| Abstract: | Src family kinases are central regulators of a large number of signaling pathways. To adapt to the idiosyncrasies of different cell types, these kinases may need a fine-tuning of their intrinsic molecular control mechanisms. Here, we describe on a molecular level how the Fyn kinase uses alternative splicing to adapt to different cellular environments. Using structural analysis, site-directed mutagenesis, and functional analysis, we show how the inclusion of either exon 7A or 7B affects the autoinhibition of Fyn and how this changes the SH3-dependent interaction and tyrosine phosphorylation of Sam68, with functional consequences for the Sam68-regulated survival of epithelial cells. Our results illustrate a novel mechanism of evolution that may contribute to the complexity of Src kinase regulation.The Src family of nonreceptor protein tyrosine kinases comprises nine members, including Src, Blk, Fgr, Fyn, Hck, Lyn, Lck, Yes, and Yrk. These kinases play crucial roles in a variety of cellular processes, such as cell cycle control, cell adhesion, cell motility, cell proliferation, and cell differentiation (41). Extensive studies indicate that the complexity of functional roles of Src kinases derives mainly from their ability to communicate with a large number of upstream receptors and downstream effectors, which vary by cell type (31). Given their critical role, diverse mechanisms of autoregulation have evolved, and their importance is highlighted by the implication of elevated Src expression levels and/or activity in epithelial cancers (for a review, see reference 48). The autoregulatory mechanisms depend on the composition and order of various domains and on posttranslational modification sites in the linker segments that connect the domains (35). From the N to C terminus, Src contains a myristoyl group attached to a unique domain, an Src homology 3 (SH3) domain that typically binds left-handed polyproline type II sequence motifs, an SH2 domain that binds to tyrosine-phosphorylated protein motifs, a protein-tyrosine kinase domain (SH1), and a C-terminal regulatory segment. Early biochemical studies suggested that these domains were critical for keeping Src catalytic activity under control (4, 23, 39, 40). The validation of the autoinhibitory role of these regulatory moieties came from the structures of Src and Hck kinases (36, 37, 43, 46, 47). The structures showed how interdomain interactions, stabilized by the binding of the SH2 domain to the tyrosine-phosphorylated C terminus (pTyr528), lock the molecule in a closed conformation. They further showed the unanticipated finding that residues in the linker region between the SH2 domain and the kinase domain, the SH2-kinase linker, make direct contact with the catalytic domain and adopts a polyproline type II helix conformation that docks onto the SH3 domain. This intramolecular interaction hinders the formation of a salt bridge that is crucial for the kinase activity, thereby eliciting an inhibitory effect. However, these interactions are suboptimal, and other phosphotyrosine- or polyproline-containing sequences can compete favorably with Src''s own sequences for SH2 or SH3 domain binding (3, 25). These binding events lead to the stimulation of Src kinase activity by disrupting the intramolecular constraints imposed on the kinase domain. Once released from the repressed state, the autophosphorylation of tyrosine residue Tyr416 (pTyr416) in the activation loop rapidly occurs, resulting in a conformational change that releases a fully active kinase.Remarkably, recent advances have highlighted the crucial role of linker regions in establishing the structural and functional assembly of multimodular proteins in signal transduction, and Src kinases are influential in our understanding of these mechanisms (13). The nine Src family members are very similar in terms of sequence identity, with, for example, the strong conservation of the SH3 binding surface and the cores of the kinase domain (44). Nevertheless, high sequence variability is noted in the SH2-kinase linker segment, except that the overall hydrophobicity is conserved. The interactions that this linker makes with both the SH3 domain and the back of the kinase domain probably result in a high-specificity binding. Indeed, the activities of chimeras in which the SH3 domain of Src kinases have been swapped show altered regulation (12, 14, 16). Furthermore, in contrast to deletion or point mutations in the SH3 domain, Src mutants in the linker segment or in the linker-interacting surface on the catalytic domain can transform fibroblast, suggesting specific function(s) (14).Src kinases originated by the duplication and diversification of the same ancestral gene with an original 10-intron structure before the separations of Teleostei from Tetrapoda (6). One of the Src-related kinases, Fyn, possesses two kinds of exon 7, exon 7A and exon 7B, essentially encoding for the SH2-kinase linker segment and the N terminus of the SH1 domain. The alternative splicing of exon 7A or 7B yields two major Fyn isoforms, FynB (exon 7A) and FynT (exon 7B) (7). Exon 7A shows a different evolutionary pattern from that of the other parts of the gene, suggesting that it is derived by a recombinatorial event with another gene (33). The newly captured exon, encoding FynB, was coopted by the central nervous system and possibly other tissues, while the ancestral isoform, encoding FynT, is expressed mainly in the hematopoietic system (7, 32). Whether this diversification process generated intrinsic biochemical functional novelty in addition to the differential tissue distribution and related functional divergence currently is unknown. Since the alternatively spliced exon that distinguishes the two isoforms essentially encodes for the SH2-kinase linker segment, it is possible that it confers distinct regulatory features. Thus, this evolutionary divergence in the SH2-linker segment of FynT and FynB, which maintain identical SH2, SH3, and kinase domains, offers the unique opportunity to explore the specific functions that this linker segment may impose on Src kinase function and/or regulation.Here, we have investigated how exons 7A and 7B affect the functional interaction of Fyn with the RNA-binding protein Sam68. Sam68 is known to activate Fyn by binding to its SH3 domain and also to serve as a substrate for phosphorylation by Fyn. We show that FynT and FynB display a distinct capacity to bind and phosphorylate Sam68. This differential interaction with a substrate is functionally relevant, because it allows the specific phosphorylation-mediated regulation of the Sam68-dependent alternative splicing of Bcl-x by FynT and results in the differential regulation of apoptosis in epithelial cells. Swapping experiments identify core residues of the exon 7A- or 7B-encoded SH2-kinase linker segment as both required and sufficient to confer this distinct function. In agreement with structural models, our data show that exon 7B reinforces the autoinhibitory lock that the SH2-linker region imposes onto the kinase domain and on SH3 domain accessibility. These results uncover a novel specific function that the SH2-kinase linker segment can play in Src biology and highlight the importance of alternative splicing for the acquisition of fine-tuning regulatory functions during evolution. |
