scholarly journals Two isoforms of murine hck, generated by utilization of alternative translational initiation codons, exhibit different patterns of subcellular localization

1991 ◽  
Vol 11 (9) ◽  
pp. 4363-4370
Author(s):  
P Lock ◽  
S Ralph ◽  
E Stanley ◽  
I Boulet ◽  
R Ramsay ◽  
...  
Keyword(s):  
Tyrosine Kinases ◽  
Molecular Basis ◽  
Mutational Analysis ◽  
Myeloid Cell ◽  
Cell Types ◽  
Subcellular Fractionation ◽  
Hemopoietic Cell ◽  
Translational Initiation ◽  
B Lymphoid ◽  
Triton X

Mammalian hck, a member of the src family of tyrosine kinases, is expressed predominantly in cells of the myeloid and B-lymphoid lineages. Using mutational analysis, we have investigated the molecular basis of two immunoreactive forms of murine hck of 56 and 59 kDa found in numerous hemopoietic cell types. Our results indicate that translation of murine p59hck initiates from a CTG codon located 21 codons 5' of an ATG that is utilized to generate p56hck. We provide evidence that two human hck isoforms are generated by the same mechanism. Subcellular fractionation studies reveal that while p59hck and p56hck are associated with membranes of various murine B-lymphoid and myeloid cell lines, p59hck alone is also located in the cytosol. In contrast to membrane-associated p59hck, which is metabolically labeled with [3H]myristic acid and exhibits amphiphilic properties in Triton X-114 detergent, cytosolic p59hck is hydrophilic, suggesting that it is not acylated. Possible mechanisms are proposed to account for these observations.

scholarly journals Two isoforms of murine hck, generated by utilization of alternative translational initiation codons, exhibit different patterns of subcellular localization.

1991 ◽  
Vol 11 (9) ◽  
pp. 4363-4370 ◽  
Author(s):  
P Lock ◽  
S Ralph ◽  
E Stanley ◽  
I Boulet ◽  
R Ramsay ◽  
...  
Keyword(s):  
Tyrosine Kinases ◽  
Molecular Basis ◽  
Mutational Analysis ◽  
Myeloid Cell ◽  
Cell Types ◽  
Subcellular Fractionation ◽  
Hemopoietic Cell ◽  
Translational Initiation ◽  
B Lymphoid ◽  
Triton X

Mammalian hck, a member of the src family of tyrosine kinases, is expressed predominantly in cells of the myeloid and B-lymphoid lineages. Using mutational analysis, we have investigated the molecular basis of two immunoreactive forms of murine hck of 56 and 59 kDa found in numerous hemopoietic cell types. Our results indicate that translation of murine p59hck initiates from a CTG codon located 21 codons 5' of an ATG that is utilized to generate p56hck. We provide evidence that two human hck isoforms are generated by the same mechanism. Subcellular fractionation studies reveal that while p59hck and p56hck are associated with membranes of various murine B-lymphoid and myeloid cell lines, p59hck alone is also located in the cytosol. In contrast to membrane-associated p59hck, which is metabolically labeled with [3H]myristic acid and exhibits amphiphilic properties in Triton X-114 detergent, cytosolic p59hck is hydrophilic, suggesting that it is not acylated. Possible mechanisms are proposed to account for these observations.

scholarly journals Synthesis of p36 and p35 is increased when U-937 cells differentiate in culture but expression is not inducible by glucocorticoids.

1989 ◽  
Vol 9 (1) ◽  
pp. 232-240 ◽  
Author(s):  
C M Isacke ◽  
R A Lindberg ◽  
T Hunter
Keyword(s):  
Tyrosine Kinases ◽  
Myeloid Cell ◽  
Cell Types ◽  
Culture Conditions ◽  
Actin Binding ◽  
Myeloid Cell Line ◽  
Related Proteins ◽  
Cortical Cytoskeleton ◽  
Inflammatory Action ◽  
Human Specific

p36 and p35 are distinct but related proteins that share many structural and biochemical features which were first identified as major substrates for protein-tyrosine kinases. Subsequently, both proteins have been shown to be Ca2+-, phospholipid-, and F-actin-binding proteins that underlie the plasma membrane and are associated with the cortical cytoskeleton. Recent reports have claimed that these proteins function as lipocortins, i.e., phospholipase A2 inhibitors that mediate the anti-inflammatory action of glucocorticoids. To investigate this possibility and to learn more about the functions of p36 and p35, we used human-specific anti-p36 and anti-p35 monoclonal antibodies to determine whether the expression or secretion of either protein was inducible by dexamethasone in the human U-937 myeloid cell line and in other human cell types. Additionally, we examined the levels of mRNA for both proteins. No effect of dexamethasone was observed on p36 or p35 expression at either the mRNA or protein level, nor were these proteins secreted under any of the culture conditions investigated. However, it was observed that in these cells the rate of synthesis and accumulation of both proteins was increased when the U-937 cells were induced to differentiate in culture to adherent macrophagelike cells. This offers a model system with which to study the control of p36 and p35 expression.

scholarly journals Overexpression of the Csk homologous kinase (Chk tyrosine kinase) induces multinucleation: a possible role for chromosome-associated Chk in chromosome dynamics

2001 ◽  
Vol 114 (9) ◽  
pp. 1631-1641 ◽  
Author(s):  
N. Yamaguchi ◽  
Y. Nakayama ◽  
T. Urakami ◽  
S. Suzuki ◽  
T. Nakamura ◽  
...  
Keyword(s):  
Tyrosine Kinase ◽  
Tyrosine Phosphorylation ◽  
Tyrosine Kinases ◽  
Subcellular Fractionation ◽  
Mitotic Spindles ◽  
Mitotic Chromosomes ◽  
Chromosome Dynamics ◽  
Phosphorylated Proteins ◽  
Triton X

The Csk family of non-receptor-type tyrosine kinases consists of Csk and the Csk homologous kinase Chk. Each enzyme suppresses the catalytic activity of Src family kinases by phosphorylating their C-terminal negative regulatory tyrosine residues. Ectopic and transient expression of Chk in COS-1 cells showed nuclear localization of Chk and growth inhibition. To further explore the role of Chk in cell growth, we overexpressed Chk in human immature myeloid KMT-2 cells. Chk overexpression brought about growth retardation and aberrant chromosome movement leading to multinucleation, and these events were accompanied by insufficient formation of mitotic spindles. In vitro kinase assays showed that Chk overexpression suppressed the tyrosine kinase activity of Lyn, a member of the Src family, immunoprecipitated from Triton X-100 lysates. Subcellular fractionation studies revealed that fractions of Chk and Lyn, resistant to Triton X-100 solubilization, are associated with mitotic chromosome scaffolds and spindles. Chk overexpression induced a decrease in autophosphorylation of Lyn and concomitant changes in levels of tyrosine phosphorylation of proteins associated with both fractions. These results indicate that Chk, Lyn and the tyrosine-phosphorylated proteins localize to mitotic chromosomes and spindles, suggesting that Chk-dependent tyrosine phosphorylation, presumably through Lyn, may be involved in chromosome dynamics.

Synthesis of p36 and p35 is increased when U-937 cells differentiate in culture but expression is not inducible by glucocorticoids

1989 ◽  
Vol 9 (1) ◽  
pp. 232-240
Author(s):  
C M Isacke ◽  
R A Lindberg ◽  
T Hunter
Keyword(s):  
Tyrosine Kinases ◽  
Myeloid Cell ◽  
Cell Types ◽  
Culture Conditions ◽  
Actin Binding ◽  
Myeloid Cell Line ◽  
Related Proteins ◽  
Cortical Cytoskeleton ◽  
Inflammatory Action ◽  
Human Specific

p36 and p35 are distinct but related proteins that share many structural and biochemical features which were first identified as major substrates for protein-tyrosine kinases. Subsequently, both proteins have been shown to be Ca2+-, phospholipid-, and F-actin-binding proteins that underlie the plasma membrane and are associated with the cortical cytoskeleton. Recent reports have claimed that these proteins function as lipocortins, i.e., phospholipase A2 inhibitors that mediate the anti-inflammatory action of glucocorticoids. To investigate this possibility and to learn more about the functions of p36 and p35, we used human-specific anti-p36 and anti-p35 monoclonal antibodies to determine whether the expression or secretion of either protein was inducible by dexamethasone in the human U-937 myeloid cell line and in other human cell types. Additionally, we examined the levels of mRNA for both proteins. No effect of dexamethasone was observed on p36 or p35 expression at either the mRNA or protein level, nor were these proteins secreted under any of the culture conditions investigated. However, it was observed that in these cells the rate of synthesis and accumulation of both proteins was increased when the U-937 cells were induced to differentiate in culture to adherent macrophagelike cells. This offers a model system with which to study the control of p36 and p35 expression.

scholarly journals Glycolipid-anchored proteins in neuroblastoma cells form detergent-resistant complexes without caveolin.

1995 ◽  
Vol 129 (3) ◽  
pp. 619-627 ◽  
Author(s):  
A Gorodinsky ◽  
D A Harris
Keyword(s):  
G Proteins ◽  
Tyrosine Kinases ◽  
Protein Complexes ◽  
Buoyant Density ◽  
Neuroblastoma Cells ◽  
Cell Types ◽  
Heterotrimeric G Proteins ◽  
Glycosyl Phosphatidylinositol ◽  
Src Family Tyrosine Kinases ◽  
Triton X

It has been known for a number of years that glycosyl-phosphatidylinositol (GPI)-anchored proteins, in contrast to many transmembrane proteins, are insoluble at 4 degrees C in nonionic detergents such as Triton X-100. Recently, it has been proposed that this behavior reflects the incorporation of GPI-linked proteins into large aggregates that are rich in sphingolipids and cholesterol, as well as in cytoplasmic signaling molecules such as heterotrimeric G proteins and src-family tyrosine kinases. It has been suggested that these lipid-protein complexes are derived from caveolae, non-clathrin-coated invaginations of the plasmalemma that are abundant in endothelial cells, smooth muscle, and lung. Caveolin, a proposed coat protein of caveolae, has been hypothesized to be essential for formation of the complexes. To further investigate the relationship between the detergent-resistant complexes and caveolae, we have characterized the behavior of GPI-anchored proteins in lysates of N2a neuroblastoma cells, which lack morphologically identifiable caveolae, and which do not express caveolin (Shyng, S.-L., J. E. Heuser, and D. A. Harris. 1994. J. Cell Biol. 125:1239-1250). We report here that the complexes prepared from N2a cells display the large size and low buoyant density characteristic of complexes isolated from sources that are rich in caveolae, and contain the same major constituents, including multiple GPI-anchored proteins, alpha and beta subunits of heterotrimeric G proteins, and the tyrosine kinases fyn and yes. Our results argue strongly that detergent-resistant complexes are not equivalent to caveolae in all cell types, and that in neuronal cells caveolin is not essential for the integrity of these complexes.

scholarly journals Genomic, Transcriptional and Mutational Analysis of the Mouse microphthalmia Locus

Genetics ◽  
2000 ◽  
Vol 155 (1) ◽  
pp. 291-300 ◽  
Author(s):  
Jón H Hallsson ◽  
Jack Favor ◽  
Colin Hodgkinson ◽  
Tom Glaser ◽  
M Lynn Lamoreux ◽  
...  
Keyword(s):  
Rna Processing ◽  
Molecular Basis ◽  
Mutational Analysis ◽  
Cell Types ◽  
Functional Domains ◽  
Allelic Series ◽  
Intron Structure ◽  
Microphthalmia Transcription Factor ◽  
First Time

Abstract Mouse microphthalmia transcription factor (Mitf) mutations affect the development of four cell types: melanocytes, mast cells, osteoclasts, and pigmented epithelial cells of the eye. The mutations are phenotypically diverse and can be arranged in an allelic series. In humans, MITF mutations cause Waardenburg syndrome type 2A (WS2A) and Tietz syndrome, autosomal dominant disorders resulting in deafness and hypopigmentation. Mitf mice thus represent an important model system for the study of human disease. Here we report the complete exon/intron structure of the mouse Mitf gene and show it to be similar to the human gene. We also found that the mouse gene is transcriptionally complex and is capable of generating at least 13 different Mitf isoforms. Some of these isoforms are missing important functional domains of the protein, suggesting that they might play an inhibitory role in Mitf function and signal transduction. In addition, we determined the molecular basis for six microphthalmia mutations. Two of the mutations are reported for the first time here (Mitf mi-enu198 and Mitf mi-x39), while the others (Mitf mi-ws, Mitf mi-bws, Mitf mi-ew, and Mitf mi-di) have been described but the molecular basis for the mutation not determined. When analyzed in terms of the genomic and transcriptional data presented here, it is apparent that these mutations result from RNA processing or transcriptional defects. Interestingly, three of the mutations (Mitf mi-x39, Mitf mi-bws, and Mitf mi-ws) produce proteins that are missing important functional domains of the protein identified in in vitro studies, further confirming a biological role for these domains in the whole animal.

scholarly journals Cell Activation Mediated by Glycosylphosphatidylinositol-Anchored or Transmembrane Forms of CD14

1998 ◽  
Vol 66 (3) ◽  
pp. 1174-1180 ◽  
Author(s):  
J. Pugin ◽  
V. V. Kravchenko ◽  
J.-D. Lee ◽  
L. Kline ◽  
R. J. Ulevitch ◽  
...  
Keyword(s):  
Tyrosine Kinases ◽  
Protein Tyrosine Kinase ◽  
Cell Activation ◽  
Myeloid Cell ◽  
Microbial Pathogens ◽  
Membrane Microdomains ◽  
Cross Linking ◽  
Calcium Signal ◽  
Protein Tyrosine ◽  
Triton X

ABSTRACT CD14 is a glycosylphosphatidylinositol (GPI)-anchored membrane glycoprotein which functions as a receptor on myeloid cells for ligands derived from microbial pathogens such as lipopolysaccharide (LPS). We have studied the importance of the GPI tail of CD14 in signalling with the promonocytic cell line THP-1 expressing recombinant CD14 in a GPI-anchored form (THP1-wtCD14 cells) or in a transmembrane form (THP1-tmCD14). We found that, like other GPI-anchored molecules, GPI-anchored CD14 was recovered mainly from a Triton X-100-insoluble fraction, whereas transmembrane CD14 was fully soluble in Triton X-100. LPS induced cell activation of THP1-wtCD14 and of THP1-tmCD14 (protein tyrosine kinase phosphorylation, NF-κB activation, and cytokine production) in a very similar manner. However, anti-CD14 antibody-induced cross-linking caused a rapid calcium mobilization signal only in GPI-anchored CD14 cells. Studies with pharmacologic inhibitors of intracellular signalling events implicate phospholipase C and protein tyrosine kinases in the genesis of this antibody-induced calcium signal. Our results suggest that GPI anchoring and CD14 targeting to glycolipid-rich membrane microdomains are not required for LPS-mediated myeloid cell activation. GPI anchoring may however be important for other signalling functions, such as those events reflected by antibody cross-linking.

scholarly journals Three distinct tolerogenic CD14+ myeloid cell types to actively manage autoimmune disease: Opportunities and challenges

2021 ◽  
Vol 120 ◽  
pp. 102645
Author(s):  
Glenn F. van Wigcheren ◽  
Daphne Roelofs ◽  
Carl G. Figdor ◽  
Georgina Flórez-Grau
Keyword(s):  
Autoimmune Disease ◽  
Myeloid Cell ◽  
Cell Types

scholarly journals Identification of a C3bi-specific membrane complement receptor that is expressed on lymphocytes, monocytes, neutrophils, and erythrocytes

1982 ◽  
Vol 155 (1) ◽  
pp. 96-110 ◽  
Author(s):  
GD Ross ◽  
JD Lambris
Keyword(s):  
Peripheral Blood ◽  
Myeloid Cell ◽  
Fluid Phase ◽  
Cell Types ◽  
Peripheral Blood Lymphocytes ◽  
Polymorphonuclear Cells ◽  
Coated Particles ◽  
Blood Lymphocytes ◽  
T Cell Antigens ◽  
D Region

Cells expressing a membrane C receptor (CR(3)) specific for C3b-inactivator- cleaved C3b (C3bi) were identified by rosette assay with C3bi-coated sheep erythrocytes (EC3bi) or C3bi-coated fluorescent microspheres (C3bi-ms). C3bi- ms, probably because of their smaller size, bound to a higher proportion of cells than did EC3bi. C3bi-ms bound to greater than 90 percent of mature neutrophils, 85 percent of monocytes, 92 percent of erythrocytes, and 12 percent of peripheral blood lymphocytes. Binding of C3bi-ms to neutrophils, monocytes, and erythrocytes was inhibited by fluid-phase C3bi, Fab anti-C3c, or Fab anti-C3d but was not inhibited by F(ab')(2) anti-CR(1) (C3b receptor) or F(ab')(2) anti-CR(2) (C3d receptor) nor by fluid-phase C3b, C3c, or C3d. This indicated that monocytes, neutrophils, and erythrocytes expressed C3bi receptors (CR(3)) that were separate and distinct from CR(1) and CR(2) and specific for a site in the C3 molecule that was only exposed subsequently to cleavage of C3b by C3b inactivator and that was either destroyed, covered, or liberated by cleavage of C3bi into C3c and C3d fragments. Lymphocytes differed from these other cell types in that they expressed CR2 in addition to CRa. Lymphocyte C3bi-ms rosettes were inhibited from 50 to 84 percent by F(ab')(2)-anti-CR(2) or fluid-phase C3d, whereas C3d-ms rosettes were inhibited completely by F(ab')(2) anti-CR(2), fluid-phase C3bi, or fluid- phase C3d. Thus, with lymphocytes, C3bi was bound to CR(3), and in addition was bound to CR(2) by way of the intact d region of the C3bi molecule. In studies of the acquisition of C receptors occurring during myeloid cell maturation, the ability to rosette with C3bi-coated particles was detected readily with immature low-density cells, whereas this ability was nearly undetectable with high density mature polymorphonuclear cells. This absence of C3bi binding to polymorphs was not due to a loss of the CR(3) but instead was due to the maturation-linked acquisition of the abiity to secrete elastase that cleaved reagent particle-bound C3bi into CR(3)-unreactive C3d. Neither neutrophils nor monocytes bound C3d-coated particles at any stage of maturation. Assay of CR(3) with mature neutrophils required inhibition of neutrophil elastase with either soybean trypsin inhibitor or anti-elastase antibodies, and the amounts of these elastase inhibitors required to allow EC3bi rosette formation increased with neutrophil maturation. Because lymphocytes bound C3bi to CR(2) as well as to CR(3), specific assay of lymphocyte CR(3) required saturation of membrane CR(2) with Fab' anti-CR(2) before assay for rosettes with C3bi-ms. Only 3.5 percent of anti-CR(2)- treated peripheral blood lymphocytes bound C3bi-ms. Therefore, among normal blood lymphocytes the majority of the 12 percent C3bi-ms-binding cells expressed only CR(2) (8.5 percent), and the small proportion of C3bi-ms- binding cells that expressed CR(3) (3.5 percent) represented a distinct subset from the CR2(+) cells. Double-label assay indicated that 3.0 percent out of 3.5 percent of these CR(3)-bearing lymphocytes were B cells because they expressed membrane immunoglobulins. Of the remaining CR(3)(+) cells, 0.2 percent expressed either Leu-1 or 3A1 T cell antigens, and 0.6 percent expressed the OKM-1 monocyte-null lymphocyte determinant.

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