Identification of the human pim-1 gene product as a 33-kilodalton cytoplasmic protein with tyrosine kinase activity

1988 ◽  
Vol 8 (4) ◽  
pp. 1498-1503
Author(s):  
A Telerman ◽  
R Amson ◽  
R Zakut-Houri ◽  
D Givol
Keyword(s):  
Tyrosine Kinase ◽  
Tyrosine Kinases ◽  
Kinase Activity ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Kinase Assay ◽  
In Vitro Translation ◽  
Tyrosine Kinase Activity

The human pim-1 gene was recently identified as a new putative oncogene located on chromosome 6p21, a region showing karyotypic abnormalities in particular leukemias. In the present work we characterized the pim protein product. In vitro translation of positively selected poly(A)+ mRNA indicates that this gene encodes a 33-kilodalton protein. Anti-pim antibodies were raised against a fused TrpE-pim protein induced in a bacterial expression vector. This antibody immunoprecipitated a 33-kilodalton protein from in vivo [35S]methionine-labeled K562 and KCl myelogenous origin cell lines. This protein was localized to the cytoplasm, and in vivo labeling as well as in vitro kinase assay suggests that it is a phosphoprotein with tyrosine kinase activity. This was further confirmed by performing autophosphorylation directly on a p33pim-containing gel band cut out after sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The results imply that the tyrosine kinase activity of pim can be recovered after boiling the pim-1 protein in sample buffer: a feature not described yet for this class of protein. These results suggest that pim-1 is a new member of the subgroup of oncogenes encoding tyrosine kinases.

scholarly journals Identification of the human pim-1 gene product as a 33-kilodalton cytoplasmic protein with tyrosine kinase activity.

1988 ◽  
Vol 8 (4) ◽  
pp. 1498-1503 ◽  
Author(s):  
A Telerman ◽  
R Amson ◽  
R Zakut-Houri ◽  
D Givol
Keyword(s):  
Tyrosine Kinase ◽  
Tyrosine Kinases ◽  
Kinase Activity ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Kinase Assay ◽  
In Vitro Translation ◽  
Tyrosine Kinase Activity

The human pim-1 gene was recently identified as a new putative oncogene located on chromosome 6p21, a region showing karyotypic abnormalities in particular leukemias. In the present work we characterized the pim protein product. In vitro translation of positively selected poly(A)+ mRNA indicates that this gene encodes a 33-kilodalton protein. Anti-pim antibodies were raised against a fused TrpE-pim protein induced in a bacterial expression vector. This antibody immunoprecipitated a 33-kilodalton protein from in vivo [35S]methionine-labeled K562 and KCl myelogenous origin cell lines. This protein was localized to the cytoplasm, and in vivo labeling as well as in vitro kinase assay suggests that it is a phosphoprotein with tyrosine kinase activity. This was further confirmed by performing autophosphorylation directly on a p33pim-containing gel band cut out after sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The results imply that the tyrosine kinase activity of pim can be recovered after boiling the pim-1 protein in sample buffer: a feature not described yet for this class of protein. These results suggest that pim-1 is a new member of the subgroup of oncogenes encoding tyrosine kinases.

scholarly journals Novel yeast protein kinase (YPK1 gene product) is a 40-kilodalton phosphotyrosyl protein associated with protein-tyrosine kinase activity.

1990 ◽  
Vol 10 (12) ◽  
pp. 6244-6256 ◽  
Author(s):  
D Dailey ◽  
G L Schieven ◽  
M Y Lim ◽  
H Marquardt ◽  
T Gilmore ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Tyrosine Kinase ◽  
Tyrosine Kinases ◽  
Protein Tyrosine Kinase ◽  
Kinase Activity ◽  
Protein Tyrosine Kinases ◽  
Tyrosine Kinase Activity ◽  
Protein Tyrosine ◽  
Protein Tyrosine Kinase Activity

Extracts of bakers' yeast (Saccharomyces cerevisiae) contain protein-tyrosine kinase activity that can be detected with a synthetic Glu-Tyr copolymer as substrate (G. Schieven, J. Thorner, and G.S. Martin, Science 231:390-393, 1986). By using this assay in conjunction with ion-exchange and affinity chromatography, a soluble tyrosine kinase activity was purified over 8,000-fold from yeast extracts. The purified activity did not utilize typical substrates for mammalian protein-tyrosine kinases (enolase, casein, and histones). The level of tyrosine kinase activity at all steps of each preparation correlated with the content of a 40-kDa protein (p40). Upon incubation of the most highly purified fractions with Mn-ATP or Mg-ATP, p40 was the only protein phosphorylated on tyrosine. Immunoblotting of purified p40 or total yeast extracts with antiphosphotyrosine antibodies and phosphoamino acid analysis of 32P-labeled yeast proteins fractionated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis indicated that the 40-kDa protein is normally phosphorylated at tyrosine in vivo. 32P-labeled p40 immunoprecipitated from extracts of metabolically labeled cells by affinity-purified anti-p40 antibodies contained both phosphoserine and phosphotyrosine. The gene encoding p40 (YPK1) was cloned from a yeast genomic library by using oligonucleotide probes designed on the basis of the sequence of purified peptides. As deduced from the nucleotide sequence of YPK1, p40 is homologous to known protein kinases, with features that resemble known protein-serine kinases more than known protein-tyrosine kinases. Thus, p40 is a protein kinase which is phosphorylated in vivo and in vitro at both tyrosine and serine residues; it may be a novel type of autophosphorylating tyrosine kinase, a bifunctional (serine/tyrosine-specific) protein kinase, or a serine kinase that is a substrate for an associated tyrosine kinase.

Detection of c-abl tyrosine kinase activity in vitro permits direct comparison of normal and altered abl gene products

1985 ◽  
Vol 5 (11) ◽  
pp. 3116-3123
Author(s):  
J B Konopka ◽  
O N Witte
Keyword(s):  
Tyrosine Kinase ◽  
Kinase Activity ◽  
Myelogenous Leukemia ◽  
Gene Products ◽  
Tyrosine Kinase Activity ◽  
Abl Kinase ◽  
Abl Tyrosine Kinase ◽  
Assay Conditions

The v-abl transforming protein P160v-abl and the P210c-abl gene product of the translocated c-abl gene in Philadelphia chromosome-positive chronic myelogenous leukemia cells have tyrosine-specific protein kinase activity. Under similar assay conditions the normal c-abl gene products, murine P150c-abl and human P145c-abl, lacked detectable kinase activity. Reaction conditions were modified to identify conditions which would permit the detection of c-abl tyrosine kinase activity. It was found that the Formalin-fixed Staphylococcus aureus formerly used for immunoprecipitation inhibits in vitro abl kinase activity. In addition, the sodium dodecyl sulfate and deoxycholate detergents formerly used in the cell lysis buffer were found to decrease recovered abl kinase activity. The discovery of assay conditions for c-abl kinase activity now makes it possible to compare P150c-abl and P145c-abl kinase activity with the altered abl proteins P160v-abl and P210c-abl. Although all of the abl proteins have in vitro tyrosine kinase activity, they differ in the way they utilize themselves as substrates in vitro. Comparison of in vitro and in vivo tyrosine phosphorylation sites of the abl proteins suggests that they function differently in vivo. The development of c-abl kinase assay conditions should be useful in elucidating c-abl function.

p145, a protein with associated tyrosine kinase activity in a human gastric carcinoma cell line

1988 ◽  
Vol 8 (8) ◽  
pp. 3510-3517
Author(s):  
S Giordano ◽  
M F Di Renzo ◽  
R Ferracini ◽  
L Chiadò-Piat ◽  
P M Comoglio
Keyword(s):  
Gastric Carcinoma ◽  
Tyrosine Kinase ◽  
Cell Line ◽  
Kinase Activity ◽  
Carcinoma Cell ◽  
Tyrosine Kinase Activity ◽  
Gastric Carcinoma Cell ◽  
Gastric Carcinoma Cell Line

A protein with an Mr of 145,000 (p145) was detected by antibodies to phosphotyrosine by Western blot (immunoblot) analysis. This protein was phosphorylated on tyrosine in a gastric carcinoma cell line. In cells that were metabolically labeled with 32Pi, this protein was phosphorylated on tyrosine and serine. p145 is a cysteine-rich transmembrane glycoprotein. The extracellular domain could be labeled by 125I under nonpermeating conditions and was cleaved by mild trypsin treatment of intact cells. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis under nonreducing conditions revealed a shift of p145 mobility to an apparent Mr of 190,000. After immunoprecipitation with phosphotyrosine antibodies, p145 displayed a strong associated protein kinase activity in vitro, becoming phosphorylated on tyrosine. There was no immunological cross-reaction between p145 and known tyrosine kinases. Both in vivo and in vitro tyrosine phosphorylations were unaffected by the addition of known growth factors. However, p145 was rapidly dephosphorylated in vivo when cells were exposed to low pH, a condition that is known to dissociate ligands from their receptors. These data suggest that p145 is associated with a protein tyrosine kinase activity which, in the tumor cell line studied, is activated by an as yet unidentified factor.

Novel yeast protein kinase (YPK1 gene product) is a 40-kilodalton phosphotyrosyl protein associated with protein-tyrosine kinase activity

1990 ◽  
Vol 10 (12) ◽  
pp. 6244-6256
Author(s):  
D Dailey ◽  
G L Schieven ◽  
M Y Lim ◽  
H Marquardt ◽  
T Gilmore ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Tyrosine Kinase ◽  
Tyrosine Kinases ◽  
Protein Tyrosine Kinase ◽  
Kinase Activity ◽  
Protein Tyrosine Kinases ◽  
Tyrosine Kinase Activity ◽  
Protein Tyrosine ◽  
Protein Tyrosine Kinase Activity

Extracts of bakers' yeast (Saccharomyces cerevisiae) contain protein-tyrosine kinase activity that can be detected with a synthetic Glu-Tyr copolymer as substrate (G. Schieven, J. Thorner, and G.S. Martin, Science 231:390-393, 1986). By using this assay in conjunction with ion-exchange and affinity chromatography, a soluble tyrosine kinase activity was purified over 8,000-fold from yeast extracts. The purified activity did not utilize typical substrates for mammalian protein-tyrosine kinases (enolase, casein, and histones). The level of tyrosine kinase activity at all steps of each preparation correlated with the content of a 40-kDa protein (p40). Upon incubation of the most highly purified fractions with Mn-ATP or Mg-ATP, p40 was the only protein phosphorylated on tyrosine. Immunoblotting of purified p40 or total yeast extracts with antiphosphotyrosine antibodies and phosphoamino acid analysis of 32P-labeled yeast proteins fractionated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis indicated that the 40-kDa protein is normally phosphorylated at tyrosine in vivo. 32P-labeled p40 immunoprecipitated from extracts of metabolically labeled cells by affinity-purified anti-p40 antibodies contained both phosphoserine and phosphotyrosine. The gene encoding p40 (YPK1) was cloned from a yeast genomic library by using oligonucleotide probes designed on the basis of the sequence of purified peptides. As deduced from the nucleotide sequence of YPK1, p40 is homologous to known protein kinases, with features that resemble known protein-serine kinases more than known protein-tyrosine kinases. Thus, p40 is a protein kinase which is phosphorylated in vivo and in vitro at both tyrosine and serine residues; it may be a novel type of autophosphorylating tyrosine kinase, a bifunctional (serine/tyrosine-specific) protein kinase, or a serine kinase that is a substrate for an associated tyrosine kinase.

scholarly journals Detection of c-abl tyrosine kinase activity in vitro permits direct comparison of normal and altered abl gene products.

1985 ◽  
Vol 5 (11) ◽  
pp. 3116-3123 ◽  
Author(s):  
J B Konopka ◽  
O N Witte
Keyword(s):  
Tyrosine Kinase ◽  
Kinase Activity ◽  
Myelogenous Leukemia ◽  
Gene Products ◽  
Tyrosine Kinase Activity ◽  
Abl Kinase ◽  
Abl Tyrosine Kinase ◽  
Assay Conditions

The v-abl transforming protein P160v-abl and the P210c-abl gene product of the translocated c-abl gene in Philadelphia chromosome-positive chronic myelogenous leukemia cells have tyrosine-specific protein kinase activity. Under similar assay conditions the normal c-abl gene products, murine P150c-abl and human P145c-abl, lacked detectable kinase activity. Reaction conditions were modified to identify conditions which would permit the detection of c-abl tyrosine kinase activity. It was found that the Formalin-fixed Staphylococcus aureus formerly used for immunoprecipitation inhibits in vitro abl kinase activity. In addition, the sodium dodecyl sulfate and deoxycholate detergents formerly used in the cell lysis buffer were found to decrease recovered abl kinase activity. The discovery of assay conditions for c-abl kinase activity now makes it possible to compare P150c-abl and P145c-abl kinase activity with the altered abl proteins P160v-abl and P210c-abl. Although all of the abl proteins have in vitro tyrosine kinase activity, they differ in the way they utilize themselves as substrates in vitro. Comparison of in vitro and in vivo tyrosine phosphorylation sites of the abl proteins suggests that they function differently in vivo. The development of c-abl kinase assay conditions should be useful in elucidating c-abl function.

scholarly journals p145, a protein with associated tyrosine kinase activity in a human gastric carcinoma cell line.

1988 ◽  
Vol 8 (8) ◽  
pp. 3510-3517 ◽  
Author(s):  
S Giordano ◽  
M F Di Renzo ◽  
R Ferracini ◽  
L Chiadò-Piat ◽  
P M Comoglio
Keyword(s):  
Gastric Carcinoma ◽  
Tyrosine Kinase ◽  
Cell Line ◽  
Kinase Activity ◽  
Carcinoma Cell ◽  
Tyrosine Kinase Activity ◽  
Gastric Carcinoma Cell ◽  
Gastric Carcinoma Cell Line

A protein with an Mr of 145,000 (p145) was detected by antibodies to phosphotyrosine by Western blot (immunoblot) analysis. This protein was phosphorylated on tyrosine in a gastric carcinoma cell line. In cells that were metabolically labeled with 32Pi, this protein was phosphorylated on tyrosine and serine. p145 is a cysteine-rich transmembrane glycoprotein. The extracellular domain could be labeled by 125I under nonpermeating conditions and was cleaved by mild trypsin treatment of intact cells. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis under nonreducing conditions revealed a shift of p145 mobility to an apparent Mr of 190,000. After immunoprecipitation with phosphotyrosine antibodies, p145 displayed a strong associated protein kinase activity in vitro, becoming phosphorylated on tyrosine. There was no immunological cross-reaction between p145 and known tyrosine kinases. Both in vivo and in vitro tyrosine phosphorylations were unaffected by the addition of known growth factors. However, p145 was rapidly dephosphorylated in vivo when cells were exposed to low pH, a condition that is known to dissociate ligands from their receptors. These data suggest that p145 is associated with a protein tyrosine kinase activity which, in the tumor cell line studied, is activated by an as yet unidentified factor.

scholarly journals Insulin resistance in adult rat offspring associated with maternal dietary fat and alcohol consumption

2002 ◽  
Vol 173 (1) ◽  
pp. 63-71 ◽  
Author(s):  
CW Elton ◽  
JS Pennington ◽  
SA Lynch ◽  
FM Carver ◽  
SN Pennington
Keyword(s):  
Insulin Sensitivity ◽  
Tyrosine Kinase ◽  
Glucose Uptake ◽  
Dietary Fat ◽  
Kinase Activity ◽  
In Utero ◽  
Whole Body ◽  
Tyrosine Kinase Activity

Maternal diet during pregnancy has been reported to alter the offspring's ability to respond to a glucose challenge. The current studies report changes in basal and insulin-stimulated, in vitro glucose uptake in red (soleus) and white (extensor digitorum longus) muscle fiber types, as well as whole body insulin responsiveness of adult rat offspring associated with their mother's dietary fat and alcohol content during pregnancy. The offspring of Harlan-derived Sprague-Dawley female rats, dosed during pregnancy with ethanol (ETOH) via a liquid diet (35% of calories as ETOH) with either 12% or 35% of calories as fat, were compared with offspring from litters whose mothers were pair-fed an isocaloric amount of the liquid diet without ETOH. Maternal access to the liquid diets was terminated on day 20 of the pregnancies (sperm plug=day 0). The offspring were surrogate fostered within 48 h of birth to mothers which had consumed commercial chow throughout their pregnancy. Following weaning at 21 days of age, the offspring consumed only commercial rat chow and they were examined over the next 14 months for changes in glucose homeostasis as a consequence of in utero exposure to maternal dietary fat and/or alcohol. The 35% maternal fat diet resulted in both in vivo and in vitro decreases in insulin sensitivity. Thus, compared with adults whose mother's diet contained 12% fat, significant, in vitro muscle and in vivo whole body insulin resistance (measured by hyperinsulinemic-euglycemic clamping) was observed in adult rats whose mothers consumed 35% of dietary calories as fat. The addition of ethanol to the maternal 35% fat diet further reduced the offspring's red muscle tissues in vitro response to insulin, but did not affect whole body insulin sensitivity. Muscle basal and insulin-stimulated receptor tyrosine kinase activity were significantly decreased (approximately -50%) by the 35% fat maternal diet but there was no compensatory increase in serum insulin or glucose levels. Based upon both in vivo and in vitro data, these studies suggested that in utero exposure to 35% fat has a sustained effect on the adult offspring's glucose uptake/insulin sensitivity and that the effect is paralleled, at least in part, by decreased insulin receptor tyrosine kinase activity. In utero ETOH exposure resulted in the loss of basal and insulin-stimulated, in vitro glucose uptake in red muscle fibers but maternal dietary ETOH had no detectable effect on either in vivo insulin sensitivity or muscle tyrosine kinase activity.

scholarly journals Intracellular delivery of anti-BCR/ABL antibody by PLGA nanoparticles suppresses the oncogenesis of chronic myeloid leukemia cells

2021 ◽  
Vol 14 (1) ◽  
Author(s):  
Guoyun Jiang ◽  
Zhenglan Huang ◽  
Ying Yuan ◽  
Kun Tao ◽  
Wenli Feng
Keyword(s):  
Chronic Myeloid Leukemia ◽  
Tyrosine Kinase ◽  
Myeloid Leukemia ◽  
Kinase Activity ◽  
Kinase Inhibitors ◽  
Fusion Gene ◽  
Intracellular Delivery ◽  
Tyrosine Kinase Activity

Abstract Background The pathogenesis of chronic myeloid leukemia (CML) is the formation of the BCR/ABL protein, which is encoded by the bcr/abl fusion gene, possessing abnormal tyrosine kinase activity. Despite the wide application of tyrosine kinase inhibitors (TKIs) in CML treatment, TKIs drug resistance or intolerance limits their further usage in a subset of patients. Furthermore, TKIs inhibit the tyrosine kinase activity of the BCR/ABL oncoprotein while failing to eliminate the pathologenic oncoprotein. To develop alternative strategies for CML treatment using therapeutic antibodies, and to address the issue that antibodies cannot pass through cell membranes, we have established a novel intracellular delivery of anti-BCR/ABL antibodies, which serves as a prerequisite for CML therapy. Methods Anti-BCR/ABL antibodies were encapsulated in poly(d, l-lactide-co-glycolide) nanoparticles (PLGA NPs) by a double emulsion method, and transferrin was labeled on the surface of the nanoparticles (Ab@Tf-Cou6-PLGA NPs). The characteristics of nanoparticles were measured by dynamic light scattering (DLS) and transmission electron microscopy (TEM). Cellular uptake of nanoparticles was measured by flow cytometry (FCM). The effect of nanoparticles on the apoptosis and proliferation of CML cells was testified by FCM and CCK-8 assay. In addition, the anti-cancer impact of nanoparticles was evaluated in mouse models of CML. Results The results demonstrated that the Ab@Tf-Cou6-PLGA NPs functioned as an intracellular deliverer of antibodies, and exhibited an excellent effect on degrading BCR/ABL oncoprotein in CML cells via the Trim-Away pathway. Treatment with Ab@Tf-Cou6-PLGA NPs inhibited the proliferation and induced the apoptosis of CML cells in vitro as well as impaired the oncogenesis ability of CML cells in vivo. Conclusions In conclusion, our study indicated that this approach achieved safe and efficient intracellular delivery of antibodies and degraded BCR/ABL oncoprotein via the Trim-Away pathway, which provides a promising therapeutic strategy for CML patients, particularly those with TKI resistance.

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