The role of alternative splicing (AS) in eliciting immune responses is poorly understood. We used quantitative AS microarray profiling to survey changes in AS during activation of Jurkat cells, a leukemia-derived T-cell line. Our results indicate that ;10%-15% of the profiled alternative exons undergo a >10% change in inclusion level during activation. The majority of the genes displaying differential AS levels are distinct from the set of genes displaying differential transcript levels. These two gene sets also have overlapping yet distinct functional roles. For example, genes that show differential AS patterns during T-cell activation are often closely associated with cell-cycle regulation, whereas genes with differential transcript levels are highly enriched in functions associated more directly with immune defense and cytoskeletal architecture. Previously unknown AS events were detected in genes that have important roles in T-cell activation, and these AS level changes were also observed during the activation of normal human peripheral CD4+ and CD8+ lymphocytes. In summary, by using AS microarray profiling, we have discovered many new AS changes associated with T-cell activation. Our results suggest an extensive role for AS in the regulation of the mammalian immune response.

ALS2 is the gene mutated in a recessive juvenile form of amyotrophic lateral sclerosis (ALS2). ALS2 encodes a large protein termed alsin, which contains a number of predicted cell signaling and protein trafficking sequence motifs. To gain insight into the overall function of alsin and to begin to evaluate its role in motor neuron maintenance, we examined the subcellular localization of alsin and the biochemical activities associated with its individual subdomains. We found that the Vps9p domain of alsin has Rab5 guanine nucleotide exchange activity. In addition, alsin interacted specifically with and acted as a guanine nucleotide exchange factor for Rac1. Immunofluorescence and fractionation experiments in both fibroblasts and neurons revealed that alsin is a cytosolic protein, with a significant portion associated with small, punctate membrane structures. Many of these membrane structures also contained Rab5 or Rac1. Upon overexpression of full-length alsin, the overexpressed material was largely cytosolic, indicating that the association with membrane structures could be saturated. We also found that alsin was present in membrane ruffles and lamellipodia. These data suggest that alsin is involved in membrane transport events, potentially linking endocytic processes and actin cytoskeleton remodeling.Amyotrophic lateral sclerosis (ALS) 1 is a heterogeneous neurological disorder characterized by progressive degeneration of motor neurons, usually causing death as a result of respiratory paralysis (1, 2). Although mostly sporadic in nature, a genetic link has been established in 5-10% of ALS cases (3). Chromosomal mapping studies have identified six independent loci associated with the familial forms of ALS (Refs. 4 -10; reviewed in Refs. 11 and 12). Molecular genetic analysis identified two genes that, when mutated, lead to ALS. The first discovered was the gene coding for Cu-Zn superoxide dismutase 1 (SOD1) (10). Initially, mutations in SOD1 were thought to result in defective free radical scavenger activity. However, it is now generally accepted that the alterations in SOD1 that lead to ALS are the result of an unknown, but toxic gain-of-function. The second gene identified is mutated in a juvenile form of ALS, ALS2 (13,14). Mutations in this gene lead to a rare recessive form of ALS that presents early in life and progresses much more slowly than the classical form (6, 15). Two small deletions in ALS2 were originally associated with the disease (13,14). Each is expected to severely truncate the predicted protein product of ALS2. In addition, mutations in ALS2 have recently been associated with two other neurodegenerative disorders, juvenile-onset primary lateral sclerosis (14) and infantile-onset hereditary spastic paralegia (16 -18). Like the original mutations identified, these mutations are predicted to generate prematurely truncated forms of the protein product.The protein encoded by ALS2, alsin, is predicted to contain numerous domains implicating roles in cell signaling, membrane localization, and protein...

The human CD45 gene encodes a protein-tyrosine phosphatase that exhibits differential isoform expression in resting and activated T cells due to alternative splicing of three variable exons. Previously, we have used biochemical methods to identify two regulatory proteins, hnRNP L and PSF, which contribute to the activation-induced skipping of CD45 via the ESS1 regulatory element in variable exon 4. Here we report the identification of a third CD45 regulatory factor, hnRNP L-like (hnRNP LL), via a cell-based screen for clonal variants that exhibit an activation-like phenotype of CD45 splicing even under resting conditions. Microarray analysis of two splicing-altered clones revealed increased expression of hnRNP LL relative to wild-type cells. We further demonstrate that both the expression of hnRNP LL protein and its binding to ESS1 are up-regulated in wild-type cells upon activation. Forced overexpression of hnRNP LL in wild-type cells results in an increase in exon repression, while knockdown of hnRNP LL eliminates activation-induced exon skipping. Interestingly, analysis of the binding of hnRNP L and hnRNP LL to mutants of ESS1 reveals that these proteins have overlapping, but distinct binding requirements. Together, these data establish that hnRNP LL plays a critical and unique role in the signal-induced regulation of CD45 and demonstrate the utility of cell-based screens for the identification of novel splicing regulatory factors.

Calcium has been implicated in regulating vesicle fusion reactions, but its potential role in regulating other aspects of protein transport, such as vesicle assembly, is largely unexplored. We find that treating cells with the membrane-permeable calcium chelator, 1,2-bis(2-aminophenoxy)ethane-N,N,N,N-tetraacetic acid tetrakis(acetoxymethyl ester) (BAPTA-AM), leads to a dramatic redistribution of the vesicle coat protein, coatomer, in the cell. We have used the cell-free reconstitution of coatprotomer I (COPI) vesicle assembly to characterize the mechanisms of this redistribution. We find that the recovery of COPI-coated Golgi vesicles is inhibited by the addition of BAPTA to the cell-free vesicle budding assay. When coatomer-coated membranes are incubated in the presence of calcium chelators, the membranes "uncoat," indicating that calcium is necessary for maintaining the integrity of the coat. This uncoating is reversed by the addition of calcium. Interestingly, BAPTA, a calcium chelator with fast binding kinetics, is more potent at uncoating the coatomer-coated membrane than EGTA, suggesting that a calcium transient or a calcium gradient is important for stabilizing COPI vesicle coat. The primary target for the effects of calcium on coatomer recruitment is a step that occurs after ADP-ribosylation factor binding to the membrane. We suggest that a calcium gradient may serve to regulate the timing of vesicle uncoating.

Rab5GTPases are key regulators of protein trafficking through the early stages of the endocytic pathway. The yeast Rab5 ortholog Vps21p is activated by its guanine nucleotide exchange factor Vps9p. Here we show that Vps9p binds ubiquitin and that the CUE domain is necessary and sufficient for this interaction. Vps9p ubiquitin binding is required for efficient endocytosis of Ste3p but not for the delivery of the biosynthetic cargo carboxypeptidase Y to the vacuole. In addition, Vps9p is itself monoubiquitylated. Ubiquitylation is dependent on a functional CUE domain and Rsp5p, an E3 ligase that participates in cell surface receptor endocytosis. These findings define a new ubiquitin binding domain and implicate ubiquitin as a modulator of Vps9p function in the endocytic pathway.Rab proteins are critical regulators of the vesicle targeting and fusion events (reviewed in Refs. 1 and 2). Discrete classes of these small GTPases mediate very specific transport events, showing little if any functional overlap. For example, the Rab5 family members appear to be involved exclusively in targeting events within the early stages of the endocytic pathway (3, 4). The activation of Rab5 GTPases like all Rab proteins is dependent on the state of bound nucleotide, GDP or GTP. Two classes of proteins that modulate the Rab nucleotide occupancy are the GTPase activating proteins (GAPs) 1 and the guanine nucleotide exchange factors (GEFs). GAPs stimulate GTP hydrolysis, leaving the Rab in the GDP-bound, inactive state; conversely, GEFs initiate GDP release to permit GTP binding and thereby Rab activation. Multiple GAPs and GEFs for the Rab proteins have been identified, and an interesting distinction has been observed (reviewed in Ref. 5). The Rab GAPs share a conserved sequence motif and exhibit substrate promiscuity among the Rab families (6, 7) (reviewed in Ref. 8). In contrast, the GEF proteins for different Rab families are dissimilar at the sequence level and show great specificity for their cognate Rab proteins. Consequently, the GEFs appear to be the primary mechanism to control specific Rab activity.A number of exchange factors for Vps21p/Rab5 family members have been identified in mammalian and yeast systems. In yeast, Vps9p is the exchange factor for the Rab5 ortholog Vps21p (9). VPS9 was initially identified in genetic screens for mutants defective in vacuolar protein sorting (10). In vitro reconstitution of Vps9p-stimulated GDP release and GTP loading onto Vps21p demonstrated that Vps9p is the GEF for Vps21p (9). Concurrently, Rabex5 was identified as a Rab5-binding protein and demonstrated to exhibit in vitro GEF activity (11). In addition to conserved functions in activating Rab5 proteins, yeast Vps9p (451 amino acids) and human Rabex5 (491 amino acids) share 27% overall sequence identity (11). A peptide comprised of residues 158 -347 of Vps9p was identified as the domain necessary and sufficient for GEF catalytic activity. 2 Although Vps9p is the only known GEF for Vps21p in yeast, six human proteins have been identifi...