The role of the CD45-associated phosphoprotein (LPAP / CD45-AP) during an immune response remains unclear. To understand better the function of LPAP we generated LPAP-deficient mice by disrupting exon 2 of the LPAP gene. LPAP-null mice were healthy and did not show gross abnormalities compared to their wild-type littermates. However, immunofluorescence analysis of T and B lymphocytes revealed a reduced expression of CD45, which did not affect a particular subpopulation. In contrast to a recent report (Matsuda et al., J. Exp. Med. 1998. 187: 1863 - 1870) we neither observed significant alterations of the assembly of the CD45 / lck-complex nor of polyclonal T-cell responses. However, lymphnodes from LPAP-null mice showed increased cellularity, which could indicate that expression of LPAP might be required to prevent expansion of lymphocytes in particular lymphatic organs rather than potentiating immune responses.

Previous work has demonstrated that SLP-76, a Grb2-associated tyrosine-phosphorylated protein, augments Interleukin-2 promoter activity when overexpressed in the Jurkat T cell line. This activity requires regions of SLP-76 that mediate protein-protein interactions with other molecules in T cells, suggesting that SLP-76-associated proteins also function to regulate signal transduction. Here we describe the molecular cloning of SLAP-130, a SLP-76-associated phosphoprotein of 130 kDa. We demonstrate that SLAP-130 is hematopoietic cell-specific and associates with the SH2 domain of SLP-76. Additionally, we show that SLAP-130 is a substrate of the T cell antigen receptor-induced protein tyrosine kinases. Interestingly, we find that in contrast to SLP-76, overexpression of SLAP-130 diminishes T cell antigen receptor-induced activation of the interleukin-2 promoter in Jurkat T cells and interferes with the augmentation of interleukin-2 promoter activity seen when SLP-76 is overexpressed in these cells. These data suggest that SLP-76 recruits a negative regulator, SLAP-130, as well as positive regulators of signal transduction in T cells.

Cytosolic extracts from a human lymphoblastoid B-cell line, RPMI-8392, established from a patient with acute lymphocytic leukemia, contain two major forms of cyclic nucleotide phosphodiesterase (PDE): Ca2+-calmodulin dependent PDE (PDE1) and cAMP-specific PDE (PDE4). In contrast, normal quiescent human peripheral blood lymphocytes (HPBL) are devoid of PDE1 activity [Epstein, P. M., Moraski, S., Jr., and Hachisu, R. (1987) Biochem. J. 243, 533-539]. Using reverse transcription-polymerase chain reaction (RT-PCR), we show that the mRNA encoding the 63-kDa form of PDE1 (PDE1B1) is expressed in RPMI-8392 cells, but not in normal, resting HPBL. This mRNA is, however, induced in HPBL following mitogenic stimulation by phytohemagglutinin (PHA). Also using RT-PCR, the full open reading frame for human PDE1B1 cDNA was cloned from RPMI-8392 cells and it encodes a protein of 536 amino acids with 96% identity to bovine, rat, and mouse species. RT-PCR also identifies the presence of PDE1B1 in other human lymphoblastoid and leukemic cell lines of B- (RPMI-1788, Daudi) and T-(MOLT-4, NA, Jurkat) cell origin. Inhibition of PDE1 or PDE4 activity by selective inhibitors induced RPMI-8392 cells, as well as the other cell lines, to undergo apoptosis. Culture of RPMI-8392 cells with an 18-bp phosphorothioate antisense oligodeoxynucleotide, targeted against the translation initiation region of the RPMI-8392 mRNA, led to a specific reduction in the amount of PDE1B1 mRNA after 1 day, and its disappearance after 2 days, and induced apoptosis in these cells in a sequence specific manner. This suggests that PDEs, particularly PDE1B1, because its expression is selective, may be useful targets for inducing the death of leukemic cells.

IL-1 beta-converting enzyme (ICE) cleaves pro-IL-1 beta to generate mature IL-1 beta. ICE is homologous to other proteins that have been implicated in apoptosis, including CED-3 and Nedd-2/lch-1. We generated ICE-deficient mice and observed that they are overtly normal but have a major defect in the production of mature IL-1 beta after stimulation with lipopolysaccharide. IL-1 alpha production is also impaired. ICE-deficient mice are resistant to endotoxic shock. Thymocytes and macrophages from the ICE-deficient animals undergo apoptosis normally. ICE therefore plays a dominant role in the generation of mature IL-1 beta, a previously unsuspected role in production of IL-1 alpha, but has no autonomous function in apoptosis.

Interleukin-1 beta converting enzyme (ICE) is a cytoplasmic cysteine protease required for generating the bioactive form of the interleukin-1 beta cytokine from its inactive precursor. We report the identification of ICH-2, a novel human gene encoding a member of the ICE cysteine protease family, and characterization of its protein product. ICH-2 mRNA is widely expressed in human tissues in a pattern similar to, but distinct from, that of ICE. Overexpression of ICH-2 in insect cells induces apoptosis. Purified ICH-2 is functional as a protease in vitro. A comparison of the inhibitor profiles and substrate cleavage by ICH-2 and ICE shows that the enzymes share catalytic properties but may differ in substrate specificities, suggesting that the two enzymes have different functions in vivo.

A cDNA clone for cyclic GMP Stimulated Phosphodiesterase (cGSPDE; PDE2) was isolated from a rat brain cDNA library. The cDNA has an open reading frame which encodes a protein of 928 amino acids of which 829 are identical with the reported bovine adrenal gland cGSPDE cDNA (Sonnenburg, W.K., Mullaney, P.J., and Beavo, J.A. (1991) J. Biol. Chem. 266, 17655-17661). Although the overall homology of these two cDNAs is high, they are distinctly different in their 5′ ends, with the N-terminal 37 amino acids of the rat brain protein showing no homology with the N-terminal end of the bovine adrenal protein. Hydrophilicity plots show that in contrast to the bovine adrenal cGSPDE, the N-terminal end of the rat brain cGSPDE is highly hydrophobic. Isolation and analysis of a genomic clone for cGSPDE from a rat genomic library shows the presence of an exon/intron junction at the Gln39 codon. The cGSPDE cDNA we have isolated and that of Sonnenburg et al. represent aternatively spliced mRNA products from the same gene, with the brain isoform designed to be targeted to membranes.

Interleukin-1 beta-converting enzyme (ICE) proteolytically cleaves pro-IL-1 beta to its mature, active form. The crystal structure at 2.5 Angstrom resolution of a recombinant human ICE-tetrapeptide chloromethylketone complex reveals that the holoenzyme is a homodimer of catalytic domains, each of which contains a p20 and a p10 subunit. The spatial separation of the C-terminus of p20 and the N-terminus of pin in each domain suggests two alternative pathways of assembly and activation in vivo. ICE is homologous to the C. elegans cell death gene product, CED-3, and these may represent a novel class of cytoplasmic cysteine proteases that are important in programmed cell death (apoptosis). Conservation among members of the ICE/CED-3 family of the amino acids that form the active site region of ICE supports the hypothesis that they share functional similarities.

c-abl is the normal cellular homolog of the v-abl transforming gene of Abelson murine leukemia virus. By constructing recombinants between c- and v-abl retroviruses, we show that a point mutation in c-Abl is sufficient to change the myristoylated form of c-Abl into a protein able to transform fibroblasts, but not capable of transforming bone marrow or inducing Abelson disease. This activating mutation, which changes the phenylalanine at amino acid 420 to valine (F420V) found in the homologous position of v-Abl, is positioned outside of the SH3 domain, a region typically modified in transforming alleles of abl. Phenylalanine 420 is perfectly conserved among tyrosine kinases with N-terminal SH3 domains (the Src and Abl families). The equivalent position in other protein tyrosine kinases is a conserved hydrophobic residue that predicts the specific family to which that kinase belongs. Mutation of phenylalanine 420 to other hydrophobic residues activates c-Abl. Unlike other transforming variants of Abl, the F420V mutant protein is not highly phosphorylated on tyrosine. Mutation of the nearby proposed autophosphorylation site, tyrosine 412, shows that this tyrosine is not strictly required for fibroblast transformation in either F420V or SH3-deleted variants of c-Abl (IV).

The c-abl gene in mice is transcribed into two major and at least two minor mRNAs which have different 5'-ends, but are otherwise colinear. Here we show that the two major mRNAs are initiated by separate promoters and that the minor transcripts arise by alternative splicing. Like in the human gene, one of the alternative murine 5' c-abl exons lies far upstream of the remaining exons. The major mRNA that begins with this exon has about 1275 nucleotides upstream of the abl coding region. Interestingly, this unusually long upstream mRNA segment contains multiple short open reading frames both in mouse and man and is highly conserved in sequence between these species. The 5'-most c-abl promoter contains several sequence motifs that are highly conserved between mouse and man. The downstream promoter is much less conserved.

The c-abl protooncogene is unusual in two respects; it has multiple, widely space N-terminal coding exons transcribed by different promoters, and it is the target of the translocations that form the Philadelphia chromosome found in cells of chronic myelogenous leukemia patients. To understand the organization of the gene in normal and chronic myelogenous leukemia patient DNA we have mapped c-abl by pulsed field gradient gel electrophoresis. We find that one of the alternative 5' exons of the gene lies at least 200 kilobases upstream of the remaining c-abl exons, posing formidable transcription and splicing problems. The 5'-most c-abl exon includes an unusually long 1,276-base-pair segment that contains 15 ATG codons and multiple short open reading frames, upstream of the abl initiator codon. Its peculiar structure suggests that c-abl may be decapitated in most chronic myelogenous leukemia patients, and we demonstrate that this is the case in the chronic myelogenous leukemia cell line K562.

The cellular abl proto-oncogene encodes a proteintyrosine kinase and is expressed in many cell types in two or three mRNA size species. Four types of mouse c-abl cDNAs have been cloned from 70Z/3 lymphoid cells that have different 5′ sequences encoding predicted N-terminal regions of 20–45 amino acids. One of the four cDNAs has a predicted N-terminal sequence of met-gly-gln in common with the gag N terminus of v-abl. The 5′ heterogeneity appears to be generated by alternative addition of 5′ exons onto a common set of 3′ exons. Alternative splicing occurs at the same site at which bcr sequences join to abl sequences in the Philadelphia chromosome translocation.

The Philadelphia chromosome, observed in greater than 90% of patients with chronic myelogenous leukemia, results from a reciprocal translocation between chromosomes 9 and 22. The translocation breakpoint on chromosome 9 occurs near the ABL gene and correlates with the production of a chronic myelogenous leukemia-specific 8.5-kilobase ABL-related mRNA species accompanied by a structurally altered ABL protein (P210c-abl). The N-terminal sequence of the protein is derived from the BCR gene on chromosome 22. We have isolated overlapping cDNA clones from the K-562 cell line corresponding to approximately 8.5 kilobases of mRNA and have sequenced 2550 nucleotides at the 5' end. Our results indicate that the 5' end of the 8.5-kilobase mRNA consists of greater than 400 nucleotides of noncoding sequence that are greater than 80% G + C rich. Based on our sequence analysis, we propose that initiation of translation occurs at nucleotide 471, such that the initial 927 amino acids of P210c-abl are derived from BCR sequences. Our cDNA clones thus define the complete coding sequences for the P210c-abl gene product.

The variable regions of immunoglobulin heavy chains are encoded in the germ line by three discrete DNA segments: VH (variable) elements, D (diversity) elements and JH (joining) elements. During the differentiation of B lymphocytes, individual segments from each group are brought together by recombination to form the complete VHDJH variable region. To understand these processes better, we have now isolated and sequenced molecular clones representing intermediates (DJH fusions) and final products (VH-to-DJH joins) of heavy-chain gene rearrangement in two cell lines that represent analogues of cells at early stages of B-lymphocyte differentiation. Heavy-chain gene assembly in one cell line but not in the other is accompanied by the appearance of short nucleotide insertions at the recombinational junctions. The generation of such insertions is positively correlated with the expression of terminal deoxynucleotidyl transferase in these lines.

The most JH-proximal VH gene segments are used highly preferentially to form VHDJH rearrangements in pre-B-cell lines. This result demonstrates that the rate at which immunoglobulin VH gene segments recombine is influenced by their chromosomal organization, and that the initial repertoire of VH genes expressed in pre-B cells is strikingly different from that seen in mature populations.

Circular duplex reverse transcripts of the genome of a strain of Abelson murine leukemia virus that encodes a 160,000-molecular-weight protein were isolated, cleaved with HindIII restriction endonuclease, and cloned into the unique HindIII site of lambda phage Charon 21A. Recombinant phage clones, some of which were infectious in transfection assays, were found to contain a 789-base-pair region specific for Abelson murine leukemia virus; this region is not found in other strains of this virus. The extra sequence was localized by restriction endonuclease and electron microscopic heteroduplex analysis. Sequence analysis showed no homology at the ends of the extra sequence, implying that it was deleted by an event that did not utilize sequence homology. The sequence of this unique region has an open reading frame through its entirety.

Studies of the murine lambda light chains produced by myeloma cells provided the first evidence for somatic point mutation of germ-line variable (V) region genes. An examination of the variable regions of 19 lambda 1 chains revealed seven which differed from a common sequence by one to three amino acid substitutions. Subsequently, one of these presumed somatic variants of the single lambda 1 V gene was characterized by DNA sequence analysis of the rearranged functional gene. The predicted DNA sequence alteration was observed and no silent mutation was evident. These studies of lambda chain variants suggested that the hypervariable, complementarity-determining regions (CDRs) ht be a preferred site of somatic mutation because all seven characterized variants contained substitutions only in these regions. By contrast, comparisons of closely related kappa chain variable region amino acid sequences, and more recently VK and VH genes, have suggested that somatic mutation probably occurs in codons for both framework and CDR residues. To examine this apparent discrepancy between the sites of somatic mutations in lambda and kappa genes, we have determined the nucleotide sequence of two lambda 1 gene from hybridomas and a lambda 2 gene from a myeloma. These sequences demonstrate that somatic mutation in lambda genes can occur in both the framework and CDR residues.