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S Desiderio

Publications and source records attributed to S Desiderio.

At least 37 records · Page 2Linked to original sources
PubMed1996

A conserved degradation signal regulates RAG-2 accumulation during cell division and links V(D)J recombination to the cell cycle.

The proteins RAG-1 and RAG-2 are essential for initiation of V(D)J recombination. In dividing cells, RAG-2 accumulates during G1 and is undetectable during the S and G2/M cell cycle phases. A conserved degradation signal, including an essential CDK phosphorylation site at Thr-490, regulates RAG-2 accumulation during cell division and links V(D)J recombination to the cell cycle. Mutations within this signal abolish periodic degradation of RAG-2 protein in dividing cells. In mice expressing endogenous or wild-type transgenic RAG-2, V(D)J recombination intermediates accumulate preferentially in G0/G1 thymocytes; this restriction is relieved by mutation of Thr-490 to alanine (T490A). Thus, periodic destruction of RAG-2 protein couples V(D)J recombination to cell cycle phase. Using transgenic mice expressing the T490A RAG-2 mutant and a functional T cell receptor beta chain, we demonstrate that coupling of V(D)J recombination to the cell cycle is not essential for enforcement of allelic exclusion.

Amino Acid Sequence↗
PubMed1995

An SH3-binding site conserved in Bruton's tyrosine kinase and related tyrosine kinases mediates specific protein interactions in vitro and in vivo.

Mutations in Bruton's tyrosine kinase (Btk) have been associated with immunodeficiencies in man and in the mouse. Btk and two related proteins, Itk and Tec, are members of a distinct family of tyrosine kinases. These kinases are believed to function in various receptor-mediated signaling pathways, but their specific functions are as yet undefined. Btk and its homologues share extensive sequence similarity, including a conserved region, the Tec-homology (TH) domain, that has been proposed to mediate specific intermolecular or intramolecular interactions. The TH region of Btk contains a functional SH3-binding site at residues 189-192. SH3 binding is selective: Btk is retained by the SH3 domain of Fyn but not by that of Blk, another Src-type kinase. TH-SH3 binding in vitro is abolished by specific, single amino acid substitutions within the Btk TH domain or the Fyn SH3 domain. We provide two lines of evidence that the SH3-binding site in the Btk TH domain mediates protein interactions in intact cells. First, treatment of cells with pervanadate induces an increase in the phosphotyrosine content of kinase-inactive Btk; this response is substantially reduced by a mutation that inactivates the SH3-binding site in the Btk TH domain. Second, in cell lysates Btk is found in association with an as yet unidentified 72-kDa phosphotyrosine-containing protein; this interaction requires a functional SH3-binding site in the TH domain. The TH domain may therefore interact in vivo with other proteins that regulate the phosphorylation state of Btk.

Agammaglobulinaemia Tyrosine Kinase↗
PubMed1995

V(D)J recombination and the cell cycle.

V(D)J recombination is a major source of antigen receptor diversity and represents the only known form of site-specific DNA rearrangement in vertebrates. V(D)J recombination is initiated by specific DNA cleavage at recombinational signal sequences and requires components of the general machinery used for double-strand (DS)-break repair. The involvement of DS cleavage and repair mechanisms suggests that V(D)J recombination might be coupled to the cell cycle, as introduction or persistence of DS breaks during DNA replication or mitosis could interfere with faithful transmission of genetic information to daughter cells. Here, Weei-Chin Lin and Stephen Desiderio review recent evidence indicating that this is indeed the case and consider some biological implications of this linkage.

Animals↗
PubMed1994

A cyclin-dependent kinase homologue, p130PITSLRE is a phosphotyrosine-independent SH2 ligand.

Src-homology 2 (SH2) domains are conserved, globular protein modules that mediate assembly of multicomponent signaling complexes. Phosphoproteins from the B-lymphoid cell line A20 were isolated by SH2 affinity chromatography; the peptide sequence from one of these proteins was used to molecularly clone several related complementary DNAs whose predominant protein product, p130PITSLRE, is an abundant serine/threonine kinase with ubiquitous expression in murine tissues. The sequence of a previously described cyclin-dependent kinase homologue, p58clk-1, is entirely contained within the p130PITSLRE sequence. Specific binding of p130PITSLRE to SH2 domains is mediated by a serine- and glutamic acid-rich cluster of amino acids in the N-terminal region. This interaction is dependent on serine/threonine phosphorylation but independent of tyrosine phosphorylation. Binding is inhibited by free phosphotyrosine and by a phosphotyrosine-containing peptide from polyoma middle T antigen, suggesting that the p130PITSLRE binding site in the SH2 domain overlaps the region that binds phosphotyrosine-containing peptides. Bacterially expressed p130PITSLRE fragments acquire the ability to bind an SH2 domain when phosphorylated in vitro with casein kinase II. A subset of casein kinase II phosphorylation sites may therefore constitute a phosphotyrosine-independent class of SH2 ligands.

Amino Acid Sequence↗
PubMed1994

Mapping of the gene for the tyrosine kinase Itk to a region of conserved synteny between mouse chromosome 11 and human chromosome 5q.

The protein-tyrosine kinase gene Itk is expressed preferentially in T lymphoid cells of the mouse and is induced by IL-2. A related gene, Btk, is expressed in the murine B lymphoid and myeloid lineages. Because mutations in Btk and the corresponding human gene are associated with X-linked immunodeficiency syndromes, it was of interest to map Itk and its human counterpart. By Southern blot analysis of DNA from the progeny of two multilocus crosses, murine Itk was mapped to Chromosome 11. By fluorescence in situ hybridization, human ITK was mapped to 5q32-q33. Murine Itk and its human homologue lie within regions of conserved synteny that include several growth factor and growth factor receptor genes. This region in humans is frequently deleted in the myelodysplastic syndrome, suggesting possible involvement of ITK in this disorder.

Agammaglobulinaemia Tyrosine Kinase↗
PubMed1994

Specific recognition of the blk promoter by the B-lymphoid transcription factor B-cell-specific activator protein.

Several genes of the src family encode protein-tyrosine kinases that associate with the B-cell antigen receptor complex and are activated upon receptor cross-linking, including blk, lyn, and fyn. The blk gene is the only member of this family whose expression is restricted to B-lymphoid cells. In the B lineage, blk is developmentally regulated: blk transcripts are first detected in pro-B-cells and persist until the differentiation of mature B-cells to plasma cells. We have found that the blk promoter is a target for a specific DNA-binding protein whose activity in B-lymphoid cell lines is positively correlated with blk expression. By three criteria, we have identified this DNA-binding protein as the transcription factor B-cell-specific activator protein (BSAP): 1) oligonucleotides containing known BSAP recognition sites were found to compete specifically with blk for binding to the protein detected in B-lymphoid extracts; 2) authentic BSAP was shown to bind the same site within the blk promoter as the protein identified in B-lymphoid extracts; and 3) the specific DNA-protein complex formed in B-lymphoid extracts was shown to react with an anti-BSAP antiserum. BSAP has been implicated previously in the transcriptional regulation of CD19, whose pattern of expression in B-cell development coincides with that of blk. These observations, and the correlation between expression of BSAP and expression of blk, suggest that BSAP is a positive regulator of blk transcription.

B-Lymphocytes↗
PubMed1994

Cell cycle regulation of V(D)J recombination-activating protein RAG-2.

The antigen receptors of B and T lymphocytes are encoded in multiple germ-line DNA segments that are joined during lymphocyte development. The recombination-activating proteins RAG-1 and RAG-2 are both essential for this process, termed V(D)J rearrangement. Phosphorylation of the RAG-2 protein at Thr-490 by one or more cyclin-dependent kinases is associated with its rapid degradation. In an immature B-cell line and in normal thymocytes, RAG-2 protein accumulates preferentially in the G0/G1 phases of the cell cycle and declines by at least 20-fold before cells enter S phase. The amount of RAG-2 protein remains low throughout the S, G2, and M phases. The amount of RAG-1 protein shows considerably less fluctuation. The variation in RAG-2 protein is likely to be established, at least in part, by a posttranscriptional mechanism. These observations suggest that V(D)J rearrangement occurs entirely or preferentially within G0/G1.

Animals↗
PubMed1994

The B cell antigen receptor in B-cell development.

During B-cell development, immature and mature forms of the B cell antigen receptor complex are deployed in a regulated fashion; thus, B cell antigen receptor complexes play essential roles in the transit of cells through ontogeny. The past year has seen progress in our understanding of how antigen receptor gene assembly is controlled and in defining the requirements for antigen receptor mediated signaling at specific developmental stages. The discovery that a defective protein tyrosine kinase is responsible for X-linked agammaglobulinemia in man and X-linked immunodeficiency in the mouse is particularly interesting, as it may provide the means to link a specific intracellular signaling pathway with a particular step in B-cell development.

Amino Acid Sequence↗
PubMed1993

SH2 domains of the protein-tyrosine kinases Blk, Lyn, and Fyn(T) bind distinct sets of phosphoproteins from B lymphocytes.

Several members of the Src family, including Blk, Lyn, Fyn(T), and Lck, are expressed in B cells. These kinases associate with the antigen receptor complex, and the activities of Blk, Fyn(T), and Lyn increase upon receptor engagement. Differences in the amino acid sequences and patterns of expression of these kinases suggest that they serve distinct functions. In this communication it is shown that the SH2 domains from Blk, Lyn, and Fyn(T) preferentially bind distinct sets of phosphoproteins from the mature B cell line A20. These interactions were found to depend on recognition of phosphotyrosine. The Blk SH2 domain bound more than 10 distinct phosphoprotein species, most of which reacted with an antiphosphotyrosine antibody; the phosphotyrosine content of these proteins was increased if surface immunoglobulin was cross-linked before extracts were made. Phosphoproteins of 72, 76, 115, and 130 kDa bound to the SH2 domains of Blk, Lyn, and Fyn(T). Phosphoamino acid analysis of these four proteins revealed that each contained phosphoserine, phosphothreonine, and phosphotyrosine. Proteins of 90 kDa, 130 kDa, and 150 kDa were preferentially bound by the Blk SH2 domain, while the Fyn(T) SH2 domain showed preferential binding to proteins of 76 and 180 kDa. The Lyn SH2 binding profile resembled that of Blk, but differences in the binding specificities of these kinases were also observed. Thus, among proteins that exhibit increased tyrosine phosphorylation following antigen receptor cross-linking, several have been identified that bind preferentially to SH2 domains of Blk, Fyn(T), or Lyn, suggesting that these kinases serve distinct functions. In addition, chimeric Fyn(T)-Blk SH2 domains were shown to be functional in binding assays and to exhibit binding specificities intermediate between those of the parent domains, consistent with the interpretation that the differences we observe in phosphoprotein binding by Fyn(T) and Blk SH2 domains reflect differences in their native structures.

Amino Acid Sequence↗
PubMed1993

Tandem SH2 domains of ZAP-70 bind to T cell antigen receptor zeta and CD3 epsilon from activated Jurkat T cells.

A proximal and critical biochemical event upon T cell antigen receptor (TCR) stimulation is the activation of a protein tyrosine kinase (PTK) pathway. ZAP-70, a PTK of the p72syk family, associates with phosphorylated TCR subunits upon TCR stimulation. Here we report that the tandem SH2 domains of ZAP-70, expressed as a fusion protein, bind to tyrosine-phosphorylated CD3 epsilon and TCR zeta from activated Jurkat T cell lysates. The single N- and C-terminal SH2 domains of ZAP-70, expressed separately, do not bind these TCR subunits. In comparison to fusion proteins containing SH2 domains from other proteins, the tandem SH2 domains of ZAP-70 demonstrate a remarkably restricted repertoire of protein binding, binding only TCR zeta and CD3 epsilon. ZAP-70 is also recovered in the binding assay, but this is likely to be a consequence of its interaction with multiple SH2 binding sites on the zeta-zeta and CD3 epsilon-containing dimers.

Amino Acid Sequence↗
PubMed1993

Regulation of V(D)J recombination activator protein RAG-2 by phosphorylation.

Antigen receptor genes are assembled by site-specific DNA rearrangement. The recombination activator genes RAG-1 and RAG-2 are essential for this process, termed V(D)J rearrangement. The activity and stability of the RAG-2 protein have now been shown to be regulated by phosphorylation. In fibroblasts RAG-2 was phosphorylated predominantly at two serine residues, one of which affected RAG-2 activity in vivo. The threonine at residue 490 was phosphorylated by p34cdc2 kinase in vitro; phosphorylation at this site in vivo was associated with rapid degradation of RAG-2. Instability was transferred to chimeric proteins by a 90-residue portion of RAG-2. Mutation of the p34cdc2 phosphorylation site of the tumor suppressor protein p53 conferred a similar phenotype, suggesting that this association between phosphorylation and degradation is a general mechanism.

3T3 Cells↗
PubMed1993

A V(D)J recombinase-inducible B-cell line: role of transcriptional enhancer elements in directing V(D)J recombination.

Rapid analysis of mechanisms that regulate V(D)J recombination has been hampered by the lack of appropriate cell systems that reproduce aspects of normal prelymphocyte physiology in which the recombinase is activated, accessible antigen receptor loci are rearranged, and rearrangement status is fixed by termination of recombinase expression. To generate such a system, we introduced heat shock-inducible V(D)J recombination-activating genes (RAG) 1 and 2 into a recombinationally inert B-cell line. Heat shock treatment of these cells rapidly induced high levels of RAG transcripts and RAG proteins that were accompanied by a parallel induction of V(D)J recombinase activity, strongly suggesting that RAG proteins have a primary role in V(D)J recombination. Within hours after induction, these cells began to rearrange chromosomally integrated V(D)J recombination substrates but only if the substrates contained an active transcriptional enhancer; substrates lacking an enhancer were not efficiently rearranged. Activities necessary to target integrated substrates for rearrangement were provided by two separate lymphoid-specific transcriptional enhancers, as well as an active nonlymphoid enhancer, unequivocally demonstrating that such elements enhance both transcription and V(D)J recombinational accessibility.

Animals↗