Alternative pre-mRNA splicing is a process by which multiple functionally distinct proteins may be encoded from a single gene through the variable inclusion of individual exons. In general, variable exon inclusion is regulated by proteins which bind to sequences within the regulated gene and influence the recognition of splice sites by the basic splicing machinery, or spliceosome (for recent reviews, see references 1
, and 18
). Frequently, alternative splicing is regulated in a tissue- or developmental stage-specific fashion through the activity of tissue-specific factors (17
). In addition, there have been several documented examples of exon inclusion being regulated in response to extracellular stimuli, including stimulation of the T-cell receptor (TCR) (10
). However, the mechanisms by which such signaling events can influence pre-mRNA splicing are largely unexplored.
Engagement of the TCR by ligand initiates a signal transduction cascade within the T cell which ultimately results in a number of morphological and functional changes in the cell (11
). One such change is a dramatic alteration in the cell surface expression of isoforms of the transmembrane protein tyrosine phosphatase CD45 (for reviews see references 57
). The gene encoding CD45 encompasses 33 exons. Most of these exons, including those which encode the intracellular phosphatase domains, are constitutively included in the mature mRNA. However, three of the exons which encode part of the extracellular domain (exons 4, 5, and 6) are variably excluded from the mRNA. The peptide sequences encoded by the CD45 variable exons are rich in O-linked glycosylation sites; thus, a change in inclusion of these exons from the mRNA results in a dramatic change in the size and structure of the resulting CD45 protein (33
No ligand has yet been identified for CD45; however, the use of chimeric molecules has demonstrated that activity of the phosphatase domain of CD45 is influenced by homodimerization, suggesting that ligand binding might regulate CD45 phosphatase activity (14
). Moreover, the largest and smallest CD45 isoforms have been shown to differ in the ability to associate with the TCR (26
). In T cells, CD45 functions to maintain Lck in an active conformation by removing an inhibitory phosphate from this kinase (34
). Because Lck function is critical for early events in T-cell development and for activation, CD45 phosphatase activity is likewise required for both activation and development (7
). Thus, although the functional consequence of alternative isoform expression of CD45 is not yet clear, it is likely that the various isoforms differentially influence T-cell function due to a difference in their abilities to interact with ligand, with one another, or with the TCR.
CD45 is expressed in all nucleated hematopoietic cell types. Whereas CD45 surface expression changes frequently in T cells during development and upon activation, B cells only ever express the largest CD45 isoforms (58
). The difference in CD45 surface expression between B cells and thymocytes (which express predominantly the smallest CD45 isoforms) has clearly been shown to be a result of regulated alternative splicing. These two cell types, and cell lines derived from each, show a marked difference in their expression of CD45 mRNA variants (39
). Furthermore, this difference in mRNA expression requires sequences within and flanking the variable exons and is mediated by some, yet undefined, cell-specific factors (39
). An additional conclusion from these studies is that although exons 4, 5, and 6 are all variably included in CD45 mRNA, only the inclusion of exons 4 and 6 appears to be tightly regulated. Inclusion of exon 5, by contrast, is most likely a stochastic event.
Despite the progress in understanding the tissue-specific regulation of CD45 expression, characterization of activation-mediated changes in CD45 isoform expression in T cells has been significantly more limited. One reason for the limited understanding of activation-induced changes in CD45 expression is that all previous studies have analyzed primary T cells, which are not easily propagated or transfected and do not represent a homogeneous population (2
). In this report we describe a cell line-based assay which faithfully reproduces the activation-induced alternative splicing of CD45 which is observed in primary T cells. Using this cell line, we show that activation-induced exclusion of the CD45 variable exons is mediated via a protein kinase C (PKC)-dependent signaling pathway, which can be mimicked by constitutive activation of Ras. We rule out the possibility that CD45 alternative splicing is a general result of stimulation of PKC by demonstrating that treatment of several B cell lines with phorbol myristate acetate (PMA) has no effect on CD45 splicing, indicating some level of specificity to the response in T cells. In addition, synthesis of an activation-specific factor(s) is required for CD45 regulation, as indicated by the inhibition of the activation-induced alternative splicing of CD45 by treatment with cycloheximide. Last, we demonstrate that sequences within and flanking exon 4, which have previously been shown to be important for the cell-type-specific regulation of CD45 splicing, are similarly sufficient for mediating activation-induced splicing. In the future, this cell line should allow for a more detailed mechanistic study of CD45 splicing than is possible with primary cells, thereby leading to a greater understanding of how T-cell activation, and signaling events in general, may regulate alternative splicing.