In this study, we have identified the complete signaling pathway that leads from T cell receptor engagement to RNA binding of PSF and CD45 exon exclusion (). We show that TRAP150, a protein of largely unknown function, and GSK3, a versatile signaling mediator, are key components of this pathway. Our data also provide a molecular explanation for the built-in “time lag” that is a hallmark of CD45 signal-induced splicing. This study thus reveals substantial new insight into the mechanism for signal induced alternative splicing in T cells, and extends the known cellular functions of TRAP150 and GSK3.
While numerous examples of signal-induced alternative splicing have been described in a variety of cell types, the signaling pathways that connect membrane-bound receptors to the nuclear RNA processing machinery have been identified in only very few cases (Lynch, 2007
; Shin and Manley, 2004
). Some notable examples in T cells are the Sam68-mediated splicing of CD44 or the TIA-1 regulated Fas alternative splicing (Izquierdo and Valcárcel, 2007
; Matter et al., 2002
). In these cases, direct phosphorylation of Sam68 or TIA-1 was shown to occur rapidly upon activation of signaling pathways and to directly increase the activity of these proteins bound to RNA. Such a direct effect of phosphorylation on splicing activity is markedly different from the mechanism we demonstrate here in which phosphorylation of PSF indirectly regulates its accessibility to the target RNA via protein-protein interactions. Therefore, the GSK3-dependent regulation of a mutually exclusive interaction between PSF and either TRAP150 or CD45 RNA is a unique paradigm for connecting intracellular signaling pathways to RNA processing events.
In our model, the crucial regulatory point is the phosphorylation dependent interaction of PSF with TRAP150, which prevents PSF from binding to the ESS1 RNA. We were able to map the functionally-relevant phosphorylation in PSF to a single threonine residue, T687, as a T687A point mutant shows substantially reduced interaction with TRAP150 and bypasses the regulatory pathway leading to CD45 exon exclusion in resting cells. We do detect a residual interaction of the PSF T687A mutant with TRAP150, suggesting that phosphorylation strongly facilitates this interaction, but is not an absolute requirement. Furthermore, the finding that deletion of the C-terminal 40 amino acids does not prevent interaction of PSF with TRAP150, argues against direct interaction of TRAP150 with the phosphorylated T687 residue. Rather, our data suggest that phosphorylation of T687 drives conformational changes regulating the interaction with TRAP150. In such a model, a small proportion of the T687A mutant or the non-phosphorylated WT PSF could still be in the conformation permissive to TRAP150 interaction thereby explaining a basal level of interaction in the non-phosphorylated state. Future studies to characterize the interface between TRAP150 and PSF are thus predicted to uncover complex and important intra- and intermolecular interactions.
A further broad implication of the data presented here is in characterizing the regulation of PSF T687 phosphorylation, and defining PSF as a substrate of GSK3. In many cases GSK3-mediated phosphorylation requires a priming phosphate located 4 amino acids towards the C-terminus from the target site (Cohen and Frame, 2001
). PSF does contain a tyrosine at position 691, which could potentially serve as a priming phosphate of GSK3-mediated T687 phosphorylation. However, as GSK3 phosphorylates PSF purified from bacteria, we conclude that a priming phosphate is not strictly required, limiting the potential functional relevance of Y691. Finally, as PSF is a mostly nuclear protein and GSK3 resides mostly in the cytoplasm, the simplest model for GSK3-mediated PSF phosphorylation would be that it occurs immediately upon translation, prior to nuclear import, although we cannot rule out other possible models for the location of this activity.
Interestingly, reduced GSK3 activity has been previously shown to regulate the CD28 costimulatory signal in activated T cells (Diehn et al., 2002
). This data, combined with our work suggest that a decrease in GSK3 activity is required early during T cell activation to initiate proliferation, but that it later acts on CD45 alternative splicing to increase the threshold for activation, thereby leading to T cell attenuation. Obviously it is important for proper immune function to have the attenuation step temporally delayed with respect to initial activation. In general, direct phosphorylation or dephosphorylation of proteins occurs rapidly upon receptor engagement and are thus not suited to exert a delayed response. In our model the appropriate temporal response is accomplished by a mostly stable phosphorylation of PSF and its interaction with TRAP150, which is only released by de novo synthesis of PSF in conditions with reduced GSK3 activity.
Our data regarding the requirement for de novo protein synthesis in order to induce reduced PSF phosphorylation and CD45 exon skipping is fully consistent with the model we have proposed. We cannot formally rule out that cycloheximide blocks synthesis of a phosphatase required to dephosphorylate PSF under activated conditions. However, as we have demonstrated that changing the activity of GSK3 alone is sufficient to change the phosphorylation state of PSF in the absence of any phosphatase inhibitors (, ), we favor a model in which the predominant factor determining the phosphorylation state of PSF is GSK3. This notion is further supported by our data showing that experimental decrease of GSK3 activity in resting conditions is sufficient to induce a CD45 splicing pattern resembling that of activated cells. In addition, if PMA stimulation would mainly act through increasing expression of a phosphatase, treatment of cells simultaneously with PMA and SB216763 should have an additive effect. The fact that this is not the case again suggests that the contribution of a phosphatase, if any, is minor. Nevertheless, it is possible that there may be alternative models that are consistent with our data and further studies are necessary to work out the complete details of the molecular interactions between GSK3, PSF and TRAP150.
It is interesting to note that we have previously identified a large number of genes that are alternatively splicing in response to T cell activation, several of which are regulated in an ESS1 dependent manner in concert with CD45 (Ip et al., 2007
; Rothrock et al., 2003
). Consistent with PSF binding specifically to the ESS1 sequence motif, we have shown PSF to be involved in the regulation of several of the ESS1-containing exons studied thus far ((Motta-Mena et al., 2010
) and data not shown). PSF is also known to have multiple functions in the nucleus, including roles in transcription and nuclear retention of RNA (Shav-Tal and Zipori, 2002
). It therefore is likely that in addition to influencing CD45 alternative splicing in T cells, the regulation of PSF by GSK3 and TRAP150 influences a wide spectrum of PSF-mediated gene expression events impinging on numerous cellular functions. In light of the many roles GSK3 has been shown to play in a variety of cell types, it will be interesting to elucidate the contribution of PSF in mediating these functions.