scholarly journals Platelet-derived growth factor induces phosphorylation of multiple JAK family kinases and STAT proteins.

1996 ◽  
Vol 16 (4) ◽  
pp. 1759-1769 ◽  
Author(s):  
M L Vignais ◽  
H B Sadowski ◽  
D Watling ◽  
N C Rogers ◽  
M Gilman
Keyword(s):  
Growth Factor ◽  
Tyrosine Kinase ◽  
Tyrosine Kinases ◽  
Kinase Activity ◽  
Human Fibroblasts ◽  
Platelet Derived Growth Factor ◽  
Interferon Signaling ◽  
Tyrosine Kinase Activity ◽  
Protein Tyrosine ◽  
Stat Proteins

Receptors for interferons and other cytokines signal through the action of associated protein tyrosine kinases of the JAK family and latent cytoplasmic transcription factors of the STAT family. Genetic and biochemical analysis of interferon signaling indicates that activation of STATs by interferons requires two distinct JAK family kinases. Loss of either of the required JAKs prevents activation of the other JAK and extinguishes STAT activation. These observations suggest that JAKs provide interferon receptors with a critical catalytic signaling function and that at least two JAKs must be incorporated into an active receptor complex. JAK and STAT proteins are also activated by ligands such as platelet-derived growth factor (PDGF), which act through receptors that possess intrinsic protein tyrosine kinase activity, raising questions about the role of JAKs in signal transduction by this class of receptors. Here, we show that all three of the ubiquitously expressed JAKs--JAK1, JAK2, and Tyk2--become phosphorylated on tyrosine in both mouse BALB/c 3T3 cells and human fibroblasts engineered to express the PDGF-beta receptor. All three proteins are also associated with the activated receptor. Through the use of cell lines each lacking an individual JAK, we find that in contrast to interferon signaling, PDGF-induced JAK phosphorylation and activation of STAT1 and STAT3 is independent of the presence of any other single JAK but does require receptor tyrosine kinase activity. These results suggests that the mechanism of JAK activation and JAK function in signaling differs between receptor tyrosine kinases and interferon receptors.

scholarly journals Development of the Sensing Platform for Protein Tyrosine Kinase Activity

Biosensors ◽  
2021 ◽  
Vol 11 (7) ◽  
pp. 240
Author(s):  
Lan-Yi Wei ◽  
Wei Lin ◽  
Bey-Fen Leo ◽  
Lik-Voon Kiew ◽  
Chia-Ching Chang ◽  
...  
Keyword(s):  
Tyrosine Kinase ◽  
Tyrosine Kinases ◽  
Protein Tyrosine Kinase ◽  
Kinase Activity ◽  
Significant Inhibition ◽  
Tyrosine Kinase Activity ◽  
Protein Tyrosine ◽  
Sensing Platform ◽  
Relative Standard ◽  
Protein Tyrosine Kinase Activity

A miniature tyrosinase-based electrochemical sensing platform for label-free detection of protein tyrosine kinase activity was developed in this study. The developed miniature sensing platform can detect the substrate peptides for tyrosine kinases, such as c-Src, Hck and Her2, in a low sample volume (1–2 μL). The developed sensing platform exhibited a high reproducibility for repetitive measurement with an RSD (relative standard deviation) of 6.6%. The developed sensing platform can detect the Hck and Her2 in a linear range of 1–200 U/mL with the detection limit of 1 U/mL. The sensing platform was also effective in assessing the specificity and efficacies of the inhibitors for protein tyrosine kinases. This is demonstrated by the detection of significant inhibition of Hck (~88.1%, but not Her2) by the Src inhibitor 1, an inhibitor for Src family kinases, as well as the significant inhibition of Her2 (~91%, but not Hck) by CP-724714 through the platform. These results suggest the potential of the developed miniature sensing platform as an effective tool for detecting different protein tyrosine kinase activity and for accessing the inhibitory effect of various inhibitors to these kinases.

scholarly journals Topology of Streptococcus pneumoniae CpsC, a Polysaccharide Copolymerase and Bacterial Protein Tyrosine Kinase Adaptor Protein

2014 ◽  
Vol 197 (1) ◽  
pp. 120-127 ◽  
Author(s):  
Jonathan J. Whittall ◽  
Renato Morona ◽  
Alistair J. Standish
Keyword(s):  
Streptococcus Pneumoniae ◽  
Tyrosine Kinase ◽  
Tyrosine Kinases ◽  
Kinase Activity ◽  
Tyrosine Phosphatase ◽  
Adaptor Protein ◽  
Tyrosine Kinase Activity ◽  
Content Type ◽  
Protein Tyrosine ◽  
C Terminus

In Gram-positive bacteria, tyrosine kinases are split into two proteins, the cytoplasmic tyrosine kinase and a transmembrane adaptor protein. InStreptococcus pneumoniae, this transmembrane adaptor is CpsC, with the C terminus of CpsC critical for interaction and subsequent tyrosine kinase activity of CpsD. Topology predictions suggest that CpsC has two transmembrane domains, with the N and C termini present in the cytoplasm. In order to investigate CpsC topology, we used a chromosomal hemagglutinin (HA)-tagged Cps2C protein inS. pneumoniaestrain D39. Incubation of both protoplasts and membranes with carboxypeptidase B (CP-B) resulted in complete degradation of HA-Cps2C in all cases, indicating that the C terminus of Cps2C was likely extracytoplasmic and hence that the protein's topology was not as predicted. Similar results were seen with membranes fromS. pneumoniaestrain TIGR4, indicating that Cps4C also showed similar topology. A chromosomally encoded fusion of HA-Cps2C and Cps2D was not degraded by CP-B, suggesting that the fusion fixed the C terminus within the cytoplasm. However, capsule synthesis was unaltered by this fusion. Detection of the CpsC C terminus by flow cytometry indicated that it was extracytoplasmic in approximately 30% of cells. Interestingly, a mutant in the protein tyrosine phosphatase CpsB had a significantly greater proportion of positive cells, although this effect was independent of its phosphatase activity. Our data indicate that CpsC possesses a varied topology, with the C terminus flipping across the cytoplasmic membrane, where it interacts with CpsD in order to regulate tyrosine kinase activity.

scholarly journals Role of tyrosine kinase and membrane-spanning domains in signal transduction by the platelet-derived growth factor receptor.

1988 ◽  
Vol 8 (12) ◽  
pp. 5126-5131 ◽  
Author(s):  
J A Escobedo ◽  
P J Barr ◽  
L T Williams
Keyword(s):  
Growth Factor ◽  
Tyrosine Kinase ◽  
Kinase Activity ◽  
Growth Factor Receptor ◽  
Platelet Derived Growth Factor ◽  
Tyrosine Kinase Activity ◽  
Down Regulation ◽  
Atp Binding Site ◽  
Membrane Spanning ◽  
Membrane Spanning Domains

Three types of mutations were introduced into the platelet-derived growth factor (PDGF) receptor to cause a loss of PDGF-stimulated tyrosine kinase activity: (i) a point mutation of the ATP-binding site, (ii) a deletion of the carboxyl-terminal region, and (iii) replacement of the membrane-spanning sequences by analogous transmembrane sequences of other receptors. Transfectants expressing mutated receptors bind, 125I-labeled PDGF with a high affinity but had no PDGF-sensitive tyrosine kinase activity, phosphatidylinositol turnover, increase in the intracellular calcium concentration, change in cellular pH, or stimulation of DNA synthesis. However, PDGF-induced receptor down regulation was normal in the mutant cells. These results indicate that the transmembrane sequence has a specific signal-transducing function other than merely serving as a membrane anchor and that the receptor kinase activity is necessary for most responses to PDGF but is not required for receptor down regulation.

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.

scholarly journals Platelet-derived growth factor induces multisite phosphorylation of pp60c-src and increases its protein-tyrosine kinase activity.

1988 ◽  
Vol 8 (8) ◽  
pp. 3345-3356 ◽  
Author(s):  
K L Gould ◽  
T Hunter
Keyword(s):  
Protein Kinase C ◽  
Growth Factor ◽  
Protein Kinase ◽  
Tyrosine Kinase ◽  
Protein Tyrosine Kinase ◽  
Kinase Activity ◽  
Platelet Derived Growth Factor ◽  
Tyrosine Kinase Activity ◽  
Kinase C ◽  
Protein Tyrosine Kinase Activity

We have shown previously that pp60c-src is a substrate for protein kinase C in vivo and that the target of protein kinase C phosphorylation in mammalian pp60c-src is serine 12. We now demonstrate that in addition to tumor promoters, all activators of phosphatidylinositol turnover that we have tested in fibroblasts (platelet-derived growth factor, fibroblast growth factor, serum, vasopressin, sodium orthovanadate, and prostaglandin F2 alpha) lead to the phosphorylation of pp60c-src at serine 12. In addition to stimulating serine 12 phosphorylation in pp60c-src, platelet-derived growth factor treatment of quiescent fibroblasts induces phosphorylation of one or two additional serine residues and one tyrosine residue within the N-terminal 16 kilodaltons of the enzyme and activates its immune complex protein-tyrosine kinase activity.

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.

Inhibition by 5′-methylthioadenosine of cell growth and tyrosine kinase activity stimulated by fibroblast growth factor receptor in human gliomas

1995 ◽  
Vol 83 (4) ◽  
pp. 690-697 ◽  
Author(s):  
Katsuya Miyaji ◽  
Eiichi Tani ◽  
Atsuhisa Nakano ◽  
Hideyasu Ikemoto ◽  
Keizo Kaba
Keyword(s):  
Growth Factor ◽  
Cell Growth ◽  
Tyrosine Kinase ◽  
Tyrosine Phosphorylation ◽  
Protein Tyrosine Kinase ◽  
Kinase Activity ◽  
Glioma Cells ◽  
Protein Tyrosine Phosphorylation ◽  
Tyrosine Kinase Activity ◽  
Protein Tyrosine

✓ Stimulation of three human glioma cell lines with basic fibroblast growth factor (bFGF) led to the enhancement of cell growth and the rapid tyrosine phosphorylation of cellular proteins, including major substrates of 90 kD. A methyltransferase inhibitor, 5′-methylthioadenosine (MTA), inhibited dose dependently the bFGF-stimulated cell growth and protein tyrosine phosphorylation in glioma cells by blocking both receptor autophosphorylation and substrate phosphorylation, as shown by immunoblotting with antiphosphotyrosine antibodies and cross-linking bFGF to receptors. The antiproliferative activity of MTA correlated quantitatively with its potency as an inhibitor of bFGF-stimulated protein tyrosine kinase activity. The methyltransferase inhibitor MTA had no effect on either epidermal growth factor— or platelet-derived growth factor—stimulated protein tyrosine phosphorylation in glioma cells, but inhibited specifically bFGF-stimulated protein tyrosine kinase activity. The concentration of MTA required for inhibition of protein methylation correlated well with the concentration required for inhibition of bFGF-stimulated cell growth and protein tyrosine phosphorylation. Because MTA had no effect on numbers and dissociation constants of high- and low-affinity bFGF receptors, the inhibition of bFGF-stimulated bFGF receptor tyrosine kinase activity is not likely to be the result of a reduction in bFGF receptor and bFGF binding capacity. In fact, MTA delayed and reduced the internalization and nuclear translocation of bFGF, and the internalized bFGF was submitted to a limited proteolysis that converted it to lower molecular peptides whose presence remained for at least 22 hours. The effect of MTA on bFGF-stimulated tyrosine phosphorylation was immediate and readily reversible.

Role of tyrosine kinase and membrane-spanning domains in signal transduction by the platelet-derived growth factor receptor

1988 ◽  
Vol 8 (12) ◽  
pp. 5126-5131
Author(s):  
J A Escobedo ◽  
P J Barr ◽  
L T Williams
Keyword(s):  
Growth Factor ◽  
Tyrosine Kinase ◽  
Kinase Activity ◽  
Growth Factor Receptor ◽  
Platelet Derived Growth Factor ◽  
Tyrosine Kinase Activity ◽  
Down Regulation ◽  
Atp Binding Site ◽  
Membrane Spanning ◽  
Membrane Spanning Domains

Three types of mutations were introduced into the platelet-derived growth factor (PDGF) receptor to cause a loss of PDGF-stimulated tyrosine kinase activity: (i) a point mutation of the ATP-binding site, (ii) a deletion of the carboxyl-terminal region, and (iii) replacement of the membrane-spanning sequences by analogous transmembrane sequences of other receptors. Transfectants expressing mutated receptors bind, 125I-labeled PDGF with a high affinity but had no PDGF-sensitive tyrosine kinase activity, phosphatidylinositol turnover, increase in the intracellular calcium concentration, change in cellular pH, or stimulation of DNA synthesis. However, PDGF-induced receptor down regulation was normal in the mutant cells. These results indicate that the transmembrane sequence has a specific signal-transducing function other than merely serving as a membrane anchor and that the receptor kinase activity is necessary for most responses to PDGF but is not required for receptor down regulation.

Dephosphorylation or antibody binding to the carboxy terminus stimulates pp60c-src

1986 ◽  
Vol 6 (12) ◽  
pp. 4467-4477
Author(s):  
J A Cooper ◽  
C S King
Keyword(s):  
Tyrosine Kinase ◽  
Tyrosine Kinases ◽  
Protein Tyrosine Kinase ◽  
Kinase Activity ◽  
Antibody Binding ◽  
Carboxy Terminus ◽  
Tyrosine Kinase Activity ◽  
Protein Tyrosine ◽  
Protein Tyrosine Kinase Activity

Phosphorylation of pp60c-src at Tyr-527, six residues from the carboxy terminus, has been implicated in regulation of the protein-tyrosine kinase activity of pp60c-src. Here we show that dephosphorylation of pp60c-src by phosphatase treatment in vitro caused a 10- to 20-fold increase in pp60c-src protein-tyrosine kinase activity. Binding of specific antibody to the region of pp60c-src which contains phosphotyrosine-527 also increased kinase activity. Each treatment increased phosphorylation of added substrates and of Tyr-416 within pp60c-src by a similar mechanism that involved altered interactions with ATP and increased catalytic rate. We suggest that the phosphorylated carboxy terminus acts as an inhibitor of the protein kinase domain of pp60c-src, unless its conformation is altered by either dephosphorylation or antibody binding. The antibody additionally stimulated the phosphorylation of forms of pp60c-src that had reduced gel mobility, much like those phosphorylated in kinase reactions containing pp60c-src activated by polyomavirus medium tumor antigen. These in vitro experiments provide models for the activation of pp60c-src in cells transformed by polyomavirus. We also show that autophosphorylation of pp60c-src at Tyr-527 occurs only to a very limited extent in vitro, even when Tyr-527 is made available for phosphorylation by treatment with phosphatase. This suggests that other protein-tyrosine kinases may normally phosphorylate Tyr-527 and regulate pp60c-src in the cell.

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