-null mice show growth retardation and developmental defects in various tissues [12
], which suggests a critical role of SIRT1 in the developmental stage. Based on these reports, our data could raise the possibility that zyxin is one of the downstream effectors necessary for SIRT1 to execute some biological functions in the developing brain and various adult tissues.
There is an apparent contradiction between the yeast and mammalian results. Apparently SIRT1 does not bind to full length zyxin in yeast because of some hypothesized masking of the LIM3. However, the mammalian cell experiments do show apparent association between full-length zyxin and SIRT1. As mentioned above, a previous study showed that full-length zyxin interacts with h-warts/LATS1 in vivo
but not in vitro
, raising the possibility that LIM1/2 domains are masked in full-length zyxin and some modification might be critical for their interaction [22
]. Therefore we also hypothesize some modification would be required for full-length zyxin to bind to SIRT1 in mammalian cells.
Then what is the biological function of the interaction between SIRT1 and zyxin? Cell survival effect is one of the features shared by these proteins. SIRT1 can protect neurons from oxidative stress in mammalian cells [32
], neurotoxicity in cell-based models for AD/tauopathies, ALS [16
], and Wallerian degeneration [14
]. SIRT1 expression levels are induced in mouse models of neurodegeneration [16
], and moderate heart-specific overexpression of SIRT1 in mice delays aging of the heart by conferring resistance to oxidative stress [33
]. On the other hand, nuclear-accumulated zyxin can protect cardiomyctes from oxidative stress [28
]. These reports raise the possibility that the interaction of these proteins could be implicated in cellular survival, especially in the brain and heart, during physiological senescence. Since zyxin could affect transcriptional activity [27
] and since nuclear-accumulated zyxin executes anti-apoptotic function cooperating with Akt in the nucleus [28
], it is possible SIRT1 could modulate these functions of zyxin via deacetylation. Considering that SIRT1-null mice exhibit developmental defects with frequent exencephaly and retinal phenotype with abnormal proliferation [12
], it is possible that the interaction of these proteins could be implicated in cellular survival pathways in the developmental stage.
In addition, shuttling between the cytosol and the nucleus is a characteristic feature shared by SIRT1 and zyxin. In this study, it was not determined whether the acetylation status of zyxin affects its cellular localization or whether deacetylated zyxin is retained in the nucleus. Recently, it was reported that nuclear zyxin, phosphorylated by Akt, interacts with acinus-S to prevent apoptosis and 14-3-3γ plays a pivotal role in the nuclear translocation of zyxin [29
]. The morphological studies in the present study show that zyxin is localized primarily in the cytosol with puncta and it accumulates in the nucleus only with the LMB treatment, but not with insulin-like growth factor (IGF-1) or LY294002 treatment, an inhibitor of PI 3-kinase, in COS-7 cells (data not shown). This suggests that some unknown stimuli other than the IGF-1-Akt-related pathway could play a critical role for zyxin to translocate into the nucleus. The nuclear accumulation of zyxin is not induced by co-expression with SIRT1, suggesting that it is unlikely SIRT1 enhances the accumulation and/or translocation of zyxin into the nucleus. Since the sub-cellular localization of SIRT1 differs based on cell type and differentiation [12
], it is possible that SIRT1 could interact with zyxin in the cytosol depending on the cell type and differentiation.
ECM, beyond scaffolding functions, is responsible for transmitting environmental signals into cells, thereby essentially affecting all aspects of cell life, including its proliferation, differentiation, and death [35
]. Since zyxin is considered to convey signals from ECM into the nucleus at the focal adhesion plaques, we now assume that SIRT1 could regulate the signal transmission from ECM into the nucleus by modulating zyxin's functions via deacetylation, thereby reflecting its biological functions.
Finally as described in above, several functional significances could be assumed for the interaction, including the cell survival effect especially in development and/or regulation of signal transmission from ECM into the nucleus. Actually we had performed experiments to investigate whether the interaction of these proteins could affect the apoptosis under stress condition or the transcriptional activity of zyxin in luciferase reporter gene assay (data not shown). Thus far, we could not obtain the result to indicate the biological significance of the interaction in such simple systems. Additional experiments are required to clarify the functional significance of the interaction in the future.