scholarly journals Activation of phosphatidylinositol-3 kinase by ligation of the interleukin-7 receptor is dependent on protein tyrosine kinase activity

Blood ◽  
1994 ◽  
Vol 84 (5) ◽  
pp. 1579-1586 ◽  
Author(s):  
H Dadi ◽  
S Ke ◽  
CM Roifman
Keyword(s):  
Tyrosine Kinase ◽  
Protein Tyrosine Kinase ◽  
Kinase Activity ◽  
Enzyme Activation ◽  
Dependent Manner ◽  
Protein Tyrosine ◽  
Tyrosine Residues ◽  
Interleukin 7 ◽  
Phosphatidylinositol 3

Ligation of the interleukin-7 receptor (IL-7R) results in a rapid phosphorylation of tyrosine residues on multiple substrates. In addition, we have recently shown that the IL-7R mediates activation of phosphatidylinositol-3 (PI-3) kinase. Because PI-3 kinase activity can be immunoprecipitated with antiphosphotyrosine antibodies in most receptor systems studied, it has been examined that either PI-3 kinase or an associated protein become tyrosine-phosphorylated after ligand binding. We studied here the possibility that PI-3 kinase, which is directly linked to mitogenic responses in growth factor receptors, is tyrosine-phosphorylated after stimulation of the IL-7R. Using anti-p85 alpha or anti-p85 beta antibodies raised against the p85 subunit of PI- 3 kinase for immunoprecipitation and subsequent blotting with antiphosphotyrosine clearly shows that IL-7-stimulated human precursor cells contain both p85 alpha and p85 beta proteins phosphorylated on tyrosine residues. Specific protein tyrosine kinase inhibitors such as tyrphostin AG-490 block total cell lysate phosphorylation and tyrosine phosphorylation on p85. Similar concentrations of this inhibitor also block in vitro and in vivo PI-3 kinase activity suggesting that this enzyme activation is dependent on the phosphorylation event of p85. In addition, AG-490 blocks IL-7-mediated proliferation in a dose-dependent manner, suggesting a link between the early events of PI-3 kinase phosphorylation and activation with IL-7R-induced cell growth.

scholarly journals Activation of phosphatidylinositol-3 kinase by ligation of the interleukin-7 receptor is dependent on protein tyrosine kinase activity

Blood ◽  
1994 ◽  
Vol 84 (5) ◽  
pp. 1579-1586 ◽  
Author(s):  
H Dadi ◽  
S Ke ◽  
CM Roifman
Keyword(s):  
Tyrosine Kinase ◽  
Protein Tyrosine Kinase ◽  
Kinase Activity ◽  
Enzyme Activation ◽  
Dependent Manner ◽  
Protein Tyrosine ◽  
Tyrosine Residues ◽  
Interleukin 7 ◽  
Phosphatidylinositol 3

Abstract Ligation of the interleukin-7 receptor (IL-7R) results in a rapid phosphorylation of tyrosine residues on multiple substrates. In addition, we have recently shown that the IL-7R mediates activation of phosphatidylinositol-3 (PI-3) kinase. Because PI-3 kinase activity can be immunoprecipitated with antiphosphotyrosine antibodies in most receptor systems studied, it has been examined that either PI-3 kinase or an associated protein become tyrosine-phosphorylated after ligand binding. We studied here the possibility that PI-3 kinase, which is directly linked to mitogenic responses in growth factor receptors, is tyrosine-phosphorylated after stimulation of the IL-7R. Using anti-p85 alpha or anti-p85 beta antibodies raised against the p85 subunit of PI- 3 kinase for immunoprecipitation and subsequent blotting with antiphosphotyrosine clearly shows that IL-7-stimulated human precursor cells contain both p85 alpha and p85 beta proteins phosphorylated on tyrosine residues. Specific protein tyrosine kinase inhibitors such as tyrphostin AG-490 block total cell lysate phosphorylation and tyrosine phosphorylation on p85. Similar concentrations of this inhibitor also block in vitro and in vivo PI-3 kinase activity suggesting that this enzyme activation is dependent on the phosphorylation event of p85. In addition, AG-490 blocks IL-7-mediated proliferation in a dose-dependent manner, suggesting a link between the early events of PI-3 kinase phosphorylation and activation with IL-7R-induced cell growth.

scholarly journals The p85 and p110 Subunits of Phosphatidylinositol 3-Kinase-α Are Substrates, In Vitro, for a Constitutively Associated Protein Tyrosine Kinase in Platelets

Blood ◽  
1998 ◽  
Vol 91 (3) ◽  
pp. 930-939 ◽  
Author(s):  
Norman R. Geltz ◽  
James A. Augustine
Keyword(s):  
Tyrosine Kinase ◽  
Protein Tyrosine Kinase ◽  
Kinase Activity ◽  
Protein Kinase Activity ◽  
Phosphatidylinositol 3 Kinase ◽  
Lipid Kinase ◽  
Protein Tyrosine ◽  
Dependent Protein ◽  
Phosphatidylinositol 3

Phosphatidylinositol 3-kinase (PI3K) is a heterodimer lipid kinase consisting of an 85-kD subunit bound to a 110-kD catalytic subunit that also possesses intrinsic, Mn2+-dependent protein serine kinase activity capable of phosphorylating the 85-kD subunit. Here, we examine the Mn2+-dependent protein kinase activity of PI3Kα immunoprecipitated from normal resting or thrombin-stimulated platelets, and characterize p85/p110 phosphorylation, in vitro. Phosphoamino acid analysis of phosphorylated PI3Kα showed p85 and p110 were phosphorylated on serine, but in contrast to previous results, were also phosphorylated on threonine and tyrosine. Wortmannin and LY294002 inhibited p85 phosphorylation; however, p110 phosphorylation was also inhibited suggesting p110 autophosphorylation on serine/threonine. The protein tyrosine kinase inhibitor, erbstatin analog, partially inhibited p85 and p110 phosphorylation but did not appear to affect PI3K lipid kinase activity. The in vitro phosphorylation of p85α or p110α derived from thrombin-stimulated platelets was no different than that of resting platelets, but we confirm that in thrombin receptor-stimulated platelets enhanced levels of p85α and PI3K lipid kinase activity were recovered in antiphosphotyrosine antibody immunoprecipitates. These results suggest PI3Kα can autophosphorylate on serine and threonine, and both p85α and p110α are substrates for a constitutively-associated protein tyrosine kinase in platelets.

scholarly journals The p85 and p110 Subunits of Phosphatidylinositol 3-Kinase-α Are Substrates, In Vitro, for a Constitutively Associated Protein Tyrosine Kinase in Platelets

Blood ◽  
1998 ◽  
Vol 91 (3) ◽  
pp. 930-939 ◽  
Author(s):  
Norman R. Geltz ◽  
James A. Augustine
Keyword(s):  
Tyrosine Kinase ◽  
Protein Tyrosine Kinase ◽  
Kinase Activity ◽  
Protein Kinase Activity ◽  
Phosphatidylinositol 3 Kinase ◽  
Lipid Kinase ◽  
Protein Tyrosine ◽  
Dependent Protein ◽  
Phosphatidylinositol 3

Abstract Phosphatidylinositol 3-kinase (PI3K) is a heterodimer lipid kinase consisting of an 85-kD subunit bound to a 110-kD catalytic subunit that also possesses intrinsic, Mn2+-dependent protein serine kinase activity capable of phosphorylating the 85-kD subunit. Here, we examine the Mn2+-dependent protein kinase activity of PI3Kα immunoprecipitated from normal resting or thrombin-stimulated platelets, and characterize p85/p110 phosphorylation, in vitro. Phosphoamino acid analysis of phosphorylated PI3Kα showed p85 and p110 were phosphorylated on serine, but in contrast to previous results, were also phosphorylated on threonine and tyrosine. Wortmannin and LY294002 inhibited p85 phosphorylation; however, p110 phosphorylation was also inhibited suggesting p110 autophosphorylation on serine/threonine. The protein tyrosine kinase inhibitor, erbstatin analog, partially inhibited p85 and p110 phosphorylation but did not appear to affect PI3K lipid kinase activity. The in vitro phosphorylation of p85α or p110α derived from thrombin-stimulated platelets was no different than that of resting platelets, but we confirm that in thrombin receptor-stimulated platelets enhanced levels of p85α and PI3K lipid kinase activity were recovered in antiphosphotyrosine antibody immunoprecipitates. These results suggest PI3Kα can autophosphorylate on serine and threonine, and both p85α and p110α are substrates for a constitutively-associated protein tyrosine kinase in platelets.

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.

scholarly journals Involvement of a Protein Tyrosine Kinase in Production of the Polymeric Bioemulsifier Emulsan from the Oil-Degrading Strain Acinetobacter lwoffii RAG-1

2003 ◽  
Vol 185 (3) ◽  
pp. 1001-1009 ◽  
Author(s):  
David Nakar ◽  
David L. Gutnick
Keyword(s):  
Tyrosine Kinase ◽  
Protein Tyrosine Kinase ◽  
Kinase Activity ◽  
Tyrosine Phosphatase ◽  
Gene Products ◽  
Protein Tyrosine ◽  
Tyrosine Residues ◽  
C Terminus ◽  
Nitrophenyl Phosphate ◽  
Rag 1

ABSTRACT The genes associated with the biosynthesis of the polymeric bioemulsifier emulsan, produced by the oil-degrading Acinetobacter lwoffii RAG-1 are clustered within a 27-kbp region termed the wee cluster. This report demonstrates the involvement of two genes of the wee cluster of RAG-1, wzb and wzc, in emulsan biosynthesis. The two gene products, Wzc and Wzb were overexpressed and purified. Wzc exhibited ATP-dependent autophosphorylating protein tyrosine kinase activity. Wzb was found to be a protein tyrosine phosphatase capable of dephosphorylating the phosphorylated Wzc. Using the synthetic substrate p-nitrophenyl phosphate (PNPP) Wzb exhibited a V max of 12 μmol of PNPP min−1 mg−1 and a Km of 8 mM PNPP at 30°C. The emulsifying activity of mutants lacking either wzb or wzc was 16 and 15% of RAG-1 activity, respectively, suggesting a role for the two enzymes in emulsan production. Phosphorylation of Wzc was found to occur within a cluster of five tyrosine residues at the C terminus. Colonies from a mutant in which these five tyrosine residues were replaced by five phenylalanine residues along with those of a second mutant, which also lacked Wzb, exhibited a highly viscous colony consistency. Emulsan activity of these mutants was 25 and 24% of that of RAG-1, respectively. Neither of these mutants contained cell-associated emulsan. However, they did produce an extracellular high-molecular-mass galactosamine-containing polysaccharide. A model is proposed in which subunit polymerization, translocation and release of emulsan are all associated and coregulated by tyrosine phosphorylation.

JAK3 protein tyrosine kinase mediates interleukin-7-induced activation of phosphatidylinositol-3' kinase

Blood ◽  
1995 ◽  
Vol 86 (6) ◽  
pp. 2077-2085 ◽  
Author(s):  
N Sharfe ◽  
HK Dadi ◽  
CM Roifman
Keyword(s):  
T Cells ◽  
Tyrosine Kinase ◽  
Tyrosine Phosphorylation ◽  
Protein Tyrosine Kinase ◽  
Kinase Activity ◽  
Cytokine Receptor ◽  
Kinase Activation ◽  
Protein Tyrosine ◽  
Interleukin 7 ◽  
P13 Kinase

The interleukin-7 (IL-7) receptor is expressed throughout T-cell differentiation and, although lacking a tyrosine kinase domain, mediates tyrosine phosphorylation in T cells. We have identified IL-7- induced activation of three cyoplasmic tyrosine kinases in T cells, Jak1, Jak3, and the src-like kinase p56lck. Many members of the cytokine receptor superfamily activate the Jak protein tyrosine kinase family, with resultant phosphorylation of the Stat transcriptional activator factors. We describe here a novel function of the Jak kinases, because Jak kinase activity is not only required for Stat activation but also for P13 kinase response to IL-7 in human T cells. We show that IL-7 receptor-mediated Jak activation can occur independently of p56lck activity. IL-7-induced P13 kinase activation, mediated by tyrosine phosphorylation of the P13 kinase p85 subunit, is essential to the IL-7 proliferative signal and also occurs in the absence of src family kinase activity. Jak3 is found associated with the p85 subunit of P13 kinase in an IL-7-responsive manner in T cells and appears to regulate IL-7-induced P13 kinase activation by mediating tyrosine phosphorylation of the p85 subunit. Specific inhibition of IL- 7-induced Jak kinase activity ablates p85 tyrosine phosphorylation, subsequent P13 kinase activation, and, ultimately, proliferation. The ability to regulate P13 kinase activity indicates a more generalized role for the Jak family than activation of gene transcription via the Stat family in cytokine receptor signal transduction.

scholarly journals Capture by chemical crosslinkers provides evidence that integrin α IIb β 3 forms a complex with protein tyrosine kinases in intact platelets

1995 ◽  
Vol 309 (2) ◽  
pp. 481-490 ◽  
Author(s):  
D J Dorahy ◽  
M C Berndt ◽  
G F Burns
Keyword(s):  
Tyrosine Kinase ◽  
Platelet Activation ◽  
Tyrosine Kinases ◽  
Protein Tyrosine Kinase ◽  
Kinase Activity ◽  
Spatial Association ◽  
Protein Tyrosine Kinases ◽  
Protein Tyrosine ◽  
Kinase Reaction

Platelet activation is accompanied by a cascade of kinase reactions in which numerous specific proteins are phosphorylated on tyrosine. These events are strictly dependent upon functional activation of an integrin receptor, generally alpha IIb beta 3 (also known as glycoprotein IIb-IIIa). It is not known how alpha IIb beta 3 regulates protein tyrosine kinase activation and, in particular, neither this nor any other integrin has been shown to associate with a protein tyrosine kinase. We employed chemical crosslinking of intact platelets with the bifunctional reagents dithiobis(succinimidyl propionate) (DSP) (lipid-soluble) and dithiobis(sulphosuccinimidyl propionate) (DTSSP) (lipid-insoluble) followed by in vitro kinase assays of immunoprecipitated proteins to identify kinase activity associated with alpha IIb beta 3 in intact platelets. It was found that DSP but not DTSSP crosslinked kinase activity to alpha IIb beta 3, suggesting an internal association. In these immunoprecipitates from DSP-crosslinked platelets, the in vitro kinase reaction revealed a complex of several phosphoproteins in association with alpha IIb beta 3. This association was not seen when the resting platelets were lysed before crosslinking, indicating the specificity of the reaction in crosslinking only molecules in preformed spatial association. The beta 3 subunit of alpha IIb beta 3 was identified as one of the phosphoproteins in the complex obtained after subjecting anti-beta 3 immunoprecipitates from lysates of DSP-treated platelets to an in vitro kinase reaction and SDS/PAGE analysis. Phosphorylation of this subunit is shown to be predominantly on tyrosine. Affinity purification of the crosslinked phosphoprotein complex with anti-beta 3 followed by elution and re-precipitation identified pp60c-src and pp54/58c-lyn as two protein tyrosine kinases associating with the integrin. These results suggest that, upon platelet activation, alpha IIb beta 3 may provide a transmembrane focus for proteins involved in signal transduction.

scholarly journals 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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