Biomedical subjects

C M Pleiman

Publications and source records attributed to C M Pleiman.

16 recordsLinked to original sources

PubMed1999

Selective A1-adenosine receptor antagonists identified using yeast Saccharomyces cerevisiae functional assays.

Evaluation of a biased "library" of pyrrolo[2,3-d]pyrimidines using yeast-based functional assays expressing human A1- and A2a-adenosine receptors, led to the A1 selective antagonist 4b. A direct correlation between yeast functional activity and binding data was established. Practical compounds with polar residues at C-4 of the pyrrolopyrimidine system required H-bond donor functionality for high potency.

Binding, Competitive↗

PubMed1998

pp56Lck mediates TCR zeta-chain binding to the microfilament cytoskeleton.

The TCR zeta-chain (zeta) on mature murine T lymphocytes binds to the microfilament cytoskeleton in response to Ag receptor ligation. Here, we report the role of Src family kinases in zeta-cytoskeletal binding, using mutant mice and a cell-free model system. Binding of zeta to actin in the cell-free system has a specific requirement for ATP and divalent cations, with an apparent Michaelis-Menton constant for ATP in the millimolar range, and can be disrupted by either EDTA or the microfilament poison, cytochalasin D, suggesting that microfilaments provide the structural framework for an active process involving cellular kinases. Indeed, tyrosine-phosphorylated zeta is a predominant form of the zeta-chain bound to polymerized actin, while challenge with alkaline phosphatase prevents zeta-chain association in solution and releases zeta-chain from the bound state. Phosphorylated Src-family kinase pp56Lck also associates with membrane skeleton upon TCR engagement and is a component of the reconstituted cytoskeletal pellet. Zeta-chain phosphorylation and zeta-cytoskeletal binding are abrogated in cell lysates with reduced levels of pp56Lck and in activated mutant murine T cells lacking pp56Lck, implicating pp56Lck as the kinase involved in zeta-chain tyrosine phosphorylation and zeta-cytoskeletal binding. Finally, recombinant Lck Src homology 2 domain preferentially inhibits reconstituted zeta-cytoskeleton association, suggesting that zeta-microfilament binding is dependent on interactions between phosphorylated tyrosine residues in zeta-chain activation motifs and the Src homology 2 domain of the Lck protein tyrosine kinase.

Actin Cytoskeleton↗

PubMed1997

Qualitative regulation of B cell antigen receptor signaling by CD19: selective requirement for PI3-kinase activation, inositol-1,4,5-trisphosphate production and Ca2+ mobilization.

Genetic ablation of the B cell surface glycoprotein CD19 severely impairs the humoral immune response. This requirement is thought to reflect a critical role of CD19 in signal transduction that occurs upon antigen C3dg coligation of antigen receptors with CD19 containing type 2 complement receptors (CR2). Here we show that CD19 plays a key accessory role in B cell antigen receptor signaling independent of CR2 coligation and define molecular circuitry by which this function is mediated. While CD19 is not required for antigen-mediated activation of receptor proximal tyrosines kinases, it is critical for activation of phosphatidylinositol 3-kinase (PI3-kinase). PI3-Kinase activation is dependent on phosphorylation of CD19 Y484 and Y515. Antigen-induced CD19-dependent PI3-kinase activation is required for normal phosphoinositide hydrolysis and Ca2+ mobilization responses. Thus, CD19 functions as a B cell antigen receptor accessory molecule that modifies antigen receptor signaling in a qualitative manner.

Androstadienes↗

PubMed1997

Qualitatively distinct signaling through T cell antigen receptor subunits.

T cell antigen receptors (TCR) contain several subunits including CD3gamma, delta, and epsilon, and TCRzeta and eta which are capable of mediating signal transduction. It is unclear whether the signaling function of these subunits is completely redundant. To assess the relative signaling capabilities of TCR subunits, we compared proximal events in signal transduction by wild-type TCR complexes and TCR devoid of functional zeta subunits, as well as chimeric receptors containing the cytoplasmic domains of TCRzeta or CD3epsilon. Results demonstrate that in BW5147 wild-type TCR, tail-less zeta TCR, CD3epsilon, and TCRzeta transduce signals leading to tyrosine phosphorylation of similar sets of cellular substrates, including the receptor subunits, Fyn, ZAP-70, and phospholipase Cgamma1 (PLCgamma1). Surprisingly, unlike wild-type TCR, tail-less zeta TCR, and CD3epsilon, TCRzeta was incapable of transducing signals resulting in inositol triphosphate (IP3) generation or intracellular free calcium ([Ca2+]i) mobilization. These data indicate that tyrosine phosphorylation of PLCgamma1 is not sufficient to drive IP3 production and [Ca2+]i mobilization. Most importantly, data presented indicate that TCRzeta and CD3epsilon engage partially distinct signaling pathways.

Amino Acid Sequence↗

PubMed1995

Phosphorylated immunoreceptor signaling motifs (ITAMs) exhibit unique abilities to bind and activate Lyn and Syk tyrosine kinases.

Signal transduction by T and B cell Ag receptors and certain receptors for Ig Fc regions (Fc gamma RI, hFc gamma RIIA, Fc gamma RIII, Fc alpha R, and Fc epsilon RI) involves a conserved sequence motif, termed an immunoreceptor tyrosine-based activation motif (ITAM) and found in multiple receptor chains. Phosphorylation of the two ITAM tyrosines is a critical event in signal transduction. To address the function of this phosphorylation, we assessed the ability of nonphosphorylated and biphosphorylated ((p)2ITAM) ITAM peptides to bind and modify the activity of src and syk family kinases in vivo and in vitro. All (p)2ITAMs, but not their nonphosphorylated counterparts, induced extensive protein tyrosine phosphorylation in permeabilized cells. However, the patterns of proteins phosphorylated differed among (p)2ITAMs. This phosphorylation was found to reflect activation of the src family kinase Lyn, but not Syk. In vitro studies using purified Lyn showed that src family kinase activation resulted from a direct interaction with (p)2ITAM. Binding studies demonstrated clear differences in binding specificity of (p)2ITAMs. Most strikingly, Ig alpha (p)2ITAM and TCR-zeta c and CD3 epsilon (p)2ITAMs exhibit inverse binding preferences for src and syk family kinases. Taken together, these findings demonstrate a novel mechanism by which src family tyrosine kinases are activated, and are consistent with the possibility that different ITAMs may preferentially activate distinct signaling pathways as a consequence of distinct effector Src homology 2 domain (SH2) binding preference.

Amino Acid Sequence↗

PubMed1994

Distinct p53/56lyn and p59fyn domains associate with nonphosphorylated and phosphorylated Ig-alpha.

Among the earliest detectable events in B-cell antigen receptor-mediated signal transduction are the activation of receptor-associated Src-family tyrosine kinases and the tyrosine phosphorylation of Ig-alpha and Ig-beta receptor subunits. These kinases appear to interact with resting B-cell antigen receptor complexes primarily through the Ig-alpha chain antigen receptor homology 1 (ARH1) motif. Recent studies showed a dramatic increase in the amount of Src-family kinase p59fyn bound to Ig-alpha when ARH1 motif tyrosines were phosphorylated. To explore the submolecular basis of these interactions, we conducted mutational analysis to localize sites in p53/56lyn and p59fyn that bind nonphosphorylated and phosphorylated Ig-alpha. Here we report that distinct regions within these kinases bind nonphosphorylated and phosphorylated Ig-alpha ARH1 motifs. The N-terminal 10 residues mediate binding to the nonphosphorylated Ig-alpha ARH1 motif. Association with the phosphorylated Ig-alpha ARH1 motif is mediated by Src homology 2 domains. These findings suggest a mechanism whereby ligand-induced Ig-alpha tyrosine phosphorylation initiates a change in the orientation of an associated kinase that may alter its activity and/or access to substrates and other effectors.

Amino Acid Sequence↗

PubMed1994

Activation of phosphatidylinositol-3' kinase by Src-family kinase SH3 binding to the p85 subunit.

Engagement of antigen receptor complexes induces rapid activation of Src-family kinases and association with phosphatidylinositol-3' kinase (PI-3 kinase). Here it was found that the Src homology 3 (SH3) domain of Lyn and Fyn bound to a proline-rich region (residues 84 to 99) within the 85-kilodalton subunit (p85) of PI-3 kinase. The binding of SH3 to the purified kinase led to a five- to sevenfold increase in the specific activity of PI-3 kinase. Ligand-induced receptor stimulation activated PI-3 kinase, and this activation was blocked by a peptide containing residues 84 to 99 of p85. These data demonstrate a mechanism for PI-3 kinase activation and show that binding of SH3 domains to proline-rich target sequences can regulate enzymatic activity.

Amino Acid Sequence↗

PubMed1994

Point mutations define a mIgM transmembrane region motif that determines intersubunit signal transduction in the antigen receptor.

Ag binding to the membrane Ig (mIg) substructure of the B cell Ag receptor leads to activation of cytoplasmic effector molecules including blk, fyn, lyn, and/or lck tyrosine kinases that are associated with receptor's dimeric Ig-alpha/Ig-beta transducer substructure. The structural basis of the apparent intermolecular transmission of this information within the receptor complex is unknown. Here we report that conservative point mutation of a sequence, S584-K597, at the cytoplasmic end of the predicted transmembrane spanning domain of the mIgM heavy chain (mu) ablates Ag-activated signal transduction, while having no detectable effect on association of mIgM with Ig-alpha/Ig-beta heterodimers. Specifically, mutation of serine584 to alanine, tyrosine587 to phenylalanine, threonine592 to valine, or lysine597 to isoleucine completely abrogated Ag-induced signal transduction leading to protein tyrosine phosphorylation and Ca2+ mobilization. Interestingly, mutants in the more peripheral of these residues, serine584 to alanine and lysine597 to isoleucine, remained responsive to a monoclonal antireceptor Ab (b-7-6) and all mutants remained responsive to polyclonal antireceptor Ab. These data implicate the polar sequence, -Y587STTVT592-, in transfer of information from ligand binding to transducer substructures within this heterooligomeric receptor complex. They further indicate that receptor activation by ligands that bind with high affinity and/or to constant region mIg epitopes is less dependent on the integrity of this motif.

Amino Acid Sequence↗

PubMed1994

The B-cell antigen receptor complex: structure and signal transduction.

The specificity of the immune response is determined by the interaction between the B-cell receptor (BCR) and its cognate structure, antigen. Recent studies have provided considerable insight into the compartmentalization of function within this extremely versatile hetero-oligomeric receptor complex. In this article, Christopher Pleiman, Daniele D'Ambrosio and John Cambier consolidate new findings regarding BCR structure and signal transduction.

Amino Acid Sequence↗

PubMed1994

Signal transduction by the B cell antigen receptor and its coreceptors.

B lymphocyte antigen receptors, membrane immunoglobulins (mIg), function in focusing and internalization of antigen for subsequent presentation to T cells and in transmembrane transduction of signals leading to cell activation, anergy, or deletion. Until quite recently, the ability of this receptor to transduce signals in spite of a virtual lack of cytoplasmic structure, left a significant gap in our understanding of how it is coupled to cytoplasmic signal propagators. Studies conducted during the past five years have defined a mIg-associated protein complex homologous to the CD3 complex associated with the T cell antigen receptor. Components of this disulfide linked heterodimeric complex, Ig-alpha and Ig-beta, contain an approximately 26 residue sequence motif termed ARH1, also known as TAM, which binds to cytoplasmic effectors, including src-family tyrosine kinases, and contains all structural information needed for signal transduction. Receptor associated src-family kinases which are activated following receptor cross-linking, also associate with downstream effectors, including phospholipase C gamma (PLC gamma), p21ras. GTPase activating protein (GAP), phosphatidylinositol 3-kinase (PI3-k) and microtubule associate protein kinase (MAPk2). In some cases, these associations are induced by receptor cross-linking and lead directly to effector activation. The current literature indicates that these interactions may occur in sequence and culminate in the activation of three major pathways of signal propagation including those mediated by PLC gamma, p21ras and PI3-k. This chapter reviews various molecular aspects of the B cell antigen receptor complex, including extended structure of the complex, and receptor-effector interactions and their biologic consequences. Finally, an integrated model of antigen receptor signaling is presented.

Amino Acid Sequence↗

PubMed1993

A divergence in the MAP kinase regulatory network defined by MEK kinase and Raf.

Mitogen-activated protein kinases (MAPKs) are rapidly phosphorylated and activated in response to various extracellular stimuli in many different cell types. Such regulation of MAPK results from sequential activation of a series of protein kinases. The kinases that phosphorylate MAPKs, the MAP kinase kinases (MEKs) are also activated by phosphorylation. MEKs are related in sequence to the yeast protein kinases Byr1 (from Schizosaccharomyces pombe) and Ste7 (from Saccharomyces cerevisiae), which function in the pheromone-induced signaling pathway that results in mating. Byr1 and Ste7 are in turn regulated by the protein kinases Byr2 and Ste11. The amino acid sequence of the mouse homolog of Byr2 and Ste11, denoted MEKK (MEK kinase), was elucidated from a complementary DNA sequence encoding a protein of 672 amino acid residues (73 kilodaltons). MEKK was expressed in all mouse tissues tested, and it phosphorylated and activated MEK. Phosphorylation and activation of MEK by MEKK was independent of Raf, a growth factor-regulated protein kinase that also phosphorylates MEK. Thus, MEKK and Raf converge at MEK in the protein kinase network mediating the activation of MAPKs by hormones, growth factors, and neurotransmitters.

Amino Acid Sequence↗

PubMed1993

Mapping of sites on the Src family protein tyrosine kinases p55blk, p59fyn, and p56lyn which interact with the effector molecules phospholipase C-gamma 2, microtubule-associated protein kinase, GTPase-activating protein, and phosphatidylinositol 3-kinase.

Engagement of the B-cell antigen receptor complex induces immediate activation of receptor-associated Src family tyrosine kinases including p55blk, p59fyn, p53/56lyn, and perhaps p56lck, and this response is accompanied by tyrosine phosphorylation of distinct cellular substrates. These kinases act directly or indirectly to phosphorylate and/or activate effector proteins including p42 (microtubule-associated protein kinase) (MAPK), phospholipases C-gamma 1 (PLC gamma 1) and C-gamma 2 (PLC gamma 2), phosphatidylinositol 3-kinase (PI 3-K), and p21ras-GTPase-activating protein (GAP). Although coimmunoprecipitation results indicate that the Src family protein tyrosine kinases interact physically with some of these effector molecules, the molecular basis of this interaction has not been established. Here, we show that three distinct sites mediate the interaction of these kinases with effectors. The amino-terminal 27 residues of the unique domain of p56lyn mediate association with PLC gamma 2, MAPK, and GAP. Binding to PI 3-K is mediated through the Src homology 3 (SH3) domains of the Src family kinases. Relatively small proportions of cellular PI 3-K, PLC gamma 2, MAPK, and GAP, presumably those which are tyrosine phosphorylated, bind to the SH2 domains of these kinases. Comparative analysis of binding activities of Blk, Lyn, and Fyn shows that these kinases differ in their abilities to associate with MAPK and PI 3-K, suggesting that they may preferentially bind and subsequently phosphorylate distinct sets of downstream effector molecules in vivo. Fast protein liquid chromatography Mono Q column-fractionated MAPK maintains the ability to bind bacterially expressed Lyn, suggesting that the two kinases may interact directly.

Animals↗

PubMed1992

Analysis of the fibroblast transformation potential of GTPase-deficient gip2 oncogenes.

Expression of GTPase-deficient Gi2 alpha subunit (alpha i2) mutant polypeptides and overexpression of the wild-type alpha i2 polypeptide in Rat 1a, Swiss 3T3, and NIH 3T3 fibroblasts altered normal growth regulation and induced a loss of contact inhibition. In Rat 1a cells (but not in NIH 3T3 or Swiss 3T3 cells), expression of the GTPase-deficient alpha i2 mutant polypeptides allowed colony formation in soft agar, which correlated with a loss in anchorage dependence and a decreased serum requirement. The altered growth regulatory properties of Rat 1a cells induced by expression of alpha i2 mutant polypeptides was not significantly inhibited by cotransfection with a dominant negative Ha-ras mutant polypeptide (Asn-17rasH), indicating that the activated Gi2 membrane signal transduction protein is uniquely capable of altering the regulation of Rat 1a cell growth by a predominantly c-ras-independent mechanism. The results show that GTPase-deficient alpha i2 mutant polypeptides have the properties of an oncogene that can induce the phenotypic characteristics of transformation in Rat 1a cells but that only a subset of these changes is observed with NIH 3T3 and Swiss 3T3 cells.

3T3 Cells↗

PubMed1991

Efficient gene transfer and expression in primary B lymphocytes.

In this study we have used a panel of vectors expressing the chloramphenicol acetyltransferase (CAT) reporter gene under the control of different regulatory elements to optimize gene transfer and expression in primary B lymphocytes. The Moloney murine leukemia virus long terminal repeat (MoMLV LTR) and the SV40 early region promoters, while functional in transfected plasmacytoma cell lines, did not give rise to detectable CAT activity following transfection into primary activated mouse or human B lymphocytes. In contrast, the human cytomegalovirus immediate-early (HCMV-IE) enhancer/promoter functioned in both established and primary B cells. The highest expression levels in the primary cells were obtained with vectors containing the Adenovirus 2 major late promoter or the HCMV-IE enhancer/promoter in combination with the Adenovirus 2 tripartite leader and VA genes. These latter expression cassettes were placed in a retroviral vector with the aim of combining their capacity for high-level gene expression with the efficient stable gene transfer afforded by retroviral infection. Several retroviral constructs were made, some of which were able to generate high virus titers. However all of these underwent deletions during the process of retroviral infection, as judged by Southern analysis of infected cells, indicating that they were not optimal gene transfer vectors. The HCMV enhancer/promoter, which was the most active of the other expression cassettes tested in the primary B cells, was inserted into a retroviral vector which also expressed the hph gene under the transcriptional control of the retroviral LTR. This vector did not undergo rearrangement during the process of retroviral infection, as judged by Southern analysis. The CAT gene was inserted downstream of the HCMV promoter in this vector, and a high-titer retroviral stock was generated. Primary B lymphocytes infected with this vector gave high levels of CAT activity, under conditions in which parallel experiments with the hph drug resistance marker showed that one in 20 of the cells were infected. These experiments demonstrate efficient gene transfer and expression in primary B lymphocytes in vitro.

Animals↗

PubMed1991

Organization of the murine and human interleukin-7 receptor genes: two mRNAs generated by differential splicing and presence of a type I-interferon-inducible promoter.

To better understand the regulation of interleukin-7 receptor (IL-7R) expression, we have pursued a detailed analysis of the structure of the murine and human IL-7R genes. The genes consist of eight exons, the sizes of which are conserved in mouse and human cells, spread out over 24 kbp (murine) and 19 kbp (human). A differential splicing event results in an mRNA encoding a secreted form of the human IL-7R gene. Primer extension and S1 nuclease analysis show a single transcriptional start site for the murine IL-7R gene. The 5'-flanking region of the murine IL-7R gene contains TATA- and CAAT-like sequences. The promoter region also contains a functional interferon regulatory element, to which the interferon-induced nuclear factors IRF-1 and IRF-2 are capable of binding and which is able to confer interferon-inducible expression on a heterologous gene. There are also potential binding sites for the transcription factors AP-1 and AP-2 as well as multiple glucocorticoid response elements. A fusion gene containing 2.5 kb of murine IL-7R 5' regulatory sequence linked to the bacterial chloramphenicol acetyltransferase gene directed expression of chloramphenicol acetyltransferase activity in murine pre-B-cell line 70Z/3 but not in the mouse fibroblast cell line NIH 3T3. Comparison of the murine and human IL-7R exon/intron boundaries with those of other hematopoietin receptor superfamily members whose exon/intron boundaries are also known reveals a conserved evolutionary structure.

Amino Acid Sequence↗

PubMed1991

Structure and expression of the murine hck gene.

The hck gene encodes a src-like protein-tyrosine kinase that is expressed predominantly in cells of myeloid origin. To examine the molecular basis of hck expression, we have cloned and characterized the murine hck gene and defined a set of transcriptional regulatory sequences. Transcripts from the hck gene have heterogeneous 5' ends, with one major and several minor transcriptional start sites. The promoter lacks a TATA-like sequence, but has three Sp-1 and two AP-2 binding sites. Constructs containing 2.5Kb or 0.6Kb of sequence 5' to the major transcription start site were capable of directing expression of a reporter gene transfected into fibroblasts. Expression of the former construct, but not the latter, was inducible with LPS. We conclude that LPS induction of hck transcripts, previously demonstrated in myeloid cells, results from the presence of an LPS-responsive element located within the hck promoter.

Animals↗