Gene regulation by tyrosine kinases: src protein activates various promoters, including c-fos

A promoter of the nuclear proto-oncogene fos was activated by cotransfection with the viral src gene. Ability to transactivate the c-fos promoter was dependent on tyrosine kinase activity, because (i) src mutants which have reduced tyrosine kinase activity due to mutation of Tyr-416 to Phe showed lower promoter activation, (ii) pp60c-src mutants which have increased tyrosine kinase activity due to mutation of Tyr-527 to Phe also augmented c-fos promoter induction, and (iii) mutation in the ATP-binding site of pp60v-src strongly suppressed c-fos promoter activation. Tyrosine kinase activity alone, however, was not sufficient for promoter activation, because of pp60v-src mutant which lacked its myristylation site and consequently membrane association showed no increased c-fos promoter activation. Both the tyrosine kinase- and membrane-association-defective mutants were also unable to induce transformation. Therefore, phosphorylation of membrane-associated substrates appears to be required for both gene expression and cellular transformation by the src protein. Two regions of the c-fos promoter located between positions -362 and -324 and positions -323 and -294 were responsive to src stimulation. We believe that protein tyrosine phosphorylation represents an important step of signal transduction from the membrane to the nucleus.

Download Full-text

Development of the Sensing Platform for Protein Tyrosine Kinase Activity

Biosensors ◽

10.3390/bios11070240 ◽

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.

Download Full-text

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

Journal of Bacteriology ◽

10.1128/jb.02106-14 ◽

2014 ◽

Vol 197 (1) ◽

pp. 120-127 ◽

Cited By ~ 4

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.

Download Full-text

A lysine in the ATP-binding site of P130gag-fps is essential for protein-tyrosine kinase activity.

The EMBO Journal ◽

10.1002/j.1460-2075.1986.tb04179.x ◽

1986 ◽

Vol 5 (1) ◽

pp. 69-76 ◽

Cited By ~ 42

Author(s):

G. Weinmaster ◽

M.J. Zoller ◽

T. Pawson

Keyword(s):

Tyrosine Kinase ◽

Binding Site ◽

Protein Tyrosine Kinase ◽

Kinase Activity ◽

Atp Binding ◽

Tyrosine Kinase Activity ◽

Protein Tyrosine ◽

Atp Binding Site ◽

Protein Tyrosine Kinase Activity

Download Full-text

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

Molecular and Cellular Biology ◽

10.1128/mcb.8.12.5126 ◽

1988 ◽

Vol 8 (12) ◽

pp. 5126-5131 ◽

Cited By ~ 69

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.

Download Full-text

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

Molecular and Cellular Biology ◽

10.1128/mcb.10.12.6244 ◽

1990 ◽

Vol 10 (12) ◽

pp. 6244-6256 ◽

Cited By ~ 51

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.

Download Full-text

The Paradigm of Targeting an Oncogenic Tyrosine Kinase: Lesson from BCR-ABL

10.5772/intechopen.97528 ◽

2021 ◽

Author(s):

Enrico Bracco ◽

M. Shahzad Ali ◽

Stefano Magnati ◽

Giuseppe Saglio

Keyword(s):

Tyrosine Kinase ◽

Tyrosine Kinases ◽

Molecular Mechanisms ◽

Kinase Activity ◽

Fusion Gene ◽

Lymphoblastic Leukemia ◽

Philadelphia Chromosome ◽

Protein Tyrosine Phosphatases ◽

Selective Inhibition ◽

Tyrosine Kinase Activity

The aberrant tyrosine phosphorylation, either due to constitutive tyrosine kinases (TKs) or to inactivation of protein tyrosine phosphatases (PTPs), is a widespread feature of many cancerous cells. The BCR-ABL fusion protein, which arises from the Philadelphia chromosome, is a molecular distinct and peculiar trait of some kind of leukemia, namely Chronic Myeloid and Acute Lymphoblastic Leukemia, and displays constitutive tyrosine kinase activity. In the chapter, we will highlight the milestones that had led to the identification of the BCR-ABL fusion gene and its role as the only molecular pathogenic event sufficient to elicit and sustain chronic myeloid leukemia. We will also discuss the effort made to unveil the molecular mechanisms of action of the chimeric tyrosine kinase that eventually lead to aberrant cell proliferation and impaired cell-death. Furthermore, we will also review the lesson learned from the selective inhibition of BCR-ABL which currently represent a breakthrough in the treatment of several tumors characterized by defective tyrosine kinase activity.

Download Full-text

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

Molecular and Cellular Biology ◽

10.1128/mcb.10.12.6244-6256.1990 ◽

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.

Download Full-text

N-terminal deletions in Rous sarcoma virus p60src: effects on tyrosine kinase and biological activities and on recombination in tissue culture with the cellular src gene.

Molecular and Cellular Biology ◽

10.1128/mcb.5.10.2789 ◽

1985 ◽

Vol 5 (10) ◽

pp. 2789-2795 ◽

Cited By ~ 27

Author(s):

F R Cross ◽

E A Garber ◽

H Hanafusa

Keyword(s):

Amino Acids ◽

Tyrosine Kinase ◽

Rous Sarcoma Virus ◽

Kinase Activity ◽

Biological Activities ◽

Tyrosine Kinase Activity ◽

Rous Sarcoma ◽

Src Gene ◽

Sarcoma Virus ◽

Transforming Activity

We have constructed deletions within the region of cloned Rous sarcoma virus DNA coding for the N-terminal 30 kilodaltons of p60src. Infectious virus was recovered after transfection. Deletions of amino acids 15 to 149, 15 to 169, or 149 to 169 attenuated but did not abolish transforming activity, as assayed by focus formation and anchorage-independent growth. These deletions also had only slight effects on the tyrosine kinase activity of the mutant src protein. Deletion of amino acids 169 to 264 or 15 to 264 completely abolished transforming activity, and src kinase activity was reduced at least 10-fold. However, these mutant viruses generated low levels of transforming virus by recombination with the cellular src gene. The results suggest that as well as previously identified functional domains for p60src myristylation and membrane binding (amino acids 1 to 14) and tyrosine kinase activity (amino acids 250 to 526), additional N-terminal sequences (particularly amino acids 82 to 169) can influence the transforming activity of the src protein.

Download Full-text

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

Journal of Neurosurgery ◽

10.3171/jns.1995.83.4.0690 ◽

1995 ◽

Vol 83 (4) ◽

pp. 690-697 ◽

Cited By ~ 10

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.

Download Full-text