scholarly journals Cloning of a novel, ubiquitously expressed human phosphatidylinositol 3-kinase and identification of its binding site on p85.

1993 ◽  
Vol 13 (12) ◽  
pp. 7677-7688 ◽  
Author(s):  
P Hu ◽  
A Mondino ◽  
E Y Skolnik ◽  
J Schlessinger
Keyword(s):  
Cell Cycle Progression ◽  
Phosphatidylinositol 3 Kinase ◽  
Intact Cells ◽  
Src Homology 2 ◽  
Genes Encoding ◽  
Src Homology ◽  
Acid Domain ◽  
Vacuolar Protein ◽  
Phosphatidylinositol 3

Phosphatidylinositol 3-kinase (PI 3-kinase) has been implicated as a participant in signaling pathways regulating cell growth by virtue of its activation in response to various mitogenic stimuli. Here we describe the cloning of a novel and ubiquitously expressed human PI 3-kinase. The 4.8-kb cDNA encodes a putative translation product of 1,070 amino acids which is 42% identical to bovine PI 3-kinase and 28% identical to Vps34, a Saccharomyces cerevisiae PI 3-kinase involved in vacuolar protein sorting. Human PI 3-kinase is also similar to Tor2, a yeast protein required for cell cycle progression. Northern (RNA) analysis demonstrated expression of human PI 3-kinase in all tissues and cell lines tested. Protein synthesized from an epitope-tagged cDNA had intrinsic PI 3-kinase activity and associated with the adaptor 85-kDa subunit of PI 3-kinase (p85) in intact cells, as did endogenous human PI 3-kinase. Coprecipitation assays showed that a 187-amino-acid domain between the two src homology 2 domains of p85 mediates interaction with PI 3-kinase in vitro and in intact cells. These results demonstrate the existence of different PI 3-kinase isoforms and define a family of genes encoding distinct PI 3-kinase catalytic subunits that can associate with p85.

Cloning of a novel, ubiquitously expressed human phosphatidylinositol 3-kinase and identification of its binding site on p85

1993 ◽  
Vol 13 (12) ◽  
pp. 7677-7688
Author(s):  
P Hu ◽  
A Mondino ◽  
E Y Skolnik ◽  
J Schlessinger
Keyword(s):  
Cell Cycle Progression ◽  
Phosphatidylinositol 3 Kinase ◽  
Intact Cells ◽  
Src Homology 2 ◽  
Genes Encoding ◽  
Src Homology ◽  
Acid Domain ◽  
Vacuolar Protein ◽  
Phosphatidylinositol 3

Phosphatidylinositol 3-kinase (PI 3-kinase) has been implicated as a participant in signaling pathways regulating cell growth by virtue of its activation in response to various mitogenic stimuli. Here we describe the cloning of a novel and ubiquitously expressed human PI 3-kinase. The 4.8-kb cDNA encodes a putative translation product of 1,070 amino acids which is 42% identical to bovine PI 3-kinase and 28% identical to Vps34, a Saccharomyces cerevisiae PI 3-kinase involved in vacuolar protein sorting. Human PI 3-kinase is also similar to Tor2, a yeast protein required for cell cycle progression. Northern (RNA) analysis demonstrated expression of human PI 3-kinase in all tissues and cell lines tested. Protein synthesized from an epitope-tagged cDNA had intrinsic PI 3-kinase activity and associated with the adaptor 85-kDa subunit of PI 3-kinase (p85) in intact cells, as did endogenous human PI 3-kinase. Coprecipitation assays showed that a 187-amino-acid domain between the two src homology 2 domains of p85 mediates interaction with PI 3-kinase in vitro and in intact cells. These results demonstrate the existence of different PI 3-kinase isoforms and define a family of genes encoding distinct PI 3-kinase catalytic subunits that can associate with p85.

scholarly journals Phosphatidylinositol 3-kinase associates, via its Src homology 2 domains, with the activated erythropoietin receptor

Blood ◽  
1993 ◽  
Vol 81 (12) ◽  
pp. 3204-3210 ◽  
Author(s):  
JE Damen ◽  
AL Mui ◽  
L Puil ◽  
T Pawson ◽  
G Krystal
Keyword(s):  
Erythropoietin Receptor ◽  
Regulatory Subunit ◽  
Phosphatidylinositol 3 Kinase ◽  
Protein Substrates ◽  
Sh2 Domains ◽  
Src Homology 2 ◽  
Src Homology ◽  
Specific Association ◽  
Phosphatidylinositol 3

The erythropoietin receptor (EpR) belongs to a family of hematopoietin receptors whose members lack tyrosine kinase activity. Nonetheless, within minutes of binding Ep, a number of cellular proteins become transiently phosphorylated on tyrosine residues. One of these proteins, as we and others have shown previously, is the EpR itself. To identify the remaining protein substrates, we have examined the antiphosphotyrosine immunoprecipitates of lysates from Ba/F3 cells expressing high levels of cell surface EpRs. We now present data showing that, in response to Ep, the 85-Kd regulatory subunit of phosphatidylinositol 3-kinase (PI 3-kinase) becomes immunoprecipitable with antiphosphotyrosine antibodies. This appears to be due, in large part, to the specific association of PI 3-kinase with the tyrosine- phosphorylated EpR, either directly or through a 93- or 70-Kd tyrosine- phosphorylated intermediate. The activity of this EpR associated PI 3- kinase, assessed in anti-EpR immunoprecipitates, is maximal within 2 minutes of incubation with Ep and returns almost to baseline levels by 10 minutes. In vitro studies suggest that the interaction between PI 3- kinase and the activated EpR is mediated by the N- and C-terminal SH2 domains of p85 and tyrosine-phosphorylated motifs on the EpR.

scholarly journals Rapid and efficient purification of Src homology 2 domain-containing proteins: Fyn, Csk and phosphatidylinositol 3-kinase p85

1994 ◽  
Vol 302 (3) ◽  
pp. 737-744 ◽  
Author(s):  
M Koegl ◽  
R M Kypta ◽  
M Bergman ◽  
K Alitalo ◽  
S A Courtneidge
Keyword(s):  
Tyrosine Kinases ◽  
Intramolecular Interaction ◽  
Affinity Purification ◽  
Sh2 Domain ◽  
Exchange Chromatography ◽  
Phosphatidylinositol 3 Kinase ◽  
Src Homology 2 ◽  
Src Homology ◽  
Phosphatidylinositol 3

To analyse the regulation of Src family tyrosine kinases in vitro, we have purified Fyn and Csk, a kinase capable of regulating Fyn activity by phosphorylation, from baculovirus-infected insect cells. The proteins were purified by affinity purification over a phosphotyrosine column. Highly purified proteins were eluted from the resin by a salt gradient and further purified by ion-exchange chromatography. This purification scheme was successfully applied to a third, unrelated protein that also contains the Src homology 2 (SH2) domain, namely the 85 kDa subunit of phosphatidylinositol 3-kinase, indicating that this method is versatile and should prove applicable to any protein with an accessible SH2 domain. The binding of Csk to different phosphopeptides was tested, and specificity for the autophosphorylation site of Fyn was demonstrated. Pure Csk was used to phosphorylate Fyn and down-regulate its kinase activity, and the kinetic parameters of both the active and the repressed forms of Fyn were determined. Repression of Fyn activity by Csk reduced binding of Fyn to phosphopeptides to undetectable levels, supporting the model that predicts an intramolecular interaction of the Fyn SH2 domain with a C-terminal phosphotyrosine residue.

scholarly journals Phosphatidylinositol 3-kinase associates, via its Src homology 2 domains, with the activated erythropoietin receptor

Blood ◽  
1993 ◽  
Vol 81 (12) ◽  
pp. 3204-3210 ◽  
Author(s):  
JE Damen ◽  
AL Mui ◽  
L Puil ◽  
T Pawson ◽  
G Krystal
Keyword(s):  
Erythropoietin Receptor ◽  
Regulatory Subunit ◽  
Phosphatidylinositol 3 Kinase ◽  
Protein Substrates ◽  
Sh2 Domains ◽  
Src Homology 2 ◽  
Src Homology ◽  
Specific Association ◽  
Phosphatidylinositol 3

Abstract The erythropoietin receptor (EpR) belongs to a family of hematopoietin receptors whose members lack tyrosine kinase activity. Nonetheless, within minutes of binding Ep, a number of cellular proteins become transiently phosphorylated on tyrosine residues. One of these proteins, as we and others have shown previously, is the EpR itself. To identify the remaining protein substrates, we have examined the antiphosphotyrosine immunoprecipitates of lysates from Ba/F3 cells expressing high levels of cell surface EpRs. We now present data showing that, in response to Ep, the 85-Kd regulatory subunit of phosphatidylinositol 3-kinase (PI 3-kinase) becomes immunoprecipitable with antiphosphotyrosine antibodies. This appears to be due, in large part, to the specific association of PI 3-kinase with the tyrosine- phosphorylated EpR, either directly or through a 93- or 70-Kd tyrosine- phosphorylated intermediate. The activity of this EpR associated PI 3- kinase, assessed in anti-EpR immunoprecipitates, is maximal within 2 minutes of incubation with Ep and returns almost to baseline levels by 10 minutes. In vitro studies suggest that the interaction between PI 3- kinase and the activated EpR is mediated by the N- and C-terminal SH2 domains of p85 and tyrosine-phosphorylated motifs on the EpR.

scholarly journals IRS-1 activates phosphatidylinositol 3'-kinase by associating with src homology 2 domains of p85.

1992 ◽  
Vol 89 (21) ◽  
pp. 10350-10354 ◽  
Author(s):  
M. G. Myers ◽  
J. M. Backer ◽  
X. J. Sun ◽  
S. Shoelson ◽  
P. Hu ◽  
...  
Keyword(s):  
Phosphatidylinositol 3 Kinase ◽  
Src Homology 2 ◽  
Src Homology ◽  
Phosphatidylinositol 3

scholarly journals The structure and function of p55PIK reveal a new regulatory subunit for phosphatidylinositol 3-kinase.

1995 ◽  
Vol 15 (8) ◽  
pp. 4453-4465 ◽  
Author(s):  
S Pons ◽  
T Asano ◽  
E Glasheen ◽  
M Miralpeix ◽  
Y Zhang ◽  
...  
Keyword(s):  
Insulin Receptor ◽  
Regulatory Subunit ◽  
Stable Complex ◽  
Phosphatidylinositol 3 Kinase ◽  
Sh2 Domains ◽  
Cellular Processes ◽  
Src Homology 2 ◽  
Src Homology ◽  
And Function ◽  
Phosphatidylinositol 3

Phosphatidylinositol 3-kinase (PI-3 kinase) is implicated in the regulation of diverse cellular processes, including insulin-stimulated glucose transport. PI-3 kinase is composed of a 110-kDa catalytic subunit and an 85-kDa regulatory subunit. Here, we describe p55PIK, a new regulatory subunit that was isolated by screening expression libraries with tyrosine-phosphorylated insulin receptor substrate 1 (IRS-1). p55PIK is composed of a unique 30-residue NH2 terminus followed by a proline-rich motif and two Src homology 2 (SH2) domains with significant sequence identify to those in p85. p55PIK mRNA is expressed early during development, remains abundant in adult mouse brain and testis tissue, and is detectable in adult adipocytes and heart and kidney tissues. p55PIK forms a stable complex with p110, and it associates with IRS-1 during insulin stimulation. Moreover, the activated insulin receptor phosphorylates p55PIK in Sf9 cells, and insulin stimulates p55PIK phosphorylation in CHOIR/p55PIK cells. The unique features of p55PIK suggest that it is important in receptor signaling.

scholarly journals Impact of Src Homology 2-Containing Inositol 5′-Phosphatase 2 on the Regulation of Insulin Signaling Leading to Protein Synthesis in 3T3-L1 Adipocytes Cultured with Excess Amino Acids

Endocrinology ◽  
2004 ◽  
Vol 145 (7) ◽  
pp. 3215-3223 ◽  
Author(s):  
Shihou Murakami ◽  
Toshiyasu Sasaoka ◽  
Tsutomu Wada ◽  
Kazuhito Fukui ◽  
Kiyofumi Nagira ◽  
...  
Keyword(s):  
Amino Acids ◽  
Protein Synthesis ◽  
Insulin Signaling ◽  
Phosphatidylinositol 3 Kinase ◽  
P70s6 Kinase ◽  
Ribosomal Protein S6 ◽  
Src Homology 2 ◽  
Src Homology ◽  
The Impact ◽  
Phosphatidylinositol 3

Abstract Src homology 2-containing inositol 5′-phosphatase 2 (SHIP2) possesses 5′-phosphatase activity to specifically hydrolyze the phosphatidylinositol 3-kinase product PI(3,4,5)P3 in the regulation of insulin signaling. In the present study, we examined the impact of SHIP2 on the regulation of insulin signaling leading to protein synthesis in 3T3-L1 adipocytes cultured with standard and excess concentrations of amino acids. Insulin-induced translocation of PDK1 to the plasma membrane, phosphorylation of Akt and p70S6-kinase and ribosomal protein S6, increase in the amount of 4E-BP1 γ-form, association of eIF4E with eIF4G, and protein synthesis were decreased by overexpression of wild-type SHIP2 by adenovirus-mediated gene transfer. The effect of SHIP2 overexpression on the regulation of insulin-induced phosphorylation of Akt and p70S6-kinase was somewhat augmented by the incubation with 5-fold excess concentrations of amino acids for 30 min. In contrast, the impact of SHIP2 expression was diminished in insulin-induced phosphorylation of p70S6-kinase and S6, but not of Akt, after the incubation for 16 h. Interestingly, incubation with the excess concentrations of amino acids for 30 min induced activation of phosphatidylinositol 3-kinase and phosphorylation of Akt, whereas phosphorylation of p70S6-kinase and S6 was decreased. Furthermore, although the exposure for longer time periods up to 24 h did not elicit phosphorylation of Akt, it markedly induced phosphorylation of p70S6-kinase and S6. These results indicate that SHIP2 plays an important role in the negative regulation of insulin signaling for the protein synthesis and that the impact of SHIP2 is altered, dependent on the acute or chronic exposure of excess concentrations of amino acids in culture.

Specific binding of Fyn and phosphatidylinositol 3-kinase to the B cell surface glycoprotein CD19 through their src homology 2 domains

1995 ◽  
Vol 25 (10) ◽  
pp. 2978-2984 ◽  
Author(s):  
N. Jan Chalupny ◽  
Alejandro Aruffo ◽  
James M. Esselstyn ◽  
Po-Ying Chan ◽  
Jürgen Bajorath ◽  
...  
Keyword(s):  
Cell Surface ◽  
B Cell ◽  
Specific Binding ◽  
Surface Glycoprotein ◽  
Phosphatidylinositol 3 Kinase ◽  
Cell Surface Glycoprotein ◽  
Src Homology 2 ◽  
Src Homology ◽  
Phosphatidylinositol 3
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