Despite abundant circumstantial clues to its cellular function, an understanding of the precise cellular role of Sam68 and the significance of its RNA binding ability has proved elusive. Among the processes that it appears to regulate are transcription, splicing, RNA export and translation. The possibility that Sam68 might play multiple roles in gene expression is supported by the present study in which we show that its effects on cell proliferation and death are functionally separable.
We found that Sam68 compromises cell proliferation without the need for specific RNA binding. At least part of its proliferative block resulted from cell cycle arrest in G1 phase. This involved downregulation of cyclin D1 and E transcripts, suggesting that transcription of these genes was repressed. This effect was recapitulated by an specific-RNA binding-defective mutant of Sam68, and is reminiscent of the findings of Hong et al. [21
], who showed that Sam68 expression can repress transcription of reporter constructs in an RNA-binding-independent manner. It is of course possible that cyclin RNA diminution is attributable to post-transcriptional intervention by Sam68 since modulation of RNA export, stability or translatability could result in alteration of total RNA levels. However, the effect of RNA-binding-defective mutants favors a transcriptional repression interpretation. If so, it will be important to assess whether Sam68 directly affects cyclin transcription or acts upstream of transcription. A direct effect on cyclin transcription could involve Sam68 interaction with the transcriptional co-activator CBP [21
] or with hnRNP K [22
]. So far we have not observed any effects of Sam68 overexpression on potential upstream regulators of cyclin transcription such as Ras/Erk signaling or AP-1 expression (our unpublished results).
It was previously reported that an apparent splice variant of Sam68, in which part of the KH domain was deleted, could block G1 progression and cyclin D1 accumulation in NIH 3T3 cells, whereas wt Sam68 did not [26
]. Clearly this contrasts with our findings in which both wt and mutant Sam68 block progression and cyclin D1 accumulation. Also we have been unable to detect the reported splice variant in NIH 3T3 cells. It is not clear why our results differ.
In contrast to its effect on proliferation, induction of apoptosis by Sam68 requires its RNA-binding ability. This ability is required for other Sam68 functions as well: For example, its ability to enhance the cytoplasmic utilization of reporter constructs containing retroviral RRE or CTE elements requires RNA-binding [20
]. This enhancement does not result from an effect on RNA export (as previously surmised) since it occurs without appreciable effect on nuclear export or global transcript level. It has been hypothesized that Sam68 "marks" the transcripts in a way that directs their cytoplasmic fate [20
]. It has also recently been reported that Sam68 can regulate splicing in response to phosphorylation by activated Erk [17
]. Clearly the identification of relevant RNA targets of Sam68 will be important to elucidate its precise functions. A recent report identified RNAs capable of binding to Sam68 [27
]. Many of these possessed a motif identified by in vitro
selection procedures [9
]. This lends some credence to their authenticity, but also opens the question of whether such strategies uncover true in vivo
targets or reflect in vitro
Our findings suggest that Sam68 can act as a tumor suppressor, and are consistent with a previous study showing that antisense-treatment to reduce Sam68 levels transforms 3T3 fibroblasts [24
]. Interestingly we found that at least one anti-cancer agent, TSA, potentiated the ability of Sam68 to induce apoptosis. At the concentrations used, TSA alone did not induce apoptosis, but it increased the level of apoptosis in cells overexpressing Sam68. TSA inhibits protein deacetylases and is generally assumed to exert its effects by enhancing histone acetylation, thereby relaxing transcriptional constraints. Sam68 overexpression did not have any detectable effect on the extent of histone hyperacetylation induced by TSA (data not shown), so it seems unlikely that Sam68 modulates TSA-induced transcription. Rather, pro-apoptotic signals generated by Sam68 and TSA may synergize to enforce apoptosis. Although we have not detected reactivity of Sam68 with anti-acetyl lysine antibodies, since Sam68 associates with the acetyltransferase CBP, the potential role of acetylation in regulating Sam68 function bears closer examination.
We and others have not observed cell status-dependent modulation of Sam68 expression levels; e.g., in response to cell cycle progression, growth factors or stress. We have also not detected marked differences in its expression levels in several human cancer cell lines. Thus, it would seem most likely that Sam68 activity is regulated post-translationally. This regulation could be conferred by covalent modification, allosteric modulation or changes in subnuclear localization. Sam68 is phosphorylated at C-terminal tyrosine residues by Src family kinases and the nuclear kinase Brk/Sik. These phosphorylations reduce its ability to bind to RNA in vitro, so we would predict that tyrosine phosphorylation by these kinases would decrease its ability to induce apoptosis. Indeed we have found that co-expressing activated forms of Src or Brk does suppress apoptosis in cells overexpressing Sam68. However, until we are able to generate non-phosphorylatable, yet functional, mutants of Sam68, it will not be possible to determine whether this effect is attributable to Sam68 phosphorylation.
Serine/threonine phosphorylation may also be involved in regulation of Sam68's pro-apoptotic function: it has recently been shown that phosphorylation of Sam68 by Erk enables Sam68 to regulate splice site selection in CD44 pre-RNA [17
]. Our results with the deacetylase inhibitor also raise the possibility of regulation by acetylation. Sam68 is also dimethylated on arginine residues, and this can influence its ability to interact with other proteins [3
Sam68 might also be allosterically regulated by its interactions with multiple nuclear proteins. For example, it has been reported that binding of hnRNP K inhibits Sam68's ability to enhance export of RRE-containing messages (Yang, et al. 2002). Finally, it is known that Sam68 assumes a distinctive, punctate subnuclear distribution in certain cancer cell lines (Chen et al. 1999), and this might influence its ability to execute its normal function. In light of our results showing enhancement of Sam68 pro-apoptotic ability by the anti-cancer agent TSA it will be important to find out whether, and how, the functional status of Sam68 is altered in cellular transformation.