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.

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 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 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.

Tyrosine 706 and 807 phosphorylation site mutants in the murine colony-stimulating factor-1 receptor are unaffected in their ability to bind or phosphorylate phosphatidylinositol-3 kinase but show differential defects in their ability to induce early response gene transcription

1991 ◽  
Vol 11 (9) ◽  
pp. 4698-4709
Author(s):  
P van der Geer ◽  
T Hunter
Keyword(s):  
Growth Rate ◽  
Tyrosine Kinase ◽  
Cell Morphology ◽  
Protein Tyrosine Kinase ◽  
Early Response ◽  
Wild Type ◽  
Phosphatidylinositol 3 Kinase ◽  
Protein Tyrosine ◽  
Mutant Receptors ◽  
Phosphatidylinositol 3

The receptor for colony-stimulating factor-1 (CSF-1) is a receptor protein-tyrosine kinase. To study the possible function of CSF-1 receptor autophosphorylation, two autophosphorylation sites, Tyr-706, located in the kinase insert, and Tyr-807, a residue conserved in all protein-tyrosine kinases, were changed independently to either phenylalanine or glycine. Wild-type and mutant receptors were stably expressed in Rat-2 cells. In response to CSF-1, cells expressing Phe- or Gly-706 mutant receptors showed increased growth rate and altered cell morphology. Both the Phe- and Gly-706 mutant receptors associated with and phosphorylated phosphatidylinositol-3 kinase at levels comparable with those of wild-type receptors. However, these mutant receptors differed subtly from each other and from the wild-type receptor in their ability to induce different aspects of the response to CSF-1. The Phe-706 mutant receptor was most strongly affected in its ability to increase growth rate or elevate the levels of c-fos and NGF1A mRNAs, whereas the Gly-706 mutant receptor was most markedly affected in its ability to induce a change in cell morphology or increase the levels of c-jun and NGF1A mRNAs. These findings indicate that Tyr-706 itself, or this region of the receptor, may be important for interaction of the CSF-1 receptor with different signalling pathways. Gly-807 mutant receptors lacked protein-tyrosine kinase activity, failed to respond to CSF-1, and were defective in biosynthetic processing. Phe-807 mutant receptors had 40 to 60% reduced protein-tyrosine kinase activity in vitro. Although cells expressing Phe-807 receptors were able to respond to CSF-1, the changes in growth rate and cell morphology were significantly less than seen with wild-type receptors, and the induction of early response genes was also slightly lower than for the wild-type receptor. In contrast, Phe-807 receptors were equivalent to wild-type receptors when tested for their ability to interact with phosphatidylinositol-3 kinase. These findings indicate that phosphorylation of Tyr-807 may be important for full activation of the receptor.

Regulation of phosphatidylinositol 3-kinase by insulin in rat skeletal muscle

1993 ◽  
Vol 265 (5) ◽  
pp. E736-E742 ◽  
Author(s):  
K. S. Chen ◽  
J. C. Friel ◽  
N. B. Ruderman
Keyword(s):  
Skeletal Muscle ◽  
Soleus Muscle ◽  
Protein Tyrosine Kinase ◽  
Kinase Activity ◽  
Receptor Protein ◽  
Mammalian Skeletal Muscle ◽  
Phosphatidylinositol 3 Kinase ◽  
Phosphatidylinositol 3

The presence of phosphatidylinositol 3-kinase (PI 3-kinase) in mammalian skeletal muscle and its response to insulin stimulation were investigated. PI kinase, immunoprecipitated from rat soleus muscle with antibodies directed toward its 85-kDa subunit phosphorylated PI, phosphatidylinositol 4-phosphate [PI(4)P], and phosphatidylinositol 4,5,-bisphosphate [PI(4,5)P2] to yield phosphatidylinositol 3-phosphate [PI(3)P], phosphatidylinositol 3,4,-bisphosphate, and phosphatidylinositol trisphosphate in vitro. PI 3-kinase activity was also immunoprecipitated with antiphosphotyrosine [alpha-Tyr(P)] antibodies and with antibodies raised against IRS-1, a substrate of the insulin receptor protein tyrosine kinase that associates with and activates PI 3-kinase. Incubation of the soleus with insulin in vitro, or injection of insulin into rats in vivo, produced three- to fivefold increases in alpha-Tyr(P)- and alpha-IRS-1-immunoprecipitable PI 3-kinase activity. In nonstimulated soleus muscle, PI 3-kinase activity immunoprecipitated with alpha-IRS-1 or with alpha-Tyr(P) antibodies was evenly distributed between particulate (200,000-g pellet) and soluble fractions. Insulin treatment increased immunoprecipitable PI 5-kinase activity in both fractions, but the increase in alpha-Tyr-(P)-precipitable activity was greater in the particulate fraction, whereas the increase in alpha-IRS-1-precipitable activity was greater in the soluble fraction. In intact soleus muscles incubated with 32PO4, insulin increased the labeling of PI(3)P but did not affect the labeling of PI(4)P or PI(4,5)P2. Activation of PI 3-kinase by insulin was unaffected by prior denervation of the muscle, a manipulation that has been shown to cause both insulin resistance and hypersensitivity in muscles, depending on the parameter measured.(ABSTRACT TRUNCATED AT 250 WORDS)

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