TFII-I, a candidate gene for Williams syndrome cognitive profile: parallels between regional expression in mouse brain and human phenotype.

The gene for TFII-I, a widely expressed transcription factor, has been localized to an interval of human chromosome 7q11.23 that is commonly deleted in Williams syndrome (WS). The clinical phenotype of WS includes elfin facies, infantile hypercalcemia, supravalvular aortic stenosis, hyperacusis and mental retardation. The WS cognitive profile (WSCP) is notable for the differential impairment of visual-spatial abilities with relative sparing of verbal-linguistic function. Fine mapping of individuals with WS has revealed a close association between deletion of TFII-I and the WSCP. To determine the plausibility of the hypothesis that hemizygous deletion of TFII-I contributes to the WSCP, we have examined the anatomic distribution of TFII-I RNA and protein isoforms in brains from adult and embryonic mice. Our studies show that early in development, TFII-I expression is widespread and nearly uniform throughout the brain. In adult brain, TFII-I protein is present exclusively in neurons. Highest levels of expression are observed in cerebellar Purkinje cells and in hippocampal interneurons. TFII-I immunoreactivity is distinct from that of the related protein, TFII-IRD1, which is also localized to the region of human chromosome 7 deleted in WS. The expression pattern of TFII-I in mouse brain parallels regions in human brain which have been shown to be anatomically and functionally altered in humans with WS. These observations are consistent with the hypothesis that deletion of the gene for TFII-I contributes to the cognitive impairments observed in WS.

Identification of phosphorylation sites for Bruton's tyrosine kinase within the transcriptional regulator BAP/TFII-I.

Bruton's tyrosine kinase (Btk), a member of the Tec family of cytosolic kinases, is essential for B cell development and function. BAP/TFII-I, a protein implicated in transcriptional regulation, is associated with Btk in B cells and is transiently phosphorylated on tyrosine following B cell receptor engagement. BAP/TFII-I is a substrate for Btk in vitro and is hyperphosphorylated on tyrosine upon coexpression with Btk in mammalian cells. In an effort to understand the physiologic consequences of BAP/TFII-I tyrosine phosphorylation following B cell receptor stimulation, site-directed mutagenesis and phosphopeptide mapping were used to locate the predominant sites of BAP/TFII-I phosphorylation by Btk in vitro. These residues, Tyr248, Tyr357, and Tyr462, were also found to be the major sites for Btk-dependent phosphorylation of BAP/TFII-I in vivo. Residues Tyr357 and Tyr462 are contained within the loop regions of adjacent helix-loop-helix-like repeats within BAP/TFII-I. Mutation of either Tyr248, Tyr357, or Tyr462 to phenylalanine reduced transcription from a c-fos promoter relative to wild-type BAP/TFII-I in transfected COS-7 cells, consistent with the interpretation that phosphorylation at these sites contributes to transcriptional activation. Phosphorylation of BAP/TFII-I by Btk may link engagement of receptors such as surface immunoglobulin to modulation of gene expression.

Rag-1 mutations associated with B-cell-negative scid dissociate the nicking and transesterification steps of V(D)J recombination.

Some patients with B-cell-negative severe combined immune deficiency (SCID) carry mutations in RAG-1 or RAG-2 that impair V(D)J recombination. Two recessive RAG-1 mutations responsible for B-cell-negative SCID, R621H and E719K, impair V(D)J recombination without affecting formation of single-site recombination signal sequence complexes, specific DNA contacts, or perturbation of DNA structure at the heptamer-coding junction. The E719K mutation impairs DNA cleavage by the RAG complex, with a greater effect on nicking than on transesterification; a conservative glutamine substitution exhibits a similar effect. When cysteine is substituted for E719, RAG-1 activity is enhanced in Mn(2+) but remains impaired in Mg(2+), suggesting an interaction between this residue and an essential metal ion. The R621H mutation partially impairs nicking, with little effect on transesterification. The residual nicking activity of the R621H mutant is reduced at least 10-fold upon a change from pH 7.0 to pH 8.4. Site-specific nicking is severely impaired by an alanine substitution at R621 but is spared by substitution with lysine. These observations are consistent with involvement of a positively charged residue at position 621 in the nicking step of the RAG-mediated cleavage reaction. Our data provide a mechanistic explanation for one form of hereditary SCID. Moreover, while RAG-1 is directly involved in catalysis of both nicking and transesterification, our observations indicate that these two steps have distinct catalytic requirements.

Synergistic activity of STAT3 and c-Jun at a specific array of DNA elements in the alpha 2-macroglobulin promoter.

The transcriptional activity of natural promoters is sensitive to the precise spatial arrangement of DNA elements and their incorporation into higher order DNA-protein complexes. STAT3 and c-Jun form a specific ternary complex in vitro with a synthetic DNA element containing AP1 and SIE sites. These associations are critical for synergistic activation of transcription from a synthetic promoter by STAT3 and c-Jun. Expression of the acute phase protein alpha(2)-macroglobulin is induced in vivo by interleukin-6 (IL-6)-related cytokines; we demonstrate that coordinate interactions among STAT3, c-Jun, and a specific array of DNA elements contribute to activation of the alpha(2)-macroglobulin promoter in response to IL-6 family members. At least five promoter elements are involved in activation: two AP1 sites at -113 to -107 and -152 to -140, an acute phase response element (APRE (SIE)) at -171 to -163, and two AT-rich regions at -143 to -138 and -128 to -123. Synergism between STAT3 or STAT3-C and c-Jun is impaired by mutation of the APRE (SIE) or either AP1 site, as well as by mutations that alter the AT-rich regions or their phasing. Mutations of STAT3 previously shown to disrupt physical and functional interactions with c-Jun do not impair synergy between STAT3-C and c-Jun at the alpha(2)-macroglobulin promoter in HepG2 cells, suggesting that STAT3-C and STAT3 differ with respect to their precise contacts with c-Jun.

Cyclin A/CDK2 regulates V(D)J recombination by coordinating RAG-2 accumulation and DNA repair.

Accumulation of the V(D)J recombinase protein RAG-2 is restricted to G0/G1 cells by phosphorylation-mediated degradation at the G1-S boundary. Here cyclin A/CDK2 is shown to oppose RAG-2 accumulation; conversely, RAG-2 is induced by p27Kip1 and related CDK inhibitors. Coinduction of RAG-2 and G1 delay by p27Kip1 is accompanied by strong stimulation of V(D)J recombination. Unexpectedly, induction of RAG-2 accumulation in the absence of G1 delay has no effect on recombination frequency. p27Kip1 may stimulate V(D)J recombination by coordinating accumulation of RAG-2 with prolongation of G1, when nonhomologous end joining is preferentially active. Consistent with this, enforced expression of RAG-2 throughout cell cycle is associated with accumulation of aberrant recombination products reminiscent of those formed in the absence of nonhomologous end joining.

Malignant transformation of early lymphoid progenitors in mice expressing an activated Blk tyrosine kinase.

The intracellular signals governing cellular proliferation and developmental progression during lymphocyte development are incompletely understood. The tyrosine kinase Blk is expressed preferentially in the B lineage, but its function in B cell development has been largely unexplored. We have generated transgenic mice expressing constitutively active Blk [Blk(Y495F)] in the B and T lymphoid compartments. Expression of Blk(Y495F) in the B lineage at levels similar to that of endogenous Blk induced B lymphoid tumors of limited clonality, whose phenotypes are characteristic of B cell progenitors at the proB/preB-I to preB-II transition. Expression of constitutively active Blk in the T lineage resulted in the appearance of clonal, thymic lymphomas composed of intermediate single positive cells. Taken together, these results indicate that specific B and T cell progenitor subsets are preferentially susceptible to transformation by Blk(Y495F) and suggest a role for Blk in the control of proliferation during B cell development.

Differential effects of B cell receptor and B cell receptor-FcgammaRIIB1 engagement on docking of Csk to GTPase-activating protein (GAP)-associated p62.

The stimulatory and inhibitory pathways initiated by engagement of stimulatory receptors such as the B cell receptor for antigen (BCR) and inhibitory receptors such as Fcgamma receptors of the IIB1 type (FcgammaRIIB1) intersect in ways that are poorly understood at the molecular level. Because the tyrosine kinase Csk is a potential negative regulator of lymphocyte activation, we examined the effects of BCR and FcgammaRIIB1 engagement on the binding of Csk to phosphotyrosine-containing proteins. Stimulation of a B lymphoma cell line, A20, with intact anti-IgG antibody induced a direct, SH2-mediated association between Csk and a 62-kD phosphotyrosine-containing protein that was identified as RasGTPase-activating protein-associated p62 (GAP-A.p62). In contrast, stimulation of A20 cells with anti-IgG F(ab')2 resulted in little increase in the association of Csk with GAP-A.p62. The effect of FcgammaRIIB1 engagement on this association was abolished by blockade of FcgammaRIIB1 with the monoclonal antibody 2.4G2. Furthermore, the increased association between Csk and GAP-A.p62 seen upon stimulation with intact anti-Ig was abrogated in the FcgammaRIIB1-deficient cell line IIA1.6 and recovered when FcgammaRIIB1 expression was restored by transfection. The differential effects of BCR and BCR-FcgammaRIIB1-mediated signaling on the phosphorylation of GAP-A.p62 and its association with Csk suggest that docking of Csk to GAP-A.p62 may function in the negative regulation of antigen receptor-mediated signals in B cells.

The Pax-5 gene is alternatively spliced during B-cell development.

The transcription factor Pax-5 is expressed during the early stages of B-cell differentiation and influences the expression of several B-cell-specific genes. In addition to the existing isoform (Pax-5, which we have named Pax-5a), we have isolated three new isoforms, Pax-5b, Pax-5d, and Pax-5e, from murine spleen and B-lymphoid cell lines using library screenings and polymerase chain reaction amplification. Isoforms Pax-5b and Pax-5e have spliced out their second exon, resulting in proteins with only a partial DNA-binding domain. Isoforms Pax-5d and Pax-5e have deleted the 3'-region, which encodes the transactivating domain, and replaced it with a novel sequence. The existence of alternative Pax-5 transcripts was confirmed using RNase protection assays. Furthermore, Pax-5a and Pax-5b proteins were detected using Western blot analysis. Pax-5a was detectable in pro-, pre-, and mature B-cell lines, but not in two plasmacytomas; Pax-5b was shown to be present at low levels in mature B-cell lines and, unexpectedly, in one plasma cell line, but not in pro-B-cell or T-cell lines. Mobility shift assays showed that in vitro translated Pax-5a and Pax-5d, but not Pax-5b or Pax-5e, could interact with a B-cell-specific activator protein-binding site on the blk promoter. Using this assay, we also showed that Pax-5d was present in nuclear extracts of some (but not all) B-lymphoid lines and interacts with the B-cell-specific activator protein-binding site. The pattern of differential expression of alternatively spliced Pax-5 isoforms suggests that they may be important regulators of transcription during B-cell maturation.

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.

p150TSP, a conserved nuclear phosphoprotein that contains multiple tetratricopeptide repeats and binds specifically to SH2 domains.

Src homology 2 (SH2) domains are structural modules that function in the assembly of multicomponent signaling complexes by binding to specific phosphopeptides. The tetratricopeptide repeat (TPR) is a distinct structural motif that has been suggested to mediate protein-protein interactions. Among SH2-binding phosphoproteins purified from the mouse B cell lymphoma A20, a 150-kDa species was identified and the corresponding complementary DNA (cDNA) was molecularly cloned. This protein encoded by this cDNA, which we have termed p150TSP (for TPR-containing, SH2-binding phosphoprotein), is located predominantly in the nucleus and is highly conserved in evolution. The gene encoding p150TSP (Tsp) was mapped to chromosome 7 of the mouse with gene order: centromere-Tyr-Wnt11-Tsp-Zp2. The amino-terminal two-thirds of p150TSP consist almost entirely of tandemly arranged TPR units, which mediate specific, homotypic protein interactions in transfected cells. The carboxyl-terminal third of p150TSP, which is serine- and glutamic acid-rich, is essential for SH2 binding; this interaction is dependent on serine/threonine phosphorylation but independent of tyrosine phosphorylation. The sequence and binding properties of p150TSP suggest that it may mediate interactions between TPR-containing and SH2-containing proteins.

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.

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.

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.

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.

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.

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.

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.

Molecular cloning and analysis of cDNA encoding the murine c-yes tyrosine protein kinase.

The cellular yes (c-yes) gene is a member of the class of proto-oncogenes that encode non-receptor tyrosine protein kinases. In this report we describe the isolation of cDNAs that encode the murine c-yes gene product and analysis of the nucleotide sequence of the murine c-yes cDNA clones. The reading frame encodes a protein of 541 amino acids with a calculated molecular mass of 60.63 kDa that is reactive with anti-Yes antisera and possesses protein kinase activity.