The c-kit gene encodes a tyrosine kinase receptor (KIT) that is required in normal spermatogenesis and is expressed in seminomas and dysgerminomas, a subset of human germ cell tumors (GCTs). To determine whether activating mutations of the c-kit gene occur in GCTs , primary tissue samples of 33 testicular and ovarian tumors were examined for mutations in the juxtamembrane and phosphotransferase domains by polymerase chain reaction amplification and DNA sequencing. A novel missense mutation (D816H) was found in the phosphotransferase domain in tumors of seminoma/dysgerminoma differentiation. The c-kit alleles in nonneoplastic tissues from these patients were wild type , suggesting that the mutant alleles were acquired and selected for during malignant transformation. In cell transfection experiments , the D816H mutant protein was a constitutively activated kinase and was constitutively phosphorylated on tyrosine residues. This is the first description of an activating c-kit mutation in GCTs and is evidence that the KIT signal transduction pathway is important in the pathogenesis of neoplasms with seminoma differentiation. (Am J Pathol 1999, 154:1643-1647)

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We recently reported the molecular cloning of a cytotoxic granule-associated RNA-binding protein designated TIA-1. The ability of recombinant TIA-1 to induce DNA fragmentation in permeabilized cells suggested that this protein is the granule component responsible for inducing apoptosis in cytolytic lymphocyte (CTL) targets. Here we report the characterization of a cDNA encoding a TIA-1-related protein designated TIAR. The deduced amino acid sequence of TIAR reveals it to be a 42-kDa protein possessing three RNA-binding domains and a carboxyl-terminal auxiliary domain. Although the RNA-binding domains of TIA-1 and TIAR share >85% amino acid homology, their carboxyl-terminal auxiliary domains are only 51% homologous. The carboxyl terminus of TIAR contains a lysosome-targeting motif, indicating that TUAR is probably a cytotoxic granule-associated protein. Like TIA-1, purified recombinant TIAR induced DNA fragmentation in permeabilized target cells. Although immunoblotting analysis of post-nuclear supernatants revealed TIA-1 protein to be restricted to CTLs, PCR analysis revealed the expression of TIA-1 and TIAR mRNA transcripts in a wide variety of cell types. Our data suggest that the granules of CTLs contain at least two candidate nucleolysins involved in CTL killing.

SllnullaryWe have identified a serine/threonine kinase that is rapidly activated during Fas-mediated apoptosis. Fas-activated serine/threonine kinase (FAST) is phosphorylated on serine and threonine residues in Jurkat cells. In response to Fas ligation, it is rapidly dephosphorylated and concomitantly activated to phosphorylate TIA-1, a nuclear RNA-binding protein that has been implicated as an effector of apoptosis. Phosphorylation of TIA-1 precedes the onset of DNA fragmentation, suggesting a role in signaling downstream events in the apoptotic program. Our results introduce FAST and TIA-1 as components of a molecular cascade involved in signaling Fas-mediated apoptosis.

Covalent modification of the Ran GTPase-activating protein RanGAP1 with the ubiquitin-related protein SUMO-1 promotes its association with Nup358, a component of the cytoplasmic fibrils emanating from the nuclear pore complex (1, 2). In Xenopus egg extracts, Nup358 can be found in a complex with Ubc9 (3), a structural homologue of the E2-type ubiquitin-conjugating enzymes (UBCs). Here we show that a subset of the human homologue of Ubc9 (HsUbc9) colocalizes with Ran-GAP1 at the nuclear envelope. HsUbc9 forms thiolester conjugates with recombinant SUMO-1, but not with recombinant ubiquitin, indicating that it is functionally distinct from E2-type UBCs. Finally, HsUbc9 is required for the modification of RanGAP1 by SUMO-1. These results suggest that HsUbc9 is a component of a novel enzymatic cascade that modifies RanGAP1, and possibly other substrates, with SUMO-1.The transport of selected proteins and nucleic acids across the nuclear pore complex (NPC) 1 is regulated by the small GTPase Ran/TC4 (4, 5). Ran alternates between a GTP-bound state and a GDP-bound state, a transition facilitated by a nuclear GTP-exchange factor (RCC1) and a cytoplasmic GTPase-activating protein (RanGAP1) (reviewed in Refs. 6 and 7). The differential binding of Ran-GTP and Ran-GDP to carrier proteins (e.g. importins, transportin, and NTF2) and NPC proteins provides a mechanism that allows Ran to regulate the transport of cargo across the nuclear pore complex. In this system, the subcellular localization of RCC1 and RanGAP1 are major determinants of directed nuclear transport.The localization of RanGAP1 to the nuclear pore complex is imparted by its specific association with Nup358/RanBP2 (1), a component of the cytoplasmic filaments emanating from the NPC (8, 9). This interaction requires the covalent modification of RanGAP1 with a ubiquitin-related protein designated SUMO-1 (1, 2). Recently, Ubc9, a structural homologue of the E2-type UBCs, was shown to coimmunoprecipitate with Nup358 in Xenopus egg extracts (3). Previous studies indicated that Ubc9 may be required for regulating the progression through the cell cycle in yeast (10,11). In addition, the human homologue of Ubc9 (HsUbc9) can functionally substitute for yeast Ubc9 (12,13). Although HsUbc9 has been proposed to mediate the ubiquitin-catalyzed degradation of mitotic cyclins (10 -12), no bona fide ubiquitin-conjugating activity has been demonstrated for HsUbc9.We now show that HsUbc9 differs significantly from the E2 family of UBCs in that it can form a thiolester intermediate with SUMO-1, rather than ubiquitin. This enzymatic activity is ATP-dependent and is not observed in mutants of HsUbc9, which contain a single amino acid substitution at the putative catalytic site. Finally, HsUbc9 is required for the modification of RanGAP1 by SUMO-1. MATERIALS AND METHODS Preparation of Affinity Purified Antibodies-Rabbit antiserum raisedagainst Escherichia coli-derived recombinant HsUbc9 was affinity purified by passage over a CNBr-activated Sepharose B matrix coupled to E. coli-deri...