SALL1 expression in the human pituitary-adrenal/gonadal axis.

SALL1 was originally identified on the basis of its DNA sequence homology to the region-specific homeotic gene Sal, in Drosophila melanogaster, which acts as a downstream target of hedgehog/tumor growth factor-beta-like decapentaplegic signals. The SALL1 gene has been associated with the Townes-Brocks Syndrome (TBS), a disorder characterized by multiorgan dysgenesis including renal and genital malformations. In this study, SALL1 message production was evaluated in association with the tissue localization of the protein product of SALL1, p140. SALL1 protein expression was observed in various adult and fetal tissues which elaborate reproductive endocrine hormones. The p140 was localized in specific microanatomic sites of the pituitary, adrenal cortex and the placenta. In the human pituitary, SALL1 protein expression was limited to the adenohypophysis, where it colocalized to those cells producing GH and the gonadotropins, LH and FSH. SALL1 expression was also found in most of the fetal and adult adrenal cortex in addition to the trophoblastic cells of the placenta. This pattern of expression complements prior studies demonstrating p140 in testicular fetal Leydig cells, adult Leydig and Sertoli cells, and granulosa cells of the ovary. The SALL1 protein was also shown here to be highly expressed in trophoblast tumors, which overproduce sex hormones. The expression patterns of SALL1 at multiple levels of the reproductive endocrine axis and the phenotypic effects associated with TBS suggest that SALL1 may have an important role in the interaction of the pituitary-adrenal/gonadal axis during reproduction.

Induction of the IL-13 receptor alpha2-chain by IL-4 and IL-13 in human keratinocytes: involvement of STAT6, ERK and p38 MAPK pathways.

IL-4 and IL-13 are related cytokines which induce both pro- and anti-inflammatory effects depending on the cell type they act upon and the nature of the receptors expressed. The type I receptor complex is composed of the IL-4Ralpha and gammac and only binds IL-4, whereas, in the type II receptor, IL-4Ralpha dimerizes with IL-13Ralpha1 upon either IL-4 or IL-13 binding. Another ligand binding chain potentially implicated in the IL-4/IL-13 receptor has been described, the IL-13Ralpha2, but the regulation of its expression and its role in IL-4/IL-13 transduction is poorly understood. In this study we report that IL-4 and IL-13 upregulate IL-13Ralpha2 at both the mRNA and protein levels in the keratinocyte cell line HaCaT. In these cells, IL-4 or IL-13 were shown to activate the Janus Kinases JAK1 and JAK2, the transcription factor STAT6, and the ERK and p38 mitogen-activated protein kinases. We show that IL-4 or IL-13-induced IL-13Ralpha2 mRNA expression was inhibited by the ERK inhibitor U0126, the JAK inhibitor AG490 and, to a lesser extent, the p38 MAPK inhibitor SB203580. Moreover, expression of a constitutive active mutant of STAT6 alone did not modify IL-13Ralpha2 mRNA expression, but potentiated the effects of IL-4 or IL-13 on IL-13Ralpha2 expression. The constitutive active mutants of MEK1 or MKK6 increased the level of expression of IL-13Ralpha2 mRNA even in absence of stimulation. Our findings demonstrate, for the first time, that IL-4 and IL-13 can induce IL-13Ralpha2 expression in keratinocytes, and that the ERK and p38 MAPK together with JAK2 and STAT6 play a critical role in this process.

Cloning and characterization of two promoters for the human HSAL2 gene and their transcriptional repression by the Wilms tumor suppressor gene product.

HSAL2 is a member of a gene family that encodes a group of putative developmental transcription factors. The HSAL gene complex was originally identified on the basis of DNA sequence homology to a region-specific homeotic gene (SAL) in Drosophila. This study reveals a novel, functional 5' exon for HSAL2 and demonstrates that two distinct HSAL2 gene transcripts arise from two overlapping transcription units, resulting in proteins that differ by 25 amino acids. By utilizing functional luciferase reporter assays, two distinct promoters for HSAL2, P1 for the proximal promoter (upstream of exon 1) and P2 for the distal promoter (upstream of exon 1A), were identified. Evaluation of mRNA prevalence and tissue specificity, with particular focus on adult tissues, revealed that production of mRNA from P1 was selective and relatively rare. Production of mRNA from P2 was demonstrably higher and was expressed by a greater number of tissues. In contradistinction, HSAL2 expression directed by P2 was undetectable in some malignant populations as opposed to their normal human counterparts, suggesting a potential role as a tumor suppressor gene. Consensus-binding sites were identified for several transcriptional factors, with multiple sites for WT-1, and Hox-1.3 present within both the P1 and P2 regions. In transient transfection assays, transcription from both HSAL2 P1 and P2 was strikingly repressed by the WT-1 tumor suppressor protein. These findings suggest that an intracellular WT-1/HSAL2 pathway may play a role in development and hematopoiesis.

Hsal 1 is related to kidney and gonad development and is expressed in Wilms tumor.

Townes-Brocks syndrome (TBS) is a human genetic disorder with features including urogenital, limb, anal and cardiac malformations associated with mutations of the TBS gene, Hsal 1. To begin to understand the role of the Hsal 1 protein (p140) in both normal development and disease pathogenesis, both message and protein expression were evaluated in specific tissues associated with TBS. DNA sequence information for Hsal 1 predicts that this homeotic, Drosophila homologue (Sal) encodes a zinc-finger protein consistent with a transcription factor. mRNA for Hsal 1 was highly expressed in fetal kidney and brain, with detectable production in thymus and heart. p140 was found in fetal ureteric bud, fetal and postnatal renal tubular epithelium, and renal blastema. In the 14-week fetal testis, the Hsal 1 protein was specifically expressed in the testosterone producing Leydig cells while in adult gonads Hsal 1 was also found in both Leydig and Sertoli cells, spermatogonia of the testis, and granulosa cells of the ovary. Evaluation of Wilms tumor revealed consistently high expression of the gene product in the epithelial and blastemal components. These spatial and temporal patterns of expression for Hsal 1, and the phenotypic effects associated with TBS, suggest that Hsal 1 plays an important role in the development and functional maintenance of the kidney and gonads. Furthermore, the Hsal 1 gene product may play a part in the pathogenesis of specific neoplasms occurring in these organs in addition to its specific role in Townes-Brocks syndrome.

Vitamin D-mediated gene regulation in phenotypically defined human B cell subpopulations.

Isolation of distinct subpopulations of density-fractionated normal human B lymphocytes reveals that the requirements for up-regulation of the vitamin D receptor (VDR) and initiation of 1alpha,25-dihydroxyvitamin D3 [1alpha,25-(OH)2D3]-mediated genomic trans-activation are dependent upon the state of cellular activation. The kinetics of the response differ widely among these B cell subpopulations. However, these density-fractionated B cell subpopulations are phenotypically diverse and therefore are not representative of distinct stages of B cell maturation and differentiation. To examine the role of B cell differentiation on the induction and maintenance of biological receptivity to 1,25-(OH)2D3, we purified naive, germinal center, and memory B cells based on their expression of CD38 and CD44 surface antigens and surface Ig isotype. These phenotypically defined B cell subpopulations were all found to constitutively express VDR, and all exhibited similar activation requirements and kinetics for initiation of 1,25(OH)2D3-mediated genomic trans-activation. Taken together, these results suggest that defined stages of differentiation in normal B cells are not significant predicators of VDR expression or receptivity to 1,25-(OH)2D3. Rather, the degree of cellular activation, regardless of maturation stage, determines whether the effects of this immunoregulatory hormone will influence a mature B lymphocyte.

The human B cell response to IL-13 is dependent on cellular phenotype as well as mode of activation.

Normal mature quiescent human B lymphocytes, isolated as a function of buoyant density, require activation for up-regulation of IL-13R constituents. Cell activation through a combination of surface Ig and CD40 receptor ligation leads to the most substantial message production for IL-13Ralpha1. Functional consequences of this receptor variation, in initially quiescent cells, includes demonstrable effects on cellular proliferation in response to ligand exposure. Variations in the method of surface activation, with particular emphasis on the CD40 receptor, reveals that immobilized CD40 ligand may be sufficient, in and of itself, to up-regulate IL-13Ralpha1, which may bear significance for B-lymphocyte bystander proliferation. Regulation of the IL-13Ralpha1 protein and message also differs as a function of cellular phenotype. Although values are greater in memory than naive B cells, as they are initially isolated from extirpated tonsils, variations in the magnitude of message and protein, as a function of surface stimulation, are more substantial in the naive subset. The magnitude of variation in message production in naive cells is associated with a more vigorous proliferative response to IL-13 than seen in memory lymphocytes. The cellular response to IL-13, as a function of activation and phenotype, is the converse of that demonstrated for IL-2. Evaluation of proliferation, receptor message, ligand binding protein production, and the response to putatively synergistic cytokines reveals that IL-2 is the predominant lymphokine utilized by memory cells. This is in contradistinction to IL-13, which along with IL-4, are the predominant moieties for naive lymphocytes.

Interaction between CD45-AP and protein-tyrosine kinases involved in T cell receptor signaling.

CD45-AP associates specifically with CD45, a protein-tyrosine phosphatase essential for antigen receptor-mediated signal transduction. CD45 modulates the activity of Src family protein-tyrosine kinases involved at the onset of antigen receptor-mediated signaling by dephosphorylating their regulatory tyrosyl residues. We have shown that lymphocyte responses to antigen receptor stimulation are impaired in CD45-AP-null mice. To examine the possibility that CD45-AP coordinates the interaction between CD45 and its substrates, we investigated the associations of CD45-AP with several protein-tyrosine kinases. Endogenous CD45-AP coimmunoprecipitated with Lck and ZAP-70 in both CD45-positive T cells and their CD45-negative variants after stimulation by antigen receptor ligation. Concomitantly, CD45 coimmunoprecipitated with Lck and ZAP-70 after T cell receptor-mediated stimulation of CD45-positive cells. Recombinant CD45-AP exhibited specific binding to Lck and ZAP-70 protein-tyrosine kinases, but not to Fyn or Csk, in lysates of both CD45-positive and -negative T cells. Specific interactions were demonstrated between the respective recombinant proteins as well. These results demonstrate that CD45-AP associates directly and selectively with Lck and ZAP-70 in response to T cell receptor-mediated stimulation. The associations of CD45-AP with Lck and ZAP-70 may mediate the functional interactions of these kinases with CD45 during antigen receptor stimulation.

Differential regulation of gene transcription in subpopulations of human B lymphocytes by vitamin D3.

We have previously shown that freshly extirpated normal human tonsil B cells, which are phenotypically diverse, representing different stages of cellular activation and differentiation, are refractory to the effects of 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3] and require specific activation signals for induction of responsiveness. To determine whether these diversely activated B cell populations respond to 1,25-(OH)2D3, human tonsil B cells were density fractionated and evaluated biochemically and functionally. Low density tonsil B cells, representing the centroblastic fraction, were observed to constitutively express vitamin D receptor message and protein. In contrast, high density quiescent tonsillar B cells had no detectable vitamin D receptor message or protein and required stimulation in vitro for their up-regulation. Biological responsiveness to 1,25-(OH)2D3 was assessed by messenger RNA (mRNA) expression of the vitamin D-dependent enzyme, 25-hydroxyvitamin D3 24-hydroxylase. Low density centroblastic B cells did not require exogenous surface activation for expression of 24-hydroxylase mRNA, which was detectable after 6 h of culture in the presence of 1,25-(OH)2D3. In contrast, high density tonsil B cells required in vitro activation for induction of 24-hydroxylase mRNA, and expression was not detectable for up to 48 h of culture. These observations suggest that reactivity of normal B cell populations to vitamin D is dependent upon their specific stage of activation.

Disruption of lymphocyte function and signaling in CD45-associated protein-null mice.

CD45-AP specifically associates with CD45, a protein tyrosine phosphatase essential for lymphocyte differentiation and antigen receptor-mediated signal transduction. CD45 is thought to mediate antigen receptor signaling by dephosphorylating regulatory tyrosine residues on Src family protein tyrosine kinases such as Lck. However, the mechanism for regulating CD45 protein tyrosine phosphatase activity remains unclear. CD45-AP-null mice were created to examine the role of CD45-AP in CD45-mediated signal transduction. T and B lymphocytes showed reduced proliferation in response to antigen receptor stimulation. Both mixed leukocyte reaction and cytotoxic T lymphocyte functions of T cells were also markedly decreased in CD45-AP-null mice. Interestingly, the interaction between CD45 and Lck was significantly reduced in CD45-AP-null T cells, indicating that CD45-AP directly or indirectly mediates the interaction of CD45 with Lck. Our data indicate that CD45-AP is required for normal antigen receptor signaling and function in lymphocytes.

Regulation of interleukin-13 receptor constituents on mature human B lymphocytes.

Human B cells stimulated through both their immunoglobulin and CD40 receptors up-regulate 745 +/- 51 interleukin (IL)-13 ligand binding sites with an affinity of 0.91 +/- 0.08 nM within 24 h. IL-13 binds primarily to the IL-13Ralpha1 with subsequent sequestration of the IL-4Ralpha into the complex. IL-13Ralpha1 may also be found in those receptors capable of binding IL-4. gamma chain (gammac) participates in receptors capable of binding IL-4 but is not found in association with bound IL-13. Dimeric receptors composed of the IL-4Ralpha complexed with either the IL-13Ralpha1 or gammac occur simultaneously within defined B cell populations. mRNAs for all receptor constituents are increased subsequent to immunoglobulin stimulation alone, while maximal expression of IL-13Ralpha1 is more dependent upon co-stimulation of immunoglobulin and CD40 receptors. mRNA levels for IL-13Ralpha1 vary over a wider range subsequent to surface stimulation than other receptor components. Although gammac is not bound to IL-13 in B cells under the conditions evaluated, it may influence IL-13 binding by competing with IL-13Ralpha1 for association/sequestration with the IL-4Ralpha chain. IL-13Ralpha2 does not participate in the IL-13 receptor that is up-regulated upon activation of quiescent tonsillar B lymphocytes, although mRNA for the protein may be found in the centroblastic fraction of tonsillar cells.

1alpha,25-Dihydroxyvitamin D3 modulates CD38 expression on human lymphocytes.

B cells from chronically stimulated tonsils displayed high initial mean CD38 levels that declined during in vitro culture, despite ligation of CD40 and/or the Ag receptor in the presence of IL-4. Exposure to 1alpha,25-dihydroxyvitamin D3 (1,25(OH)2D3) restored the initial CD38 expression on these stimulated cells and up-regulated the Ag on stimulated CD38-/low cells. 1,25(OH)2D3 enhanced CD38 expression by four- to sixfold on CD8- and CD8+ peripheral blood T cells following PHA activation. The EC50 values for induction were 2 to 3 nM. Although all-trans-retinoic acid also induced CD38 expression on stimulated B and T cells, it was less effective than 1,25(OH)2D3. B cell CD38 expression was augmented less by 1,25(OH)2D3 than by IFN-alpha and IFN-gamma. However, T cell CD38 expression was induced more strongly by 1,25(OH)2D3 than by IFN-alpha, and was unaffected by IFN-gamma. The CD38 density on activated 1,25(OH)2D3-treated CD38-/low B and peripheral T cells was proportional to cell size, indicating that hormonal induction depended upon entry into the cell cycle. While IFNs induced CD38 rapidly in stimulated T and B lymphocytes, 1,25(OH)2D3 exerted its effects only after initial 1- to 3-day delays, suggesting a requirement for nuclear 1,25(OH)2D3 receptor up-regulation. In HL-60 cells, which constitutively express the nuclear receptors, 1,25(OH)2D3 rapidly induced CD38 Ag and ectoenzyme activity. The CD38 density on freshly isolated unfractionated tonsillar B lymphocytes and on the activated 1,25(OH)2D3-treated cultured cells was nearly identical for cells within the same size range, indicating that in vitro hormonal exposure reconstituted in vivo CD38 expression levels.

Requirements for induction of vitamin D-mediated gene regulation in normal human B lymphocytes.

Mature human lymphocytes are unique targets of 1 alpha,25-dihydroxyvitamin D3 (1 alpha,25(OH)2D3) in that vitamin D receptors (VDR) are not constitutively expressed, and specific cellular activation signals are required for both the up-regulation of VDR and establishment of reactivity to the lipophilic ligand. Treatment of B lymphocytes with the cytokine IL-4 (IL-4), in the absence of prior activation, induces a weak up-regulation of VDR expression but fails to generate vitamin D-responsive element (VDRE)-reactive nuclear protein complexes or to initiate the genomic transcription of 25-hydroxyvitamin D3 24-hydroxylase. Stimulation of B lymphocytes by either ligation of CD40 Ag or cross-linking the Ig receptor is also insufficient to render B lymphocytes responsive to 1 alpha,25(OH)2D3. However, this apparent lack of response to the secosterol can be overcome by stimulation of B lymphocytes with a combination of these cellular activation signals, which are sufficient to lead to G1 cell cycle progression. In the presence of 1 alpha,25(OH)2D3, cellular activation associated with stimulation of such a progression appears to be sufficient for the up-regulation of VDR message and protein and necessary for the establishment of VDRE binding complexes and the induction of 24-hydroxylase message. Furthermore, biologic functions are modulated, in that the hormone inhibits proliferation in a subset of the activated B cells. These observations suggest that reactivity to 1 alpha,25(OH)2D3 is tightly regulated in B lymphocytes, requiring specific signals for its initiation.

Differential regulation of vitamin D receptors in clonal populations of a chronic myelogenous leukemia cell line.

RWLeu4 is a chronic myelogenous leukemia cell line that is sensitive to the antiproliferative and differentiation-inducing actions of 1alpha,25(OH)2-vitamin D3 (VD3). The JMRD3 cell line is a VD3-resistant variant of RWLeu4 that was selected by continuous passage of RWLeu4 in the presence of VD3. The isolation of a spontaneous VD3-resistant variant suggests that phenotypically different cells exist within the RWLeu4 cell population. Therefore, single-cell clones of RWLeu4 cells were isolated and characterized. Four clonal cell populations that fall into three groups differing in response to the antiproliferative and differentiation-inducing actions of VD3 were examined. Surprisingly, the extent of response of the clones to VD3 does not show a correlation with the basal level of the vitamin D receptor (VDR). RWLeu4-3 and RWLeu4-4 are the clones most sensitive to the antiproliferative actions of VD3 (ED50 approximately equal to 1 nM); however, RWLeu4-3 expresses basal levels of VDRs similar to those found in the parental cells and the RWLeu4-2 clone, while in RWLeu4-4, VD3 binding and VDR protein are below the limits of detection. Furthermore, RWLeu4-10 expresses the highest basal level of VDR protein but is relatively resistant to the antiproliferative actions of VD3 (ED50 > or = 30 nM). Like JMRD3, RWLeu4-10 is still capable of differentiating in response to VD3, as judged by the induction of biochemical processes and cell-surface antigen expression. Although VD3 treatment increases VDR protein levels and DNA-binding activity in all clones, altered DNA-protein complexes are detected in RWLeu4-4. Our results suggest that sensitivity to the antiproliferative and differentiation-inducing actions of VD3 is not dependent solely upon the level of VDR expressed, but may also require posttranslational modification of the VDR or complex interactions with other nuclear transcription factors.

Characterization of the interaction between CD45 and CD45-AP.

CD45, a leukocyte-specific transmembrane protein tyrosine phosphatase, is required for critical signal transduction pathways in immune responses. To elucidate the molecular interactions of CD45 with other proteins involved in CD45-mediated signal transduction pathways, we have recently cloned a 30-kDa phosphorylated protein, CD45-AP, which specifically associates with CD45. Binding analysis employing several deleted or chimeric forms of CD45-AP and CD45 demonstrated that the potential transmembrane segment of CD45-AP bound to the transmembrane portion of CD45. CD45-AP was found in particulate fractions of lymphocytes along with CD45, indicating that it is likely to be a transmembrane protein. In addition, CD45-AP was resistant to proteolysis by tosylphenylalanyl chloromethyl ketone-treated trypsin applied to intact cells. This is consistent with the most likely membrane orientation of CD45-AP predicted from the amino acid sequence, that is, only a short amino-terminal segment of CD45-AP is extracellular. We propose that CD45-AP interacts with CD45 at the plasma membrane and that the bulk of CD45-AP located in the cytoplasm act as an adapter which directs the interaction between CD45 and other molecules involved in CD45-mediated signal transduction pathways.

Differential regulation of JunD by dihydroxycholecalciferol in human chronic myelogenous leukemia cells.

1,25-Dihydroxyvitamin D3 inhibits the proliferation of the chronic myelogenous leukemia cell line RWLeu-4 but not the resistant variant, JMRD3. Although these cells exhibit no detectable differences in the vitamin D receptor, alterations in the interaction of nuclear extracts with the osteocalcin-1,25-dihydroxyvitamin D3-response element are noted. It is shown herein that the 1,25-dihydroxyvitamin D3 receptor binds to the osteocalcin-1,25-dihydroxyvitamin D3-response element along with activator protein-1 (AP-1) complexes and that the DNA binding activities of members of the Jun and Fos proto-oncogene families, which make up the AP-1 transcription factor, are differentially regulated by 1,25-dihydroxyvitamin D3. It is shown that JunD DNA binding activity is enhanced by 1,25-dihydroxyvitamin D3 during cell cycle arrest in the sensitive cells but is decreased in the resistant cells. These results suggest that the level of JunD DNA binding activity may be a critical factor in the regulation of proliferation.

Characterization of a vitamin D3-resistant human chronic myelogenous leukemia cell line.

A variant of the chronic myelogenous leukemia cell line, RWLeu-4, that is resistant to the antiproliferative effects of vitamin D3 was established. Although RWLeu-4 proliferation is inhibited by 1 nmol/L vitamin D3, the resistant cells (JMRD3) continue to proliferate in the presence of 100 nmol/L vitamin D3. Both cells express similar patterns of differentiation-specific antigens after treatment with vitamin D3, and both express the retinoblastoma gene product (p110Rb). Vitamin D3 treatment of the sensitive RWLeu-4 cells decreased the level of the p110Rb protein, as well as its phosphorylation. In contrast, vitamin D3 treatment of JMRD3 had no effect on p110Rb expression or phosphorylation. Both RWLeu-4 and JMRD3 express similar vitamin D3 receptors and vitamin D3-inducible enzyme activities. Differences were detected in the DNA binding characteristics of the vitamin D3 receptors as determined by electrophoretic mobility shift studies. However, sequence analysis of the DNA-binding domain and immunoblot analysis showed no differences in the receptors. We conclude that some process subsequent to vitamin D3 receptor activation is altered in JMRD3 that partially separates vitamin D3-induced inhibition of proliferation from the induction of differentiation.

Functional block for 1 alpha,25-dihydroxyvitamin D3-mediated gene regulation in human B lymphocytes.

Elements necessary for the steroid hormone 1 alpha,25-dihydroxyvitamin D3 (1 alpha,25-(OH)2D3) to induce a biological response include the presence of specific intracellular receptors (vitamin D3 receptors (VDR)) and modulation of gene expression via hormone-activated receptor binding to regulatory regions of target genes. These parameters were examined in normal and Epstein-Barr virus-immortalized human B cells and compared with 1 alpha,25-(OH)2D3-responsive cells of the T and monocytic lineages. Although resting tonsillar B cells did not express VDR mRNA, activation of these cells with interleukin-4 induced VDR in the absence of exogenously supplemented 1 alpha,25-(OH)2D3. As indicators of hormone-mediated gene regulation we analyzed modulation of CD23, a common B cell/monocyte surface antigen, and 24-hydroxylase. 1 alpha,25-(OH)2D3 inhibited CD23 expression in U937 cells, yet failed to modulate CD23 expression in B cells. Furthermore, 1 alpha,25-(OH)2D3 induced 24-hydroxylase mRNA expression and metabolic activity in both U937 cells and lectin-activated T cells, yet failed to induce 24-hydroxylase mRNA or its metabolic activity in B cells. These findings suggest that although human B lymphocytes can express VDR mRNA and protein, they exhibit a functional block for vitamin D-dependent gene regulation.

Alterations in CD45 glycosylation pattern accompanying different cell proliferation states.

CD45 is a leukocyte-specific transmembrane glycoprotein whose intracellular domain exhibits protein tyrosine phosphatase activity and plays a critical role in signal transduction. CD45 derived from stationary lymphocytes migrated faster in SDS-PAGE than that derived from exponentially growing cells. A change in N-linked saccharide structure other than the neuraminidase-sensitive terminal sialic acid portion was found to be responsible for the molecular size change in CD45. The differential glycosylation appeared to occur during late-stage posttranslational processing of CD45. We speculate that the N-glycosylation difference affects the interaction between CD45 and other factors involved in signal transduction leading to modulation of leukocyte proliferation.

Molecular cloning of the CD45-associated 30-kDa protein.

CD45, a leukocyte-specific transmembrane protein tyrosine phosphatase, mediates signal transduction pathways critical for immune responses. However, the mechanism of CD45-mediated signal transduction and the identity of CD45-associated proteins have remained unclear. A CD45-associated 30-kDA phosphorylated protein (CD45-AP) was purified by virtue of its specific association with CD45, and its mouse cDNA was cloned by using the internal amino acid sequence information. In vitro translated CD45-AP bound specifically to CD45. CD45-AP appears to be leukocyte-specific and shares no significant homology with presently known sequences. The predicted sequence contains no consensus tyrosine phosphorylation sites or conserved sequences of GTP-binding proteins. CD45-AP may act as an adapter molecule for CD45-mediated signal transduction.

Comparative analysis of NFAT (nuclear factor of activated T cells) complex in human T and B lymphocytes.

Nuclear factor of activated T cells (NFAT) is a transcriptional activator that binds to sequences in the interleukin-2 (IL-2) promoter and is thought to be largely responsible for the T cell-specific inducibility of IL-2 expression. Electrophoretic mobility shift assays (EMSA) showed that specific NFAT binding activity could also be induced in human B cells. The B cell NFAT complex, however, was not functional, since it failed to activate transcription from an NFAT-driven chloramphenicol acetyltransferase (CAT) construct. Competition with an AP-1 motif or with anti-Jun and anti-Fos antibodies abolished binding to the NFAT motif in both T and B cells, indicating that Jun and Fos are critical for NFAT complex formation in both cell types. Purified recombinant Jun and Fos proteins failed to bind directly to the NFAT motif. However, when combined with unstimulated B or T cell extracts, full-length, but not truncated, Jun/Fos heterodimers were able to form an NFAT complex, indicating the presence of a constitutively expressed nuclear factor(s) in B and T cells necessary for the formation of the NFAT complex in both cell types. An NFAT oligonucleotide carrying mutations in the 5' purine-rich part of the NFAT sequence failed to form a complex and to compete with the wild type motif for NFAT complex formation in both T and B cells. We therefore propose a model whereby a core NFAT complex consisting of Jun, Fos, and a constitutive nuclear factor is formed in both T and B cells, but an additional factor and/or post-translational modification of a factor, missing in B cells, might be required for transactivation by NFAT.