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John W Schrader

Publications and source records attributed to John W Schrader.

18 recordsLinked to original sources
PubMed2007

Distinct structural features of caprin-1 mediate its interaction with G3BP-1 and its induction of phosphorylation of eukaryotic translation initiation factor 2alpha, entry to cytoplasmic stress granules, and selective interaction with a subset of mRNAs.

Caprin-1 is a ubiquitously expressed, well-conserved cytoplasmic phosphoprotein that is needed for normal progression through the G(1)-S phase of the cell cycle and occurs in postsynaptic granules in dendrites of neurons. We demonstrate that Caprin-1 colocalizes with RasGAP SH3 domain binding protein-1 (G3BP-1) in cytoplasmic RNA granules associated with microtubules and concentrated in the leading and trailing edge of migrating cells. Caprin-1 exhibits a highly conserved motif, F(M/I/L)Q(D/E)Sx(I/L)D that binds to the NTF-2-like domain of G3BP-1. The carboxy-terminal region of Caprin-1 selectively bound mRNA for c-Myc or cyclin D2, this binding being diminished by mutation of the three RGG motifs and abolished by deletion of the RGG-rich region. Overexpression of Caprin-1 induced phosphorylation of eukaryotic translation initiation factor 2alpha (eIF-2alpha) through a mechanism that depended on its ability to bind mRNA, resulting in global inhibition of protein synthesis. However, cells lacking Caprin-1 exhibited no changes in global rates of protein synthesis, suggesting that physiologically, the effects of Caprin-1 on translation were limited to restricted subsets of mRNAs. Overexpression of Caprin-1 induced the formation of cytoplasmic stress granules (SG). Its ability to bind RNA was required to induce SG formation but not necessarily its ability to enter SG. The ability of Caprin-1 or G3BP-1 to induce SG formation or enter them did not depend on their association with each other. The Caprin-1/G3BP-1 complex is likely to regulate the transport and translation of mRNAs of proteins involved with synaptic plasticity in neurons and cellular proliferation and migration in multiple cell types.

Amino Acid Motifs↗
PubMed2006

RIVETS: the recombinant immunoglobulin and viral epitope tag system.

The human monoclonal antibody 8F9 binds to a linear 10 amino acid epitope that is present within the N-terminal region of the gB envelope glycoprotein of HCMV. Here we show that this short sequence (ETIYNTTLKY) can function as a tag for the detection of recombinant proteins using antibody 8F9. The AD-2S1 tag was recognized by 8F9 whether present at the N- or C-terminus of recombinant proteins and tagged recombinant proteins could be quantified with multiple analytical techniques such as ELISA, western blotting, immunofluorescence and flow cytometry. Production of 8F9 using different constant regions or constant regions from different species enhances the convenience and range of use of this system which we term the Recombinant Immunoglobulin and Viral Epitope Tag System or RIVETS.

Amino Acid Sequence↗
PubMed2006

Autoreactivity of primary human immunoglobulins ancestral to hypermutated human antibodies that neutralize HCMV.

The human antibody response to the AD-2S1 epitope of glycoprotein B (gB) of human cytomegalovirus (HCMV) is dominated by a family of closely related somatically mutated antibodies. These antibodies neutralize viral infectivity and the genes encoding them are derived from two commonly used germ-line variable (V) region genes, IGHV3-30 and IGKV3-11. Recombination of these V genes with the appropriate junctional diversity generates genes that encode primary immunoglobulins that bind to AD-2S1. To further understand the initial primary immunoglobulin response to AD-2S1 we synthesized the germ-line-based ancestor of one such family of antibodies and showed that it bound gB at the AD-2S1 epitope. Here we show that the germ-line ancestor of a second family of antibodies likewise binds to gB. We further show that one of the ancestral primary immunoglobulins, but not the other, also recognized autoantigens. In contrast, the hypermutated derivatives did not demonstrate autoreactivity and minor structural changes in the primary immunoglobulin were sufficient to generate or abolish autoreactivity or to change specificity. Thus, our demonstration that the ancestor of a highly mutated, non-autoreactive antiviral IgG antibody binds nuclear and cell-surface autoantigens indicates for the first time that self-reactivity is not necessarily a barrier to development into a follicular B lymphocyte that undergoes antigen-initiated affinity maturation.

Amino Acid Sequence↗
PubMed2005

Pure lipopolysaccharide or synthetic lipid A induces activation of p21Ras in primary macrophages through a pathway dependent on Src family kinases and PI3K.

Recognition of bacterial LPS by macrophages plays a critical role in host defense against infection by Gram-negative bacteria. However, when not tightly regulated, the macrophage's response to LPS can induce severe disease and septic shock. Although LPS triggers the activation of multiple signaling pathways in macrophages, it was unclear whether these include activation of the p21Ras GTPases. We report that p21Ras is rapidly and transiently activated in murine primary macrophages stimulated with an ultra-pure preparation of LPS or with synthetic lipid A. The molecular basis of this activation was investigated using a pharmacological approach. LPS-induced activation of p21Ras was inhibited in the presence of PP2, LY294002, or wortmannin, suggesting that it depends on the activity of one or more members of the Src kinase family and the subsequent activation of PI3K. In that pharmacological inhibitors of PI3K inhibited LPS-induced activation of p21Ras, but not activation of ERK, we concluded that LPS-induced activation of ERK occurs through a pathway that is not dependent on the activation of p21Ras.

Animals↗
PubMed2005

Absence of caprin-1 results in defects in cellular proliferation.

Cytoplasmic activation/proliferation-associated protein-1 (Caprin-1) is a cytoplasmic phosphoprotein that is the prototype of a novel family of highly conserved proteins. Its levels, except in the brain, are tightly correlated with cellular proliferation. We disrupted caprin-1 alleles in the chicken B lymphocyte line DT40 using homologous recombination. We readily obtained clones with one disrupted allele (31% of transfectants), but upon transfection of heterozygous cells we obtained a 10-fold lower frequency of clones with disruption of the remaining allele. Clones of caprin-1-null DT40 cells exhibited marked reductions in their proliferation rate. To obviate the problem that we had selected for caprin-1-null clones with characteristics that partially compensated for the lack of Caprin-1, we generated clones of DT40 cells heterozygous for the caprin-1 gene in which, during disruption of the remaining wild-type allele of the chicken caprin-1 gene, the absence of endogenous Caprin-1 would be complemented by conditional expression of human Caprin-1. Suppression of expression of human Caprin-1 resulted in slowing of the proliferation rate, due to prolongation of the G1 phase of the cell cycle, formally demonstrating that Caprin-1 was essential for normal cellular proliferation.

Amino Acid Sequence↗
PubMed2005

Recognition of human cytomegalovirus by human primary immunoglobulins identifies an innate foundation to an adaptive immune response.

Most primates, including humans, are chronically infected with cospecifically evolved, potentially pathogenic CMV. Abs that bind a 10-aa linear epitope (antigenic determinant 2 site 1) within the extracellular domain of human CMV glycoprotein B neutralize viral infectivity. In this study, we show that genes generated by recombinations involving two well-conserved human germline V elements (IGHV3-30 and IGKV3-11), and IGHJ4, encode primary Ig molecules that bind glycoprotein B at this key epitope. These particular V(H), J(H), and V(kappa) genes enable humans to generate through recombination and N nucleotide addition, a useful frequency of primary Igs that efficiently target this critical site on human CMV and thus confer an innate foundation for a specific adaptive response to this pathogen.

Adaptation, Physiological↗
PubMed2005

Expression of a constitutively active mutant of M-Ras in normal bone marrow is sufficient for induction of a malignant mastocytosis/mast cell leukemia, distinct from the histiocytosis/monocytic leukemia induced by expression of activated H-Ras.

Expression of constitutively activated M-Ras in normal murine bone-marrow cells was sufficient to induce the factor-independent, in vitro growth and differentiation of colonies of macrophages and neutrophils, and the generation of immortal lines of factor-independent mast cells, and, upon in vivo injection of the transduced cells, a fatal mastocytosis/mast-cell leukemia. In contrast, expression of constitutively activated H-Ras in bone-marrow cells resulted in the in vitro growth, in the absence of exogenous factors, of colonies that contained only macrophages and of lines of cells resembling dendritic cells, and, upon in vivo injection of the transduced cells, a fatal histiocytosis/monocytic leukemia. Macrophages generated by bone-marrow cells expressing activated M-Ras or activated H-Ras differed morphologically, the latter appearing more activated, a difference abrogated by an inhibitor of Erk activation. Inhibition of either Erk or PI3 kinase blocked the capacity of both activated M-Ras and activated H-Ras to support proliferation and viability. However, inhibition of p38 MAPK activity suppressed proliferation of bone-marrow cells expressing activated H-Ras, but enhanced that of bone-marrow cells expressing activated M-Ras. Thus, expression of either activated M-Ras or H-Ras in normal hematopoietic cells was sufficient for transformation but each resulted in the generation of distinct lineages of cells.

Animals↗
PubMed2005

A point mutation in the Ch3 domain of human IgG3 inhibits antibody secretion without affecting antigen specificity.

Immunoglobulins (Ig) require correct folding and assembly of both heavy (H) and light (L) chains to form a functional H2L2 dimer that is secreted from plasma cells. This process is dependent upon the endoplasmic reticulum (ER) chaperone BiP, which targets improperly, folded or assembled Ig molecules for degradation. While investigating the mechanism of low IgG3 secretion, we identified a missense mutation L368P in the Ch3 region of the human gamma3 H-chain that was associated with impaired secretion of intact and functional Ig. The non-secreted H-chains displayed slower electrophoretic migration than secreted H-chains, consistent with them being glycosylated in the ER but not fully processed in the golgi apparatus and secretory pathway. Reversion of the mutated codon to wild type restored secretion of the IgG3, which displayed the same fine specificity for antigen as non-secreted IgG3. However, the non-secreted IgG3 was not opsonic in an in vitro phagocytosis assay. The results indicate that correct IgG3 Ch3 domain folding is essential for secretion and effective function but does not affect specificity for antigen.

Amino Acid Substitution↗
PubMed2004

Activation/division of lymphocytes results in increased levels of cytoplasmic activation/proliferation-associated protein-1: prototype of a new family of proteins.

We purified from activated T lymphocytes a novel, highly conserved, 116-kDa, intracellular protein that occurred at high levels in the large, dividing cells of the thymus, was up-regulated when resting T or B lymphocytes or hemopoietic progenitors were activated, and was down-regulated when a monocytic leukemia, M1, was induced to differentiate. Expression of the protein was highest in the thymus and spleen and lowest in tissues with a low proportion of dividing cells such as kidney or muscle, although expression was high in the brain. The protein was localized to the cytosol and was phosphorylated, which is consistent with a previous report that the Xenopus laevis ortholog was phosphorylated by a mitotically activated kinase (1 ). The cDNA was previously mischaracterized as encoding p137, a 137-kDa GPI-linked membrane protein (2 ). We propose that the authentic protein encoded by this cDNA be called cytoplasmic activation/proliferation-associated protein-1 (caprin-1), and show that it is the prototype of a novel family of proteins characterized by two novel protein domains, termed homology regions-1 and -2 (HR-1, HR-2). Although we have found evidence for caprins only in urochordates and vertebrates, two insect proteins exhibit well-conserved HR-1 domains. The HR-1 and HR-2 domains have no known function, although the HR-1 of caprin-1 appeared necessary for formation of multimeric complexes of caprin-1. Overexpression of a fusion protein of enhanced green fluorescent protein and caprin-1 induced a specific, dose-dependent suppression of the proliferation of NIH-3T3 cells, consistent with the notion that caprin-1 plays a role in cellular activation or proliferation.

Amino Acid Sequence↗
PubMed2004

Expression of activated M-Ras in a murine mammary epithelial cell line induces epithelial-mesenchymal transition and tumorigenesis.

The expression of activated mutants of M-Ras (G22V or Q71L), but not wild-type M-Ras, in a murine mammary epithelial cell line, scp2, resulted in epithelial-mesenchymal transition (EMT) and oncogenic transformation. Cells expressing constitutively active M-Ras continued to grow in the absence of serum and exhibited a loss of the epithelial markers cytokeratin, E-cadherin and beta-catenin, together with a gain of the mesenchymal marker vimentin, a loss of contact inhibition in monolayer growth and a gain of the capacity for anchorage-independent growth. Moreover, unlike the parental cells, they failed to form differentiated mammospheres on Matrigel and instead formed branched networks of cells that grew and invaded the Matrigel. The expression of activated p21 Ras (G12V H-Ras or Q61K N-Ras) also resulted in EMT and tumorigenesis, although there was evidence that expression of higher levels was toxic. Tumors derived from scp2 cells expressing activated M-Ras exhibited activation of Akt and of ERK. The levels of expression of Q71L M-Ras and G12V H-Ras required for tumorigenesis were comparable, although higher levels of the weaker G22V M-Ras mutant were selected for in vivo. These data indicate that the expression of activated mutants of M-Ras was sufficient for oncogenic transformation of a murine mammary epithelial cell line.

Amino Acid Substitution↗
PubMed2004

Distinct mechanisms determine the patterns of differential activation of H-Ras, N-Ras, K-Ras 4B, and M-Ras by receptors for growth factors or antigen.

Although GTPases of the Ras family have been implicated in many aspects of the regulation of cells, little is known about the roles of individual family members. Here, we analyzed the mechanisms of activation of H-Ras, N-Ras, K-Ras 4B, and M-Ras by two types of external stimuli, growth factors and ligation of the antigen receptors of B or T lymphocytes (BCRs and TCRs). The growth factors interleukin-3, colony-stimulating factor 1, and epidermal growth factor all preferentially activated M-Ras and K-Ras 4B over H-Ras or N-Ras. Preferential activation of M-Ras and K-Ras 4B depended on the presence of their polybasic carboxy termini, which directed them into high-buoyant-density membrane domains where the activated receptors, adapters, and mSos were also present. In contrast, ligation of the BCR or TCR resulted in activation of H-Ras, N-Ras, and K-Ras 4B, but not M-Ras. This pattern of activation was not influenced by localization of the Ras proteins to membrane domains. Activation of H-Ras, N-Ras, and K-Ras 4B instead depended on the presence of phospholipase C-gamma and RasGRP. Thus, the molecular mechanisms leading to activation of Ras proteins vary with the stimulus and can be influenced by either colocalization with activated receptors or differential sensitivity to the exchange factors activated by a stimulus.

Adaptor Proteins, Signal Transducing↗
PubMed2004

Multiple aspects of the phenotype of mammary epithelial cells transformed by expression of activated M-Ras depend on an autocrine mechanism mediated by hepatocyte growth factor/scatter factor.

Multiple aspects of the transformed phenotype induced in a murine mammary epithelial cell line scp-2 by expression of activated G22V M-Ras, including maintainance of cell number at low density, anchorage-independent growth, invasion of Matrigel, and secretion of matrix metalloproteinases (MMP) 2 and 9, were dependent on an autocrine mechanism. Conditioned medium from dense cultures of scp-2 cells expressing G22V M-Ras, but not from parental cells, induced activation of Erk and Akt in cells expressing G22V M-Ras, maintained the cell number and promoted anchorage-independent growth of cells expressing G22V M-Ras (although not the parental cells), and induced scattering of MDCK cells. The latter activities were blocked by neutralizing antibodies to hepatocyte growth factor/scatter factor (HGF/SF) and could be mimicked by HGF/SF. Anti-HGF/SF antibodies also inhibited invasion of Matrigel, and the production of MMP-2 and MMP-9, together with urokinase-type plasminogen activator, was secreted by G22V M-Ras scp-2 cells but not by parental cells. Invasion of Matrigel was blocked by an inhibitor of MMPs, BB94, and by the mitogen-activated protein kinase kinase 1/2 kinase inhibitor PD98059 but was only marginally affected by the phosphatidylinositol 3-kinase inhibitor LY294002. Autocrine HGF/SF was thus critical for expression of key features of the phenotype of mammary epithelial cells transformed by expression of activated M-Ras.

Animals↗
PubMed2003

Defining the involvement of p38alpha MAPK in the production of anti- and proinflammatory cytokines using an SB 203580-resistant form of the kinase.

Despite its lack of specificity, the inhibitor SB 203580 has been widely used to implicate p38 mitogen-activated protein kinase (MAPK) in the synthesis of many cytokines. Here we show unequivocally that the production of interleukin (IL)-1beta, IL-6, IL-10, and tumor necrosis factor alpha (TNFalpha) requires p38 MAPK activity by demonstrating that the inhibitory effects of SB 203580 were reversed by expression of an SB 203580-resistant form of p38alpha (SBR-p38alpha) that fails to bind to SB 203580. This strategy established the requirement for p38 activity for the lipopolysaccharide-stimulated production of IL-10, IL-1beta, and IL-6 by the monocytic cell WEHI 274 and the production of IL-6 and TNFalpha stimulated by ligation of the Fc-gamma receptor of the mast cell MC/9. Expression of SBR-p38alpha in primary macrophages abrogated the ability of SB 203580 to inhibit the lipopolysaccharide-stimulated production of TNFalpha but not of IL-10. Expression of SBR-p38alpha in primary T lymphocytes abrogated the ability of SB 203580 to inhibit the production of interferon-gamma induced by co-ligation of CD3 and CD28 but not the production of interferon-gamma or IL-10 induced by IL-12. These results suggest that the levels of p38 MAPK activity required for maximal cytokine production vary with different cytokines and stimuli.

Animals↗
PubMed2002

Activation of Rac-1, Rac-2, and Cdc42 by hemopoietic growth factors or cross-linking of the B-lymphocyte receptor for antigen.

Interleukin-3 (IL-3)-induced activation of endogenous Rac-1, Rac-2, and Cdc42. Rac-1 was also activated by colony-stimulating factor-1 (CSF-1), Steel locus factor (SLF), granulocyte-macrophage colony-stimulating factor (GM-CSF), and IL-5 or by cross-linking the B-lymphocyte receptor for antigen (BCR). The activation of Rac-1 induced by cross-linking the BCR or by IL-3 stimulation was blocked only partially by Ly294002, with about 25% to 30% of Rac-1 activation still occurring in the absence of detectable increases in phosphatidyl-inositol-3 kinase (PI-3K) activity. Overexpression of constitutively active mutants of H-Ras, N-Ras, or M-Ras resulted in activation of coexpressed Rac-1 through an Ly29402-resistant, PI-3K-independent mechanism. Overexpression of constitutively active mutants of p21 Ras, or Rac-1, but not of PI-3K, was sufficient for activation of p38 mitogen-activated protein kinase (MAPK) in cells of hemopoietic origin. Inhibition of increases in PI-3K activity by Ly294002 had no effect on the IL-3-induced activation of p38 MAPK. In contrast, Ly294002 partially inhibited the activation of p38 MAPK induced by cross-linking of the BCR, although some p38 MAPK activation occurred in the absence of increases in the activity of Rac-1 or PI-3K. The activation of Rac-1, Rac-2, and Cdc42 by IL-3 and other hemopoietic growth factors is likely to be an important component of their actions in promoting growth, survival, and function.

Animals↗
PubMed2002

Activation of small GTPases of the Ras and Rho family by growth factors active on mast cells.

The small GTPases of the Ras and Rho families are activated by the key growth factors for mast cell development and survival, SLF and IL-3. While there are many clues that activation of Ras and Rho proteins play critical roles in growth, survival and differentiation, as well as in functions, such as migration and degranulation, limitations in the specificity of experimental tools still obscure their precise functions. There is increasing evidence that differences in subcellular localization of closely related GTPases determines important differences in their function. However, other data also point to differences in sensitivity to activation by GEF and in the effectors they engage.

Animals↗
PubMed2002

Ras and relatives--job sharing and networking keep an old family together.

Many members of the Ras superfamily of GTPases have been implicated in the regulation of hematopoietic cells, with roles in growth, survival, differentiation, cytokine production, chemotaxis, vesicle-trafficking, and phagocytosis. The well-known p21 Ras proteins H-Ras, N-Ras, K-Ras 4A, and K-Ras 4B are also frequently mutated in human cancer and leukemia. Besides the four p21 Ras proteins, the Ras subfamily of the Ras superfamily includes R-Ras, TC21 (R-Ras2), M-Ras (R-Ras3), Rap1A, Rap1B, Rap2A, Rap2B, RalA, and RalB. They exhibit remarkable overall amino acid identities, especially in the regions interacting with the guanine nucleotide exchange factors that catalyze their activation. In addition, there is considerable sharing of various downstream effectors through which they transmit signals and of GTPase activating proteins that downregulate their activity, resulting in overlap in their regulation and effector function. Relatively little is known about the physiological functions of individual Ras family members, although the presence of well-conserved orthologs in Caenorhabditis elegans suggests that their individual roles are both specific and vital. The structural and functional similarities have meant that commonly used research tools fail to discriminate between the different family members, and functions previously attributed to one family member may be shared with other members of the Ras family. Here we discuss similarities and differences in activation, effector usage, and functions of different members of the Ras subfamily. We also review the possibility that the differential localization of Ras proteins in different parts of the cell membrane may govern their responses to activation of cell surface receptors.

Animals↗