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1.
Structure ; 4(9): 1105-14, 1996 Sep 15.
Article in English | MEDLINE | ID: mdl-8805596

ABSTRACT

BACKGROUND: The Abl nonreceptor tyrosine kinase is implicated in a range of cellular processes and its transforming variants are involved in human leukemias. The N-terminal regulatory region of the Abl protein contains Src homology domains SH2 and SH3 which have been shown to be important for the regulation of its activity in vivo. These domains are often found together in the same protein and biochemical data suggest that the functions of one domain can be influenced by the other. RESULTS: We have determined the crystal structure of the Abl regulatory region containing the SH3 and SH2 domains. In general, the individual domains are very similar to those of previously solved structures, although the Abl SH2 domain contains a loop which is extended so that one side of the resulting phosphotyrosine-binding pocket is open. In our structure the protein exists as a monomer with no intermolecular contacts to which a biological function may be attributed. However, there is a significant intramolecular contact between a loop of the SH3 domain and the extended loop of the SH2 domain. This contact surface includes the SH2 loop segment that is responsible for binding the phosphate moiety of phosphotyrosine-containing proteins and is therefore critical for orienting peptide interactions. CONCLUSIONS: The crystal structure of the composite Abl SH3-SH2 domain provides the first indication of how SH2 and SH3 domains communicate with each other within the same molecule and why the presence of one directly influences the activity of the other. This is the first clear evidence that these two domains are in contact with each other. The results suggest that this direct interaction between the two domains may affect the ligand binding properties of the SH2 domain, thus providing an explanation for biochemical and functional data concerning the Bcr-Abl kinase.


Subject(s)
Fusion Proteins, bcr-abl/chemistry , Protein-Tyrosine Kinases/chemistry , Amino Acid Sequence , Binding Sites , Fusion Proteins, bcr-abl/metabolism , Molecular Sequence Data , Phosphorylation , Protein Binding , Protein-Tyrosine Kinases/metabolism , Recombinant Proteins/chemistry , Recombinant Proteins/metabolism , X-Ray Diffraction , src Homology Domains
2.
Cell ; 72(5): 767-78, 1993 Mar 12.
Article in English | MEDLINE | ID: mdl-7680959

ABSTRACT

A phosphopeptide library was used to determine the sequence specificity of the peptide-binding sites of SH2 domains. One group of SH2 domains (Src, Fyn, Lck, Fgr, Abl, Crk, and Nck) preferred sequences with the general motif pTyr-hydrophilic-hydrophilic-Ile/Pro while another group (SH2 domains of p85, phospholipase C-gamma, and SHPTP2) selected the general motif pTyr-hydrophobic-X-hydrophobic. Individual members of these groups selected unique sequences, except the Src subfamily (Src, Fyn, Lck, and Fgr), which all selected the sequence pTyr-Glu-Glu-Ile. The variability in SH2 domain sequences at likely sites of contact provides a structural basis for the phosphopeptide selectivity of these families. Possible in vivo binding sites of the SH2 domains are discussed.


Subject(s)
Phosphopeptides/metabolism , Proto-Oncogene Proteins pp60(c-src)/metabolism , Amino Acid Sequence , Binding Sites , Molecular Sequence Data , Phosphorylation
3.
Science ; 257(5075): 1404-7, 1992 Sep 04.
Article in English | MEDLINE | ID: mdl-1326789

ABSTRACT

Mitogen-activated protein (MAP) kinases are 42- and 44-kD serine-threonine protein kinases that are activated by tyrosine and threonine phosphorylation in cells stimulated with mitogens and growth factors. MAP kinase and the protein kinase that activates it (MAP kinase kinase) were constitutively activated in NIH 3T3 cells infected with viruses containing either of two oncogenic forms (p35EC12, p3722W) of the c-Raf-1 protein kinase. The v-Raf proteins purified from cells infected with EC12 or 22W viruses activated MAP kinase kinase from skeletal muscle in vitro. Furthermore, a bacterially expressed v-Raf fusion protein (glutathione S-transferase-p3722W) also activated MAP kinase kinase in vitro. These findings suggest that one function of c-Raf-1 in mitogenic signaling is to phosphorylate and activate MAP kinase kinase.


Subject(s)
Protein Kinases/metabolism , Retroviridae Proteins, Oncogenic/pharmacology , 3T3 Cells , Animals , Cell Line , Cell Line, Transformed , Enzyme Activation/drug effects , Immunosorbent Techniques , Mice , Mitogen-Activated Protein Kinase Kinases , Muscles/enzymology , Oncogene Proteins v-raf , Phosphorylation , Proto-Oncogene Proteins/pharmacology , Proto-Oncogene Proteins c-raf , Recombinant Fusion Proteins/pharmacology , Retroviridae Proteins, Oncogenic/genetics
5.
Nature ; 336(6198): 479-81, 1988 Dec 01.
Article in English | MEDLINE | ID: mdl-2461518

ABSTRACT

Lymphocytes are most reliably subdivided on the basis of their receptors for antigen at the cell surface. Three subtypes of lymphocytes are well defined: B cells that bear surface immunoglobulin and make antibody, CD4+T cells with CD3 alpha beta receptors specific for antigen associated with class II major histocompatibility complex molecules, and CD8+T cells with CD3 alpha beta receptors specific for antigen associated with class I MHC molecules. These T cells are responsible for known forms of cell-mediated immunity. The discovery of a third rearranging T-cell specific gene called gamma (refs 1 and 2) has revealed the presence of a new class of T cells bearing a new receptor type, CD3 gamma delta (refs 3-7). To date, neither the function nor the specificity of cells bearing this receptor has been determined. Because gamma delta T cells are the main lymphocyte of epidermis, it was proposed that such cells could be important in surveillance of all epithelia. We have isolated intraepithelial lymphocytes from murine small intestine, and shown that they predominantly or exclusively express CD3 gamma delta receptors. Unlike the epidermal lymphocytes, these cells also express CD8, and they use a different V lambda gene to form their receptor. This strongly suggests that gamma delta T cells home in a very specific manner to epithelia, where they presumably mediate their function.


Subject(s)
Intestine, Small/cytology , Receptors, Antigen, T-Cell/immunology , T-Lymphocytes/immunology , Animals , Antigens, Differentiation, T-Lymphocyte/immunology , Blotting, Northern , Cell Separation , DNA/analysis , Epithelial Cells , Hybridomas/immunology , Immunosorbent Techniques , Iodine Radioisotopes , Macromolecular Substances , Mice , Mice, Inbred C57BL , Mice, Inbred DBA , RNA/analysis , Receptors, Antigen, T-Cell/analysis , Receptors, Antigen, T-Cell/genetics
6.
Proc Natl Acad Sci U S A ; 84(24): 9131-4, 1987 Dec.
Article in English | MEDLINE | ID: mdl-2827170

ABSTRACT

Antigen receptors on the T-cell surface are noncovalently associated with at least four invariant polypeptide chains, CD3-gamma, -delta, -epsilon, and -zeta. The mouse CD3-gamma gene, consisting of seven exons, was found to be highly homologous to the CD3-delta gene described earlier. Both the high level of sequence homology and the exon/intron organization indicate that the CD3-gamma and -delta genes arose by gene duplication. Surprisingly, murine and human genomic DNA clones could be isolated that contained elements of both the CD3-gamma and CD3-delta genes. In fact, the putative transcription start site of the mouse CD3-gamma gene is less than 1.4 kilobases from the transcription initiation site of the mouse CD3-delta gene. Common elements that regulate the divergent transcription of the two genes are therefore proposed to be located in the intervening 1.4-kilobase DNA segment. This might contribute to the coordinate expression of the CD3-gamma and -delta genes during intrathymic maturation of T lymphocytes.


Subject(s)
Antigens, Differentiation, T-Lymphocyte/genetics , Amino Acid Sequence , Animals , Base Sequence , CD3 Complex , DNA Restriction Enzymes , Exons , Gene Expression Regulation , Genetic Linkage , Humans , Introns , Mice , Molecular Sequence Data , Transcription, Genetic
7.
J Exp Med ; 166(4): 1186-91, 1987 Oct 01.
Article in English | MEDLINE | ID: mdl-3655657

ABSTRACT

The coding sequences of the murine and human T3 gamma chains are of identical length (182 amino acids) and contain a remarkable conservation of residues. The most striking observation is the high degree of homology between the murine and human cytosolic domains (89%), suggesting that the effector function of the T3 complex may be extremely similar or identical within human and murine lymphocytes. Both murine and human T lymphocytes can express two T3 gamma mRNA transcripts, suggesting that a second polyadenylation signal is present downstream. A poly(A) tail is not found in the 3' untranslated region of the murine gamma presented here, indicating that the murine clones analyzed represent mRNA generated by reading through the overlapping poly(A) signals at position 850-860 and possibly terminating at a position that would produce the 1.0 kb transcript.


Subject(s)
Cloning, Molecular , DNA/analysis , Receptors, Antigen, T-Cell/genetics , Animals , Base Sequence , Humans , Macromolecular Substances , Mice , Molecular Sequence Data , Transcription, Genetic
8.
Proc Natl Acad Sci U S A ; 84(18): 6536-40, 1987 Sep.
Article in English | MEDLINE | ID: mdl-2957697

ABSTRACT

The gamma-chain genes of the T-cell receptors form a family of related genes that are specifically expressed and somatically rearranged in T cells. Using poly- and monoclonal anti-gamma antibodies, we studied the cell-surface expression of the gamma-chain gene products in mouse peripheral T cells as well as in the T-cell blasts generated by allogeneic mixed lymphocyte reactions. The gamma chains are expressed in the Lyt2-,L3T4- subsets of these T-cell populations as disulfide-linked heterodimers. Whereas the electrophoretic mobility and the N-glycosylation of the spleen and lymph-node gamma chains are indistinguishable from those of the reported thymocyte gamma chain, a minor fraction of the T blasts generated by allogeneic stimulation of B10 lymph-node T cells with B10.BR spleen cells seems to express gamma chains with distinct properties. This suggests that the mixed lymphocyte culture conditions exert a selective effect on the expression of gamma chains among peripheral T-cell populations.


Subject(s)
Lymphocyte Activation , Peptide Fragments/metabolism , Receptors, Antigen, T-Cell/metabolism , T-Lymphocytes/physiology , Animals , Antibodies, Monoclonal , Antigens, Differentiation, T-Lymphocyte , Antigens, Ly/analysis , Antigens, Surface/analysis , Glycosylation , Isoelectric Point , Macromolecular Substances , Mice , Receptors, Antigen, T-Cell, gamma-delta , Spleen/immunology , Thymus Gland/immunology
9.
Proc Natl Acad Sci U S A ; 84(6): 1609-13, 1987 Mar.
Article in English | MEDLINE | ID: mdl-3550794

ABSTRACT

Using a subtractive cDNA cloning method, we isolated a number of T-lymphocyte-specific genes. One of these genes, represented by the cDNA clone pT49, is expressed in cytotoxic T lymphocytes but not in helper T lymphocytes or B lymphocytes. The protein structure deduced from the nucleotide sequence showed a high degree of homology to fibrinogen beta and gamma subunits. This might indicate that evolutionarily fibrinogen has its origin in lymphocytes. In spite of the strong homology of pT49 protein to the fibrinogen subunits, the positions of the introns in the pT49 gene are totally different from those of the fibrinogen genes.


Subject(s)
DNA/analysis , Fibrinogen/genetics , T-Lymphocytes, Cytotoxic/analysis , Amino Acid Sequence , Base Sequence , Biological Evolution , Fibrinogen/analysis , Introns , Multigene Family , Peptide Hydrolases/genetics , Protein Conformation
10.
Biochem J ; 242(3): 743-7, 1987 Mar 15.
Article in English | MEDLINE | ID: mdl-3593273

ABSTRACT

Antibodies were prepared against isolated rat renal glutaminase and affinity-purified against the 65 kDa peptide contained in the purified rat brain glutaminase. The affinity-purified IgGs were then used to compare the glutaminase immunoreactive peptides contained in samples that had been subjected to SDS/polyacrylamide-gel electrophoresis and transferred to nitrocellulose. The purified brain glutaminase and isolated brain mitochondria contain 68 and 65 kDa peptides that exhibit nearly equivalent immunostaining. Partial proteolysis of the isolated 68 and 65 kDa peptides with Staphylococcus aureus V8 proteinase produced an identical pattern of immunoreactive proteolytic fragments. However, digestion of the two peptides with chymotrypsin resulted in similar, but slightly different, patterns. The pattern of immunostaining was unaltered even when the brain mitochondria were solubilized with Triton X-100 and stored for 2 days at 4 degrees C. A very similar pattern was observed when intact renal mitochondria were subjected to immunoblot analysis. However, when renal mitochondria were solubilized, the 68 kDa peptide was rapidly degraded to the 65 kDa form. At 4 degrees C this reaction occurs with apparent first-order kinetics and a t1/2 of 35 min. Degradation of the 65 kDa form of the renal glutaminase occurs with much slower kinetics, but is nearly complete after 24 h. Solubilization of mitochondria isolated from various zones of the kidney indicated that the responsible endogenous proteinase was localized primarily in the cortex. Mitochondria isolated from intestinal or renal papillary tissue contain four glutaminase immunoreactive peptides (Mr 68,000, 65,000, 61,000 and 58,000). The smallest of these peptides is identical in size with the single immunoreactive peptide observed in liver tissue.


Subject(s)
Glutaminase , Mitochondria/enzymology , Peptide Fragments/analysis , Animals , Brain/enzymology , Immunoelectrophoresis , Intestine, Small/enzymology , Kidney/enzymology , Liver/enzymology , Male , Octoxynol , Polyethylene Glycols , Rats , Rats, Inbred Strains , Solubility , Tissue Distribution
11.
Arch Biochem Biophys ; 243(1): 1-7, 1985 Nov 15.
Article in English | MEDLINE | ID: mdl-2998280

ABSTRACT

Phosphate-dependent glutaminase is associated with the inner membrane of rat renal mitochondria. The orientation of this enzyme was characterized by comparing its sensitivity in isolated mitochondria and in mitoplasts to two membrane impermeable inhibitors. Mitoplasts were prepared by repeated swelling of mitochondria in a hypotonic phosphate solution. This procedure released greater than 70% of the adenylate kinase from the intermembrane space, but less than 10 and 25% of the marker activities characteristic of the inner membrane and matrix compartments, respectively. The addition of 20 microM p-chloromercuriphenylsulfonate (pCMPS) caused a rapid inactivation of the purified glutaminase. In contrast, the glutaminase contained in isolated mitochondria and mitoplasts was only slightly affected by the addition of up to 2 mM pCMPS. Similarly, the activity in mitochondria and mitoplasts was not inhibited by the addition of an excess of inactivating Fab antibodies. However, a similar extent of inactivation occurred when either membrane fraction was incubated with concentrations of octylglucoside greater than 0.35%. Mitochondria were also treated with increasing concentrations of digitonin. At 0.4 mg digitonin/mg protein, all of the adenylate kinase was released but the glutaminase activity was either slightly inhibited or unaffected by the addition of pCMPS or the Fab antibodies, respectively. These studies establish that the glutaminase is localized on the inner surface of the inner membrane. Therefore, mitochondrial catabolism of glutamine must occur only within the matrix compartment.


Subject(s)
Glutaminase/metabolism , Kidney/ultrastructure , Mitochondria/enzymology , Phosphates/metabolism , 4-Chloromercuribenzenesulfonate/metabolism , Adenylate Kinase/metabolism , Animals , Antibodies , Cell Fractionation , Digitonin/pharmacology , Glucosides/pharmacology , Immunoglobulin Fab Fragments/immunology , Intracellular Membranes/enzymology , Male , Rats , Rats, Inbred Strains , Time Factors
12.
Biochem J ; 229(2): 399-408, 1985 Jul 15.
Article in English | MEDLINE | ID: mdl-3899104

ABSTRACT

A phosphate-dependent glutaminase was purified 1200-fold from rat brain. In the absence of a polyvalent anion, the glutaminase exists as an inactive protomer which has an estimated Mr of 126000. The addition of 100mM-phosphate causes maximal activation and a dimerization (Mr 249000) of the glutaminase. The phosphate activation is sigmoidal, with a K0.5 of 25mM and a Hill coefficient (h) of 1.5 Glutamate inhibition is competitive with respect to glutamine and is decreased by increasing the concentration of phosphate. Phosphate also decreases the Km for glutamine. The purified glutaminase contains a predominant peptide (Mr 65000) and a minor peptide (Mr 68000) that are present in an approximate ratio of 4:1 respectively. The glutaminase immunoprecipitated from freshly solubilized brain tissue or from synaptosomal and non-synaptosomal brain mitochondria contains the same distribution of the two peptides. In contrast, the glutaminase purified from rat kidney contains five to seven peptides that range in Mr value from 59000 to 48000, and immunoprecipitates derived from freshly solubilized renal tissue contain only the Mr-65000 peptide. Partial proteolysis and size fractionation of the three immunoprecipitated peptides indicate that they are structurally related. The series of peptides characteristic of the purified renal glutaminase is generated on storage of the solubilized extract of kidney tissue. The glutaminase contained in the solubilized brain extract is not degraded unless a renal extract is added. Thus the difference in the pattern of peptides associated with the two purified enzymes is due to an endogenous renal proteinase that is not present in brain.


Subject(s)
Brain/enzymology , Glutaminase/metabolism , Kidney/enzymology , Phosphates/pharmacology , Animals , Brain/drug effects , Electrophoresis, Polyacrylamide Gel , Glutaminase/immunology , Glutaminase/isolation & purification , Immunoenzyme Techniques , Male , Mitochondria/enzymology , Octoxynol , Peptide Fragments/analysis , Polyethylene Glycols/pharmacology , Rats , Rats, Inbred Strains
13.
Brain Res ; 336(1): 158-61, 1985 Jun 10.
Article in English | MEDLINE | ID: mdl-3891015

ABSTRACT

Glutamate has long been considered to be a neurotransmitter candidate in vertebrate spinal sensory nerve cells. We report here the first immunohistochemical evidence in support of this hypothesis. We find that up to 30% of the moderately small dorsal root ganglion neurons in the rat contain elevated levels of glutaminase immunoreactivity. This enzyme, which mediates the synthesis of glutamate from glutamine, is not found at these high levels in large diameter neurons of the same ganglia. In contrast, another enzyme associated with glutamate metabolism, aspartate aminotransferase, is rather uniformly distributed within neurons of the sensory ganglia. These data define a subpopulation of sensory neurons which appear to contain an elevated capacity to synthesize glutamate through the glutamine cycle and suggest that glutaminase immunoreactivity may be an indicator of glutamatergic function in some nerve cells.


Subject(s)
Ganglia, Spinal/enzymology , Glutaminase/metabolism , Animals , Fluorescent Antibody Technique , Male , Rats , Rats, Inbred Strains
14.
Brain Res ; 330(2): 225-33, 1985 Mar 25.
Article in English | MEDLINE | ID: mdl-3886076

ABSTRACT

There is considerable evidence that pathways of the hippocampus use an excitatory amino acid as transmitter. We have attempted to immunocytochemically identify excitatory amino acid neurons in the hippocampus of the rat and guinea pig using antiserum to glutaminase and antiserum to aspartate aminotransferase, which have been proposed as markers for aspartergic/glutamergic neurons. Glutaminase-like immunoreactivity was seen in granule cells in the dentate gyrus and fibers and puncta associated with the mossy fiber pathway in the hilus and stratum lucidum of the hippocampus. At the ultrastructural level, glutaminase-like immunoreactivity was observed in mossy fiber terminals in the stratum lucidum. Glutaminase-like immunoreactivity was also seen in pyramidal cells in regio inferior and regio superior and in cells in layer two of the entorhinal cortex. Schaffer collateral terminals, commissural fiber terminals and perforant pathway terminals were not seen at the light microscopic level. Glutaminase-like immunoreactivity is thus found in the cell bodies of proposed excitatory amino acid neurons of hippocampal pathways, but does not appear to label all terminals. Aspartate aminotransferase-like immunoreactivity was not seen in any cells, fibers or terminals in the rat or guinea pig hippocampus.


Subject(s)
Aspartate Aminotransferases/metabolism , Glutaminase/metabolism , Hippocampus/enzymology , Animals , Guinea Pigs , Hippocampus/cytology , Immunoenzyme Techniques , Microscopy, Electron , Neural Pathways/enzymology , Rats , Rats, Inbred Strains , Synapses/enzymology
16.
Brain Res ; 291(1): 173-8, 1984 Jan 16.
Article in English | MEDLINE | ID: mdl-6365242

ABSTRACT

The immunocytochemical localization of glutaminase, which we have proposed as a marker for excitatory amino acid neurotransmitters was determined in the guinea pig auditory nerve. Glutaminase-like immunoreactivity was seen in auditory nerve terminals in the cochlear nucleus and in the cell bodies of the auditory nerve in the cochlea. This staining was seen in type I and not type II spiral ganglion cells. Glutaminase-like immunoreactivity was also observed in granule cells in the cochlear nucleus.


Subject(s)
Glutaminase/metabolism , Pons/enzymology , Vestibulocochlear Nerve/enzymology , Animals , Aspartate Aminotransferases/metabolism , Fluorescent Antibody Technique , Guinea Pigs , Rats , Rats, Inbred Strains , Spiral Ganglion/enzymology
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