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R Seger

Publications and source records attributed to R Seger.

At least 19 recordsLinked to original sources
PubMed1992

Human T-cell mitogen-activated protein kinase kinases are related to yeast signal transduction kinases.

Mitogen-activated protein (MAP) kinase kinases, intermediates in a growth factor-stimulated protein kinase cascade, are dual specificity protein kinases that specifically phosphorylate and activate MAP kinases in response to extracellular signals. Here, we report the cloning of two forms of cDNA that encode this protein from human T-cells. MKK1a encodes a protein with predicted molecular size of 43,439 Da. Overexpression of this clone in COS cells led to elevated levels of protein and phorbol ester-stimulated MAP kinase kinase activity, confirming that MKK1a encodes the predicted protein. MKK1b, which appears to be an alternatively spliced form of the MKK1a gene, encodes a protein with predicted molecular size of 40,745 Da. Northern analysis revealed that the MKK1 cDNA hybridizes with a single 2.6-kilobase mRNA species in all human tissues examined. Sequence comparison shows homology to a group of yeast kinases that participate in signal transduction and to subdomain XI of other dual specificity kinase.

Amino Acid Sequence↗
PubMed1992

Purification and characterization of mitogen-activated protein kinase activator(s) from epidermal growth factor-stimulated A431 cells.

Two peaks of mitogen-activated protein (MAP) kinase activator activity are resolved upon ion exchange chromatography of cytosolic extracts from epidermal growth factor-stimulated A431 cells. Two forms of the activator (1 and 2) have been purified from these peaks, using chromatography on Q-Sepharose, heparin-agarose, hydroxylapatite, ATP-agarose, Sephacryl S-300, Mono S, and Mono Q. The two preparations each contained one major protein band with an apparent molecular mass of 46 or 45 kDa, respectively, on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Evidence identifying the MAP kinase activators as the 46- and 45-kDa proteins is presented. Using inactive mutants of MAP kinase as potential substrates, it was found that each preparation of MAP kinase activator catalyzes phosphorylation of the regulatory residues, threonine 188 and tyrosine 190, of Xenopus MAP kinase. These results support the concept that the MAP kinase activators are protein kinases. These MAP kinase kinases demonstrate an apparent high degree of specificity toward the native conformation of MAP kinase, although slow autophosphorylation on serine, threonine, and tyrosine residues and phosphorylation of myelin basic protein on serine and threonine residues is detected as well.

Animals↗
PubMed1992

Growth factor-stimulated phosphorylation cascades: activation of growth factor-stimulated MAP kinase.

Protein phosphorylation is an important mechanism in the response of cells to growth factors by which signals can be conveyed from cell surface receptors to intracellular targets. In addition to stimulation of protein tyrosine phosphorylation, activation of growth factor receptors having protein tyrosine kinase activity leads to dramatic alterations in the levels of protein serine/threonine phosphorylation. Several growth factor-stimulated serine/threonine-specific kinases have been identified as potential mediators of such signalling. MAP (microtubule-associated protein) kinase has emerged as a very interesting member of this group, because it activates a separate kinase, pp90rsk, which is also growth factor-stimulated. MAP kinase itself appears to be regulated by protein phosphorylation, because it can be inactivated by protein phosphatases. We have identified two 60 kDa proteins that promote the phosphorylation and full activation of MAP kinase in a manner paralleling its activation by growth factors in intact cells. These 'MAP kinase activators' are themselves stimulated by growth factors, suggesting that they function as intermediates between the MAP kinase and cell surface receptors in a growth factor-stimulated kinase cascade. Identification of the components of this protein kinase cascade reveals a mechanism by which at least some of the effects of receptor tyrosine kinases can be mediated through serine/threonine phosphorylation.

Animals↗
PubMed1992

The mitogen-activated protein kinase activator.

The mitogen-activated protein kinase appears to be regulated by another growth factor regulated kinase, the mitogen-activated protein kinase activator. In the past year, much progress has been made in purifying and characterizing the mitogen-activated protein kinase activator, in determining its primary structure, and in identifying another protein kinase that may function upstream to regulate its activity.

Animals↗
PubMed1992

Construction of nucleosome cores from defined DNA sequences of prokaryotic origin.

A procedure for the de novo construction of nucleosome core particles from defined DNA sequences of prokaryotic origin is described. Efficient de novo reconstitution without added carrier DNA is demonstrated. DNase I and exonuclease III analysis of a nucleosome core prepared from a 154 base pair fragment extending from base 853 to base 1006 of pBR322 indicates a non-random positioning of the histone core along the DNA. As bacteria have no histones, their DNA cannot be expected to have a histone core positioning signal encoded in it, the efficient formation of a uniquely positioned core particle is not self evident. The possibility that a phosphate end group positions DNA fragments on the histone is considered. The de novo reconstitution of carrier-less defined nucleosome core particles should facilitate the physicochemical study of nucleosomes on the fine structural level.

Centrifugation, Density Gradient↗
PubMed1992

Identification of an activator of the microtubule-associated protein 2 kinases ERK1 and ERK2 in PC12 cells stimulated with nerve growth factor or bradykinin.

Treatment of PC12 pheochromocytoma cells with nerve growth factor (NGF) or bradykinin leads to the activation of extracellular signal-regulated kinases ERK1 and ERK2, two isozymes of microtubule-associated protein 2 (MAP) kinase that are present in numerous cell lines and regulated by diverse extracellular signals. The activation of MAP kinase is associated with its phosphorylation on tyrosine and threonine residues, both of which are required for activity. In the present studies, we have identified a factor in extracts of PC12 cells treated with NGF or bradykinin, named MAP kinase activator, that, when reconstituted with inactive MAP kinase from untreated cells, dramatically increased MAP kinase activity. Activation of MAP kinase in vitro by this factor required MgATP and was associated with the phosphorylation of a 42- (ERK1) and 44-kDa (ERK2) polypeptide. Incorporation of 32P into ERK1 and ERK2 occurred primarily on tyrosine and threonine residues and was associated with a single tryptic peptide, which is identical to one whose phosphorylation is increased by treatment of intact PC12 cells with NGF. Thus, the MAP kinase activator identified in PC12 cells is likely to be a physiologically important intermediate in the signaling pathways activated by NGF and bradykinin. Moreover, stimulation of the activator by NGF and bradykinin suggests that tyrosine kinase receptors and guanine nucleotide-binding protein-coupled receptors are both capable of regulating these pathways.

Animals↗
PubMed1992

Cytochrome b558-negative, autosomal recessive chronic granulomatous disease: two new mutations in the cytochrome b558 light chain of the NADPH oxidase (p22-phox).

Chronic granulomatous disease (CGD) is characterized by the failure of activated phagocytes to generate superoxide. Defects in at least four different genes lead to CGD. Patients with the X-linked form of CGD have mutations in the gene for the beta-subunit of cytochrome b558 (gp91-phox). Patients with a rare autosomal recessive form of CGD have mutations in the gene for the alpha-subunit of this cytochrome (p22-phox). Usually, this leads to the absence of cytochrome b558 in the phagocytes (A22(0) CGD). We studied the molecular defect in five European patients from three unrelated families with this type of CGD. P22-phox mRNA was reverse-transcribed, and the coding region was amplified by PCR in one fragment and sequenced. Three patients from one family, with parents that were first cousins, were homozygous for a single base substitution (G-297-->A) resulting in a nonconservative amino acid change (Arg-90-->Gln). This mutation was previously found in a compound heterozygote A22(0) CGD patient. Another patient, also from first-cousin parents, was homozygous for an A-309-->G mutation in the open reading frame that predicts a nonconservative amino acid replacement (His-94-->Arg). The fifth patient was also born from a first-cousin marriage and was shown to be homozygous for the absence of exon 4 from the cDNA. In this patient, a G-->A substitution was found at position 1 of intron 4 in the genomic DNA. Therefore, the absence of exon 4 in the cDNA of this patient is due to a splicing error.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1992

[Farquhar disease. Reduced phagocyte function in a 9-year-old girl with erythrophagocytic lymphohistiocytosis].

We report phagocyte function tests in a female infant with familial hemophagozytotic lymphohistiocytosis. Chemiluminescence and killing of Saccharomyces cerevisiae by monocytes and granulocytes before chemotherapy and in remission were examined. Patients' monocytes and granulocytes showed a markedly reduced chemiluminescence and killing capacity irrespective of the stage of disease.

Antineoplastic Combined Chemotherapy Protocols↗
PubMed1991

Microtubule-associated protein 2 kinases, ERK1 and ERK2, undergo autophosphorylation on both tyrosine and threonine residues: implications for their mechanism of activation.

Microtubule-associated protein 2 kinase (MAP kinase), which exists in several forms, is a protein serine/threonine kinase that participates in a growth factor-activated protein kinase cascade in which it activates a ribosomal protein S6 kinase (pp90rsk) while being regulated itself by a cytoplasmic factor (MAP kinase activator). Experiments with recombinant MAP kinase, ERK2, purified from Escherichia coli in a nonactivated form revealed a self-catalyzed phosphate incorporation into both tyrosine and threonine residues. Another MAP kinase, ERK1, purified from insulin-stimulated cells also autophosphorylated on tyrosine and threonine residues. Autophosphorylation of ERK2 correlated with its autoactivation, although both autophosphorylation and autoactivation were slow compared to that occurring in the presence of MAP kinase activator. Therefore, we propose that autophosphorylation is probably involved in the MAP kinase activation process in vitro, but it may not be sufficient for full activation. The specificity toward tyrosine and threonine residues indicates that the MAP kinases ERK1 and ERK2 are members of a group of kinases with specificity for tyrosine as well as serine and threonine residues.

Amino Acid Sequence↗
PubMed1991

Multiple components in an epidermal growth factor-stimulated protein kinase cascade. In vitro activation of a myelin basic protein/microtubule-associated protein 2 kinase.

Epidermal growth factor stimulates the activity of several cytosolic serine/threonine protein kinases in quiescent Swiss 3T3 cells. Two of these, which use myelin basic protein (MBP) as substrate, act as kinase kinases in that they are able to activate a separate peptide kinase activity in vitro by a mechanism involving protein phosphorylation. In this study, we have identified two activities from extracts of epidermal growth factor-treated cells that stimulate an ATP-dependent activation of both of the MBP kinases, derived in their inactive precursor forms from extracts of untreated cells. The resulting MBP kinase activities are stable to further purification and can be inactivated with either tyrosine or serine/threonine protein phosphatases and then reactivated to their original levels of activity. Thus, we propose that the in vitro activation involves protein phosphorylation, stimulated by the action of novel MBP kinase activating factors that represent intermediate components in a growth factor-stimulated kinase cascade.

Amino Acid Sequence↗
PubMed1991

Unknown syndrome: ischiadic hypoplasia, renal dysfunction, immunodeficiency, and a pattern of minor congenital anomalies.

We report a 6 year old male with a pattern of malformations and anomalies including intrauterine growth retardation, microcephaly, psychomotor retardation, a pattern of craniofacial anomalies (flat face, hypertelorism, epicanthic folds, strabismus, short nose, low set ears), hypospadias and cryptorchidism, bilateral partial cutaneous syndactyly between fingers 2 to 5 and toes 2 to 4, postaxial polydactyly of the fingers and toes, severe conductive hearing loss, hypoplasia of the ischiadic bones, complex renal dysfunction, hypogammaglobulinaemia with proneness to bacterial infections of the upper and lower respiratory tract, and recurrent pseudomembranous enterocolitis. The parents are cousins of Turkish origin.

Abnormalities, Multiple↗
PubMed1991

Variant of hyper-IgE syndrome: the differentiation from atopic dermatitis is important because of treatment and prognosis.

The hyper-IgE syndrome is characterized clinically by recurrent staphylococcal abscesses of the skin, lungs and other sites from infancy. Affected patients also have a pruritic dermatitis that differs in character and distribution from lesions of atopic dermatitis. Most lack other signs of atopic disease, develop persistent pneumatoceles and have osteopenia. Laboratory abnormalities include the consistent presence of marked hyperimmunoglobulinemia E and eosinophilia of blood, sputum and tissues. They may have other inconsistent abnormalities of humoral and cellular immune responses and sometimes of phagocytic cell chemotactic responsiveness. Other clinical problems reported in such patients have included lymphomas, cryptococcal meningitis and cutaneous fungal disease. An 18-year-old male patient with a variant of the hyper-IgE syndrome, which he had acquired after a measles attack at the age of 5 years, suffered from recurrent ulcerative dermatitis and lymph node abscesses. Immunological investigation revealed an excessively elevated total serum IgE level (46,850 IU/ml), the presence of specific IgE to staphylococci, and quantitative and functional deficiency of IgG2. Skin and serological (radioallergosorbent) tests to inhalant and nutritive allergens were negative. Differentiation from atopic dermatitis should be made, because a long-term antistaphylococcal regime not only improves skin lesions but hinders the occurrence of lung abscesses and pneumatoceles.

Abscess↗
PubMed1990

Identification of the heterozygotes for deficiency of the beta-subunit of the eighth component of complement by reduced levels of C8 beta and increased amounts of free C8 alpha-gamma.

Sera from obligate heterozygotes for deficiency of the C8 beta subunit of the eighth component of human complement (C8) were analyzed for the molecular composition of C8. The C8 alpha-gamma and C8 beta subunits were separated by SDS-PAGE, visualized by immunoblotting, and the resulting bands were quantitated by laser densitometry. The laser densitometric absorption data were set to 100 arbitrary units (AU) for both subunits of pooled normal human sera. The AU values of individual normal sera ranged from 45 to 150 AU for C8 alpha-gamma (median 99 AU) and from 45 to 140 AU for C8 beta (median 101), whereas the C8 alpha-gamma/C8 beta-ratio varied from 0.7 to 1.4. Sera from C8 beta-deficient heterozygotes differed, as expected, from the normal sera for the markedly reduced levels of C8 beta (20 to 90 AU, median 55 AU) and for the higher C8 alpha-gamma/C8 beta-ratio (1.3 to 3.5). High voltage agarose gel electrophoresis was used to separate free and C8 beta-bound C8 alpha-gamma. The migration of free and C8 beta-bound C8 alpha-gamma subunit was checked by hemolytic overlay gels and by second dimension SDS-PAGE and immunoblotting. Immunochemical evaluation of C8 alpha-gamma using this system revealed about 5-14% free C8 alpha-gamma in sera with normal C8 and higher levels, from 33-71%, in the C8 beta D heterozygous sera. Functional analysis confirmed the substantial increase of free C8 alpha-gamma in the heterozygous group. We conclude that the C8 in C8 beta D heterozygous sera is characterized by increased amounts of free C8 alpha-gamma due to reduced concentrations of the C8 beta subunit. This finding may help to identify individuals heterozygous for C8 beta deficiency.

Complement C8↗
PubMed1990

Communication between protein tyrosine and protein serine/threonine phosphorylation.

In the present article, we have reviewed several promising areas of research, the results of which support the existence of enzymatic cascades linking extracellular growth factor receptor/tyrosine kinases with intracellular serine/threonine kinases. Proceeding either "upstream" from select serine/threonine kinases or "downstream" from the receptors, characterization of both the distal and proximal elements of these cascades is yielding insights into the multiple pathways of signal transduction. It is anticipated that future work will define the intermediate effectors in these cascades and provide us with a clear understanding of the biochemical mechanisms underlying intracellular responses to extracellular signals.

Animals↗
PubMed1989

[Recurrent meningitis in a familial defect of the beta-subunit of the 8th complement component].

Bacterial meningitides occurred in two members of a Yugoslavian family. In one case meningitis remained a singular event, Neisseria meningitidis being identified in the CSF. The second patient developed five episodes of recurrent purulent meningitis associated with petechiae and in one instance also with arthritis of the left knee but no causative germ was found. In both patients the Western blot technique revealed a defect of the beta-subunit of the eighth component of complement that was completely eliminated by purified C8. This proved that the C8 defect was isolated and that no other component was deficient.

Adolescent↗
PubMed1989

Studying the structure of the intracellular moiety of the insulin receptor with a kinase-splitting membranal proteinase.

A kinase-splitting membranal proteinase specifically clips the cytoplasmic moiety of the insulin receptor beta-subunit (95 kd) to yield an 84-kd fragment. Using antibodies against different domains in the receptor, cleavage is shown to remove an 11-kd 'tail' (rooted at the C-terminal end of the kinase domain) which includes tyrosines 1316 and 1322. This cleavage impairs the ability of the clustered tyrosines 1146, 1150 and 1151 to undergo autophosphorylation. Nevertheless, the clipped beta-subunit is as active as the intact subunit if its kinase activity is measured at high exogenous substrate concentrations (greater than or equal to 2 mg/ml) indicating that autophosphorylation is not obligatory for insulin-dependent phosphotransferase activity. With low substrate concentrations (e.g. 0.2 mg/ml) a severe damage to the kinase activity is detected, which may reflect an important structural contribution of the 'tail' and/or the clustered phosphotyrosines in creating the preferential affinity of the kinase for its in vivo substrate(s). The membranal proteinase strictly recognizes the native conformation of the kinase domain, and fails to cleave it after denaturation. Since such a conformation-dependent cleavage occurs also in the case of the cytoplasmic moiety of the EGF receptor and the catalytic subunit of cAMP-dependent protein kinase, it is suggested that the similarity between these three kinase domains extends beyond their reported sequence homology to reflect a similarity in conformation.

Animals↗