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C W Turck

Publications and source records attributed to C W Turck.

At least 37 records · Page 2Linked to original sources
PubMed1999

hnRNP H is a component of a splicing enhancer complex that activates a c-src alternative exon in neuronal cells.

The regulation of the c-src N1 exon is mediated by an intronic splicing enhancer downstream of the N1 5' splice site. Previous experiments showed that a set of proteins assembles onto the most conserved core of this enhancer sequence specifically in neuronal WERI-1 cell extracts. The most prominent components of this enhancer complex are the proteins hnRNP F, KSRP, and an unidentified protein of 58 kDa (p58). This p58 protein was purified from the WERI-1 cell nuclear extract by ammonium sulfate precipitation, Mono Q chromatography, and immunoprecipitation with anti-Sm antibody Y12. Peptide sequence analysis of purified p58 protein identified it as hnRNP H. Immunoprecipitation of hnRNP H cross-linked to the N1 enhancer RNA, as well as gel mobility shift analysis of the enhancer complex in the presence of hnRNP H-specific antibodies, confirmed that hnRNP H is a protein component of the splicing enhancer complex. Immunoprecipitation of splicing intermediates from in vitro splicing reactions with anti-hnRNP H antibody indicated that hnRNP H remains bound to the src pre-mRNA after the assembly of spliceosome. Partial immunodepletion of hnRNP H from the nuclear extract partially inactivated the splicing of the N1 exon in vitro. This inhibition of splicing can be restored by the addition of recombinant hnRNP H, indicating that hnRNP H is an important factor for N1 splicing. Finally, in vitro binding assays demonstrate that hnRNP H can interact with the related protein hnRNP F, suggesting that hnRNPs H and F may exist as a heterodimer in a single enhancer complex. These two proteins presumably cooperate with each other and with other enhancer complex proteins to direct splicing to the N1 exon upstream.

Alternative Splicing↗
PubMed1998

Exploiting the basis of proline recognition by SH3 and WW domains: design of N-substituted inhibitors.

Src homology 3 (SH3) and WW protein interaction domains bind specific proline-rich sequences. However, instead of recognizing critical prolines on the basis of side chain shape or rigidity, these domains broadly accepted amide N-substituted residues. Proline is apparently specifically selected in vivo, despite low complementarity, because it is the only endogenous N-substituted amino acid. This discriminatory mechanism explains how these domains achieve specific but low-affinity recognition, a property that is necessary for transient signaling interactions. The mechanism can be exploited: screening a series of ligands in which key prolines were replaced by nonnatural N-substituted residues yielded a ligand that selectively bound the Grb2 SH3 domain with 100 times greater affinity.

Adaptor Proteins, Signal Transducing↗
PubMed1998

A role for the actin-bundling protein L-plastin in the regulation of leukocyte integrin function.

Regulation of leukocyte integrin avidity is a crucial aspect of inflammation and immunity. The actin cytoskeleton has an important role in the regulation of integrin function, but the cytoskeletal proteins involved are largely unknown. Because inflammatory stimuli that activate integrin-mediated adhesion in human polymorphonuclear neutrophils (PMN) and monocytes cause phosphorylation of the actin-bundling protein L-plastin, we tested whether L-plastin phosphorylation was involved in integrin activation. L-plastin-derived peptides that included the phosphorylation site (Ser-5) rapidly induced leukocyte integrin-mediated adhesion when introduced into the cytosol of freshly isolated primary human PMN and monocytes. Substitution of Ala for Ser-5 abolished the ability of the peptide to induce adhesion. Peptide-induced adhesion was sensitive to pharmacologic inhibition of phosphoinositol 3-kinase and protein kinase C, but adhesion induced by a peptide containing a phosphoserine at position 5 was insensitive to inhibition. These data establish a novel role for L-plastin in the regulation of leukocyte adhesion and suggest that many signaling events implicated in integrin regulation act via induction of L-plastin phosphorylation.

Amino Acid Sequence↗
PubMed1998

Control of Leishmania major by a monoclonal alpha beta T cell repertoire.

Little is known regarding the diversity of the host T cell response that is required to maintain immunologic control of microbial pathogens. Leishmania major persist as obligate intracellular parasites within macrophages of the mammalian host. Immunity is dependent upon activation of MHC class II-restricted T cells to an effector state capable of restricting growth and dissemination of the organisms. We generated alpha-beta Leishmania-specific (ABLE) TCR transgenic mice with MHC class II-restricted T cells that recognized an immunodominant Leishmania Ag designated LACK. Naive T cells from ABLE mice proliferated in vitro after incubation with recombinant LACK or with Leishmania-parasitized macrophages and in vivo after injection into infected mice. Infected ABLE mice controlled Leishmania infection almost as well as wild-type mice despite a drastic reduction in the T cell repertoire. ABLE mice were crossed to mice with disruption of the TCR constant region alpha gene to create animals with a single alpha beta T cell repertoire. Although mice deficient in all alpha beta T cells (TCR-C alpha 0 mice) failed to control L. major, mice with a monoclonal alpha beta T cell repertoire (ABLE TCR-C alpha 0 mice) displayed substantial control. The immune system is capable of remarkable efficiency even when constrained to recognition of a single epitope from a complex organism.

Amino Acid Sequence↗
PubMed1998

BLNK: a central linker protein in B cell activation.

Linker or adapter proteins provide mechanisms by which receptors can amplify and regulate downstream effector proteins. We describe here the identification of a novel B cell linker protein, termed BLNK, that interfaces the B cell receptor-associated Syk tyrosine kinase with PLCgamma, the Vav guanine nucleotide exchange factor, and the Grb2 and Nck adapter proteins. Tyrosine phosphorylation of BLNK by Syk provides docking sites for these SH2-containing effector molecules that, in turn, permits the phosphorylation and/or activation of their respective signaling pathways. Hence, BLNK represents a central linker protein that bridges the B cell receptor-associated kinases with a multitude of signaling pathways and may regulate the biologic outcomes of B cell function and development.

Adaptor Proteins, Signal Transducing↗
PubMed1997

3-Phosphohistidine cannot replace phosphotyrosine in high-affinity binding to phosphotyrosine binding or Src homology 2 domains.

Posttranslational phosphorylation of proteins is an important event in many cellular processes. Phosphorylated tyrosine residues can serve as association sites for other proteins in signal transduction cascades of tyrosine kinase receptors. Formation of phosphohistidine residues in proteins has been found in eukaryotic and prokaryotic organisms. Furthermore, it has been suggested that phosphohistidine might substitute for phosphotyrosine in conferring high-affinity binding to proteins involved in signal transduction. We have analyzed the ability of 3-phosphohistidine to associate with the known phosphotyrosine-specific phosphotyrosine binding and src homology 2 protein domains. From our binding studies using synthetic peptides, we conclude that 3-phosphohistidine cannot replace phosphotyrosine in conferring high-affinity binding to the phosphotyrosine binding domain of shc or the src homology 2 domain of phospholipase C-gamma1.

Amino Acid Sequence↗
PubMed1997

The type II transforming growth factor-beta receptor autophosphorylates not only on serine and threonine but also on tyrosine residues.

The type I and type II receptors for transforming growth factor-beta (TGF-beta) are structurally related transmembrane serine/threonine kinases, which are able to physically interact with each other at the cell surface. To help define the initial events in TGF-beta signaling, we characterized the kinase activity of the type II TGF-beta receptor. A recombinant cytoplasmic domain of the receptor was purified from Escherichia coli and baculovirus-infected insect cells. Anti-phosphotyrosine Western blotting demonstrated that the type II receptor kinase can autophosphorylate on tyrosine. Following an in vitro kinase reaction, the autophosphorylation of the cytoplasmic domain and phosphorylation of exogenous substrate was shown by phosphoamino acid analysis to occur not only on serine and threonine but also on tyrosine. The dual kinase specificity of the receptor was also demonstrated using immunoprecipitated receptors expressed in mammalian cells and in vivo 32P labeling showed phosphorylation of the receptor on serine and tyrosine. In addition, the kinase activity of the cytoplasmic domain was inhibited by the tyrosine kinase inhibitor tyrphostin. Tryptic mapping and amino acid sequencing of in vitro autophosphorylated type II receptor cytoplasmic domain allowed the localization of the sites of tyrosine phosphorylation to positions 259, 336, and 424. Replacement of all three tyrosines with phenylalanines strongly inhibited the kinase activity of the receptor, suggesting that tyrosine autophosphorylation may play an autoregulatory role for the kinase activity of this receptor. These results demonstrate that the type II TGF-beta receptor can function as a dual specificity kinase and suggest a role for tyrosine autophosphorylation in TGF-beta receptor signaling.

Adenosine Triphosphate↗
PubMed1997

Molecular cloning of SLAP-130, an SLP-76-associated substrate of the T cell antigen receptor-stimulated protein tyrosine kinases.

Previous work has demonstrated that SLP-76, a Grb2-associated tyrosine-phosphorylated protein, augments Interleukin-2 promoter activity when overexpressed in the Jurkat T cell line. This activity requires regions of SLP-76 that mediate protein-protein interactions with other molecules in T cells, suggesting that SLP-76-associated proteins also function to regulate signal transduction. Here we describe the molecular cloning of SLAP-130, a SLP-76-associated phosphoprotein of 130 kDa. We demonstrate that SLAP-130 is hematopoietic cell-specific and associates with the SH2 domain of SLP-76. Additionally, we show that SLAP-130 is a substrate of the T cell antigen receptor-induced protein tyrosine kinases. Interestingly, we find that in contrast to SLP-76, overexpression of SLAP-130 diminishes T cell antigen receptor-induced activation of the interleukin-2 promoter in Jurkat T cells and interferes with the augmentation of interleukin-2 promoter activity seen when SLP-76 is overexpressed in these cells. These data suggest that SLP-76 recruits a negative regulator, SLAP-130, as well as positive regulators of signal transduction in T cells.

Adaptor Proteins, Signal Transducing↗
PubMed1997

A new regulatory protein, KSRP, mediates exon inclusion through an intronic splicing enhancer.

We have purified and cloned a new splicing factor, KSRP. KSRP is a component of a multiprotein complex that binds specifically to an intronic splicing enhancer element downstream of the neuron-specific c-src N1 exon. This 75-kD protein induces the assembly of five other proteins, including the heterogeneous nuclear ribonucleoprotein F, onto the splicing enhancer. The sequence of the KSRP cDNA indicates that the protein contains four K homology RNA-binding domains and an unusual carboxy-terminal domain. KSRP is similar to two proteins, FUSE-binding protein and P-element somatic inhibitor. KSRP is expressed in both neural and non-neural cell lines, although it is present at higher levels in neural cells. Antibodies specific for KSRP inhibit the splicing of the N1 exon in vitro. Moreover, this inhibition of N1 splicing can be rescued by the addition of purified KSRP. KSRP is likely to regulate splicing from a number of intronic splicing enhancer sequences.

Amino Acid Sequence↗
PubMed1997

Nuclear export of NF-ATc enhanced by glycogen synthase kinase-3.

The transcription factor NF-AT responds to Ca2+-calcineurin signals by translocating to the nucleus, where it participates in the activation of early immune response genes. Calcineurin dephosphorylates conserved serine residues in the amino terminus of NF-AT, resulting in nuclear import. Purification of the NF-AT kinase revealed that it is composed of a priming kinase activity and glycogen synthase kinase-3 (GSK-3). GSK-3 phosphorylates conserved serines necessary for nuclear export, promotes nuclear exit, and thereby opposes Ca2+-calcineurin signaling. Because GSK-3 responds to signals initiated by Wnt and other ligands, NF-AT family members could be effectors of these pathways.

Amino Acid Sequence↗
PubMed1997

Phosphorylation of serine residues 3, 6, 10, and 13 distinguishes membrane anchored from soluble glutamic acid decarboxylase 65 and is restricted to glutamic acid decarboxylase 65alpha.

GAD65, the smaller isoform of the gamma-aminobutyric acid-synthesizing enzyme glutamic acid decarboxylase is detected as an alpha/beta doublet of distinct mobility on SDS-polyacrylamide gel electrophoresis. Glutamic acid decarboxylase (GAD) 65 is reversibly anchored to the membrane of synaptic vesicles in neurons and synaptic-like microvesicles in pancreatic beta-cells. Here we demonstrate that GAD65alpha but not beta is phosphorylated in vivo and in vitro in several cell types. Phosphorylation is not the cause of the alpha/beta heterogeneity but represents a unique post-translational modification of GAD65alpha. Two-dimensional protein analyses identified five phosphorylated species of three different charges, which are likely to represent mono-, di-, and triphosphorylated GAD65alpha in different combinations of phosphorylated serines. Phosphorylation of GAD65alpha was located at serine residues 3, 6, 10, and 13, shown to be mediated by a membrane bound kinase, and distinguish the membrane anchored, and soluble forms of the enzyme. Phosphorylation status does not affect membrane anchoring of GAD65, nor its Km or Vmax for glutamate. The results are consistent with a model in which GAD65alpha and -beta constitute the two subunits of the native GAD65 dimer, only one of which, alpha, undergoes phosphorylation following membrane anchoring, perhaps to regulate specific aspects of GAD65 function in the synaptic vesicle membrane.

Amino Acid Sequence↗
PubMed1997

Synthesis and characterization of histidine-phosphorylated peptides.

Posttranslational phosphorylation of proteins is an important event in many cellular processes. Whereas phosphoesters of serine, threonine, and tyrosine have been studied extensively, only limited information is available for other amino acids modified by a phosphate group. The formation of phosphohistidine residues in proteins was discovered originally in prokaryotic organisms, but also has been found recently in eukaryotic cells. We describe methods for the synthesis and analysis of phosphohistidine-containing peptides, a prerequisite for the investigation of the role of this posttranslational modification in cellular processes.

Chromatography, High Pressure Liquid↗
PubMed1997

Impaired Th2 subset development in the absence of CD4.

Prior studies in CD4-deficient mice established the capacity of T helper (Th) lineage cells to mature into Th1 cells. Unexpectedly, challenge of these mice with Nippostrongylus brasiliensis, a Th2-inducing stimulus, failed to result in the development of Th2 cells. Additional studies were performed using CD4+ or CD4-CD8- (double-negative) T cell receptor (TCR) transgenic T cells reactive to LACK antigen of Leishmania major. Double-negative T cells were unable to develop into Th2 cells in vivo, and, unlike CD4+ T cells, could not be primed for interleukin-4 production in vitro. Similarly, CD4+ TCR transgenic T cells primed on antigen-presenting cells expressing mutant MHC class II molecules unable to bind CD4 did not differentiate into Th2 cells. These data suggest that interactions between the TCR, MHC II-peptide complex and CD4 may be involved in Th2 development.

Animals↗
PubMed1996

B cell antigen receptor signaling induces the formation of complexes containing the Crk adapter proteins.

Crk proteins are Src homology (SH) 2/SH3-containing adapter proteins that can mediate the formation of signaling complexes. We show that engaging the B cell antigen receptor (BCR) on the RAMOS B cell line caused both Crk-L and Crk II to associate with several tyrosine-phosphorylated proteins. We identified two of these phosphoproteins as Cas and Cbl and showed that both bound to the Crk SH2 domain after BCR engagement. BCR ligation also increased the amount of Crk proteins in the particulate fraction of the cells and induced the formation of Crk.Cas and Crk.Cbl complexes in the particulate fraction. We propose that tyrosine phosphorylation of membrane-associated Cas and Cbl creates binding sites for the Crk SH2 domain and recruits Crk complexes to cellular membranes. Thus, Crk proteins may participate in BCR signaling by using their SH2 domains to direct the interactions and subcellular localization of proteins that bind to their SH3 domains. In RAMOS cells, we found that the SH3 domains of Crk-L and Crk II bound C3G. Since C3G activates Rap, a negative regulator of the Ras pathway, Crk proteins may participate in regulation of Ras signaling by the BCR.

Adaptor Proteins, Signal Transducing↗
PubMed1996

Cysteines 153 and 154 of transmembrane transforming growth factor-alpha are palmitoylated and mediate cytoplasmic protein association.

Transforming growth factor-alpha (TGF-alpha) is synthesized as a transmembrane protein with a highly conserved, short cytoplasmic domain that is rich in cysteines. TGF-alpha is a prototype of a large family of growth factors involved in cell-cell communication. We have shown previously that transmembrane TGF-alpha associates with a kinase activity and two proteins of 106 and 86 kDa. In this study, we have used site-directed mutagenesis of the cytoplasmic domain of TGF-alpha to define the structural requirements for these protein interactions. Whereas the cytoplasmic domain of TGF-alpha was not essential for association with transmembrane p106, deletion of the C-terminal 8 amino acids, including a cysteine pair, abolished the interaction with p86 and greatly reduced the kinase activity associated with transmembrane TGF-alpha. Replacement of these 2 cysteines by serines similarly reduced the association of p86 with transmembrane TGF-alpha. Using a combination of mutational analysis and direct microsequencing, we have determined that this cysteine pair was palmitoylated. We therefore conclude that these cysteines play a critical role in the interaction of TGF-alpha with associated proteins and in the function of this protein complex. The palmitoylation of these cysteines suggests a possibly dynamic role of fatty acid modification in the integrity and function of the transmembrane TGF-alpha complex.

Animals↗
PubMed1996

A natural kinase-deficient variant of fibroblast growth factor receptor 1.

A fibroblast growth factor receptor 1 variant missing 37 amino acids from the carboxy-terminal tyrosine kinase catalytic domain was discovered in human lung fibroblasts and several other human cell lines. The receptor variant binds specifically to acidic fibroblast growth factor but has no tyrosine kinase activity. It was found that cellular transfectants expressing the fibroblast growth factor receptor 1 variant are mitogenically inactive and ligand binding to the receptor causes neither receptor autophosphorylation nor phospholipase C-gamma transphosphorylation. The fibroblast growth factor receptor 1 variant therefore represents an inactive receptor for acidic fibroblast growth factor. Since both kinase and kinase-deficient receptor forms are expressed in cells, it is conceivable that the kinase-deficient receptor plays an important role in regulating cellular responses elicited by acidic fibroblast growth factor stimulation.

Base Sequence↗
PubMed1996

Identification and cloning of centaurin-alpha. A novel phosphatidylinositol 3,4,5-trisphosphate-binding protein from rat brain.

Using an affinity resin and photoaffinity label based on phospholipid analogs of inositol 1,3,4,5-tetrakisphosphate (InsP4), we have isolated, characterized, and cloned a 46-kDa protein from rat brain, which we have named centaurin-alpha. Binding specificity was determined using displacement of 1-O-[3H](3-[4-benzoyldihydrocinnamidyl]propyl)-InsP4 photoaffinity labeling. Centaurin-alpha displayed highest affinity for phosphatidylinositol 3,4,5-trisphosphate (PtdInsP3) (IC50 = 120 nM), whereas InsP4, PtdInsP2, and InsP3 bound with 5-, 12-, and >50-fold lower affinity, respectively. Screening a rat brain cDNA library with a polymerase chain reaction product, generated using partial amino acid sequence from tryptic peptides, yielded a full-length clone. The 2,450-base pair cDNA contained an open reading frame (ORF) encoding a novel protein of 419 amino acids. Northern analysis revealed a 2.5-kilobase transcript that is highly expressed in brain. The deduced sequence contains a novel putative zinc finger motif, 10 ankyrin-like repeats, and shows homology to recently identified yeast and mammalian Arf GTPase-activating proteins. Given the specificity of binding and enrichment in brain, centaurin-alpha is a candidate PtdInsP3 receptor that may link the activation of phosphoinositide 3-kinase to downstream responses in the brain.

Adaptor Proteins, Signal Transducing↗