The human CD45 gene encodes five isoforms of a transmembrane tyrosine phosphatase that differ in their extracellular domains as a result of alternative splicing of exons 4 -6. Expression of the CD45 isoforms is tightly regulated in peripheral T cells such that resting cells predominantly express the larger CD45 isoforms, encoded by mRNAs containing two or three variable exons. In contrast, activated T cells express CD45 isoforms encoded by mRNAs lacking most or all of the variable exons. We have previously identified the sequences within CD45 variable exon 4 that control its level of inclusion into spliced mRNAs. Here we map the splicingregulatory sequences within CD45 variable exons 5 and 6. We show that, like exon 4, exons 5 and 6 each contain an exonic splicing silencer (ESS) and an exonic splicing enhancer (ESE), which together determine the level of exon inclusion in naïve cells. We further demonstrate that the primary activation-responsive silencing motif in exons 5 and 6 is homologous to that in exon 4 and, as in exon 4, binds specifically to the protein heterogeneous nuclear ribonucleoprotein L. Together these studies reveal common themes in the regulation of the CD45 variable exons and provide a mechanistic explanation for the observed physiological expression of CD45 isoforms.In order to attain proteomic and functional complexity, higher eukaryotes have developed numerous mechanisms to amplify the informational content of their relatively limited genomes. One such mechanism, alternative splicing, allows for the production of multiple unique mRNAs from a single gene through the differential inclusion of exons. Since the variant mRNAs produced by alternative splicing can each potentially encode for a functionally distinct protein, alternative splicing is recognized to be a ubiquitous and critical mechanism for regulating cellular function (1, 2).The importance of regulated alternative splicing in humans is exemplified by the CD45 gene, which encodes a transmembrane proteintyrosine phosphatase (3). CD45 phosphatase activity is critical for intracellular signaling in T cells in response to antigen stimulation, as indicated by the severe immunodeficiency observed in CD45-deficient mice and humans (4 -7). Five isoforms of the CD45 protein are expressed in humans as a result of alternative inclusion of exons 4 -6 (see Fig. 1A). In human T cells, the expression of the CD45 isoforms is tightly regulated throughout development and upon activation (3). In particular, naive peripheral T cells express significant amounts of the larger isoforms of CD45, which have high phosphatase activity and maintain the T cell receptor in a state primed for antigen recognition (8). Subsequent to antigen stimulation, however, there is a marked shift in the processing of CD45 pre-mRNA, such that the variable exons are predominantly excluded from the final message, resulting in expression of the smaller CD45 isoforms (9, 10). The smaller isoforms of CD45 are more prone to homodimerization than the larger isoforms (8, 11). Importantl...

The DEP domain of Dishevelled (Dvl) proteins transduces signals to effector proteins downstream of Dvl in the Wnt pathway. Here we report that DEP-containing mutants inhibit Wnt-induced, but not Dvl-induced, activation of the transcription factor Lef-1. This inhibitory effect is weakened by a K434M mutation. Nuclear magnetic resonance spectroscopy revealed that the DEP domain of mouse Dvl1 comprises a three-helix bundle, a β-hairpin 'arm' and two short β-strands at the Cterminal region. Lys 434 is located at the tip of the β-hairpin 'arm'. Based on our findings, we conclude that DEP interacts with regulators upstream of Dvl via a strong electric dipole on the molecule's surface created by Lys 434, Asp 445 and Asp 448; the electric dipole and the putative membrane binding site are at two different locations.The Wnt signaling pathway is a key regulatory pathway for cellular development and growth. Wnt signaling has been extensively studied in Drosophila, Caenorhabditis elegans, Xenopus, and mammalian systems [1][2][3][4][5] . Through genetic studies, a working model of the Wnt signaling pathway has been established. Secreted Wnt proteins bind to Frizzled transmembrane receptors. The receptor-ligand complex activates the Dishevelled (Dsh and Dvl) proteins, which suppress the activity of glycogen synthase kinase-3β (GSK-3β). If its activity is not suppressed, GSK-3β binds to adenomatous polyposis coli (APC) protein and axin and then phosphorylates specific Ser and Thr residues at the N-terminus of β-catenin. In turn, the ubiquitin-proteasome pathway rapidly degrades hyperphosphorylated β-catenin. Wnt activation prevents this process by promoting the cytosolic accumulation and subsequent translocation of β-catenin into the nucleus. Once in the nucleus, β-catenin binds to the transcription factors T-cell factor (Tcf) or lymphoid enhancer factor (Lef) and acts as a transcriptional coactivator 5 . The result is increased expression of Tcf or Lef regulated target genes, such as Myc 6 and the gene encoding cyclin D 7 .© 2000 Nature America Inc. Correspondence should be addressed to: J.Z. Jie.Zheng@stjude.org. HHS Public Access DEP domain interacts with upstream regulators of DvlWe and others have demonstrated that Wnt-1 or Dvl1, when expressed in NIH3T3 cells, can activate the transcription regulator 16,28,29). This activation depends on β-catenin and is suppressed by GSK-3β and axin. To characterize the role of the mDvl1 DEP domain in Wnt signaling, we examined the effect of the Dvl1 N-terminal truncation mutant, DvlC1, on Wnt-induced activation of the transcription factor Lef-1. In Lef dependent reporter gene assays, DvlC1 inhibited Wnt-1 induced Lef-1 activation but not Dvl1 induced Lef-1 activation (Fig. 2). To further characterize the specific sequences that inhibit the Wnt-induced activation, we generated two additional mutants of mDvl1: DvlDEP, which contains only the DEP domain; and DvlC2, which encompasses the sequence downstream of the DEP domain. When expressed in NIH3T3 cells, DvlDEP, like DvlC1, bloc...