Itk, a Tec family tyrosine kinase, plays an important but as yet undefined role in T cell receptor (TCR) signaling. Here we show that T cells from Itk-deficient mice have a TCR-proximal signaling defect, resulting in defective interleukin 2 secretion. Upon TCR stimulation, Itk−/− T cells release normal amounts of calcium from intracellular stores, but fail to open plasma membrane calcium channels. Since thapsigargin-induced store depletion triggers normal calcium entry in Itk−/− T cells, an impaired biochemical link between store depletion and channel opening is unlikely to be responsible for this defect. Biochemical studies indicate that TCR-induced inositol 1,4,5 tris-phosphate (IP3) generation and phospholipase C γ1 tyrosine phosphorylation are substantially reduced in Itk−/− T cells. In contrast, TCR-ζ and ZAP-70 are phosphorylated normally, suggesting that Itk functions downstream of, or in parallel to, ZAP-70 to facilitate TCR-induced IP3 production. These findings support a model in which quantitative differences in cytosolic IP3 trigger distinct responses, and in which only high concentrations of IP3 trigger the influx of extracellular calcium.
TIA-1 and TIAR are two closely related RNA recognition motif (RRM) proteins which possess three RRMtype RNA binding domains (RRMs 1, 2, and 3). Although both proteins have been implicated as effectors of apoptotic cell death, the specific functions of TIA-1 and TIAR are not known. We have performed in vitro selection/amplification from pools of random RNA sequences to identify RNAs to which TIA-1 and TIAR bind with high affinity. Both proteins selected RNAs containing one or several short stretches of uridylate residues suggesting that the two proteins have similar RNA binding specificities. Replacement of the uridylate stretch with an equal number of cytidine residues eliminates the protein-RNA interaction. Mutational analysis indicates that, for both TIA-1 and TIAR, it is the second RNA binding domain (RRM 2) which mediates the specific binding to uridylate-rich RNAs. Although RRM 2 is both necessary and sufficient for this interaction, the affinity for the selected RNA (as determined by filter binding assays) does increase when the second domain of TIAR is expressed together with the first and third domainsAlthough RRM 3 (of either TIA-1 or TIAR) does not interact with the uridylate-rich sequences selected by the full-length proteins, it is a bona fide RNA binding domain capable of affinity-precipitating a population of cellular RNAs ranging in size from 0.5 to 5 kilobases. In contrast, RRM 1 does not affinity-precipitate cellular RNA. The inability of RRM 1 to interact with RNA may be due to the presence of negatively charged amino acids within the RNP 1 octamer.RNA-binding proteins are involved in a variety of fundamental cellular processes including RNA splicing, polyadenylation, RNA transport, and translation. Specific RNA sequences with which these proteins interact have been identified in some cases, but for the majority of RNA-binding proteins, the RNA targets are unknown. TIA-1 and TIAR are two closely related members of the RNA recognition motif (RRM) 1 family of RNAbinding proteins (1, 2). The RRM (also known as the RNP motif, the RNP consensus sequence, the RNP-80, and the consensus sequence RNA-binding domain) consists of 80 -90 amino acids containing two stretches of 8 and 6 highly conserved residues called RNP 1 and RNP 2, respectively (3-5). TIA-1 and TIAR both possess three amino-terminal RRM domains and a glutamine-rich carboxyl terminus. The RRM domains of TIA-1 and TIAR are very similar with 79% amino acid identity between the first domains, 89% amino acid identity between the second domains, and 91% amino acid identity between the third domains. The carboxyl termini of the two proteins, in contrast, are only 51% identical in amino acid sequence (2). Several observations suggest that TIA-1 and TIAR are involved in signaling apoptotic cell death. The introduction of purified TIA-1 or TIAR into the cytoplasm of thymocytes permeabilized with digitonin results in fragmentation of genomic DNA into nucleosome-sized oligomers (1, 2). TIAR is translocated from the nucleus to the cytoplasm in respon...
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