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

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.

Download Full-text

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

Molecular and Cellular Biology ◽

10.1128/mcb.15.8.4453 ◽

1995 ◽

Vol 15 (8) ◽

pp. 4453-4465 ◽

Cited By ~ 170

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.

Download Full-text

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

Molecular and Cellular Biology ◽

10.1128/mcb.13.12.7677 ◽

1993 ◽

Vol 13 (12) ◽

pp. 7677-7688 ◽

Cited By ~ 171

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.

Download Full-text

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

Biochemical Journal ◽

10.1042/bj3020737 ◽

1994 ◽

Vol 302 (3) ◽

pp. 737-744 ◽

Cited By ~ 24

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.

Download Full-text

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

Molecular and Cellular Biology ◽

10.1128/mcb.13.12.7677-7688.1993 ◽

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.

Download Full-text

Using a Phage Display Library to Identify Basic Residues in A-Raf Required to Mediate Binding to the Src Homology 2 Domains of the p85 Subunit of Phosphatidylinositol 3′-Kinase

Journal of Biological Chemistry ◽

10.1074/jbc.m004720200 ◽

2000 ◽

Vol 275 (46) ◽

pp. 36450-36456 ◽

Cited By ~ 22

Author(s):

Tamela R. King ◽

Yun Fang ◽

Elizabeth S. Mahon ◽

Deborah H. Anderson

Keyword(s):

Phage Display ◽

Phage Display Library ◽

Phosphatidylinositol 3 Kinase ◽

Src Homology 2 ◽

Src Homology ◽

Phosphatidylinositol 3

Download Full-text

Genetic analysis of a phosphatidylinositol 3-kinase SH2 domain reveals determinants of specificity.

Molecular and Cellular Biology ◽

10.1128/mcb.14.9.5929 ◽

1994 ◽

Vol 14 (9) ◽

pp. 5929-5938 ◽

Cited By ~ 16

Author(s):

M Yoakim ◽

W Hou ◽

Z Songyang ◽

Y Liu ◽

L Cantley ◽

...

Keyword(s):

Phosphorylation Site ◽

Growth Factor Receptor ◽

Sh2 Domain ◽

T Antigen ◽

Site Directed Mutagenesis ◽

Phosphatidylinositol 3 Kinase ◽

Sh2 Domains ◽

Src Homology ◽

Polyomavirus Middle T Antigen ◽

Phosphatidylinositol 3

Phosphatidylinositol 3-kinase is an important element in both normal and oncogenic signal transduction. Polyomavirus middle T antigen transforms cells in a manner depending on association of its tyrosine 315 phosphorylation site with Src homology 2 (SH2) domains on the p85 subunit of the phosphatidylinositol 3-kinase. Both nonselective and site-directed mutagenesis have been used to probe the interaction of middle T with the N-terminal SH2 domain of p85. Most of the 24 mutants obtained showed reduced middle T binding. However, mutations that showed increased binding were also found. Comparison of middle T binding to that of the platelet-derived growth factor receptor showed that some mutations altered the specificity of recognition by the SH2 domain. Mutations altering S-393, D-394, and P-395 were shown to affect the ability of the SH2 domain to select peptides from a degenerate phosphopeptide library. These results focus attention on the role of the EF loop in the SH2 domain in determining binding selectivity at the third position after the phosphotyrosine.

Download Full-text

Correlation of α-Fetoprotein Expression in Normal Hepatocytes during Development with Tyrosine Phosphorylation and Insulin Receptor Expression

Molecular Biology of the Cell ◽

10.1091/mbc.9.5.1093 ◽

1998 ◽

Vol 9 (5) ◽

pp. 1093-1105 ◽

Cited By ~ 10

Author(s):

Leila Khamzina ◽

Pierre Borgeat

Keyword(s):

Insulin Receptor ◽

Tyrosine Phosphorylation ◽

Receptor Expression ◽

Regulatory Subunit ◽

Β Subunit ◽

Phosphatidylinositol 3 Kinase ◽

Fetal Hepatocytes ◽

In Cells ◽

Phosphatidylinositol 3

The molecular mechanism of hepatic cell growth and differentiation is ill defined. In the present study, we examined the putative role of tyrosine phosphorylation in normal rat liver development and in an in vitro model, the α-fetoprotein-producing (AFP+) and AFP-nonproducing (AFP−) clones of the McA-RH 7777 rat hepatoma. We demonstrated in vivo and in vitro that the AFP+ phenotype is clearly associated with enhanced tyrosine phosphorylation, as assessed by immunoblotting and flow cytometry. Moreover, immunoprecipitation of proteins with anti-phosphotyrosine antibody showed that normal fetal hepatocytes expressed the same phosphorylation pattern as stable AFP+clones and likewise for adult hepatocytes and AFP− clones. The tyrosine phosphorylation of several proteins, including the β-subunit of the insulin receptor, insulin receptor substrate-1, p85 regulatory subunit of phosphatidylinositol-3-kinase, andras-guanosine triphosphatase-activating protein, was observed in AFP+ clones, whereas the same proteins were not phosphorylated in AFP− clones. We also observed that fetal hepatocytes and the AFP+ clones express 4 times more of the insulin receptor β-subunit compared with adult hepatocytes and AFP− clones and, accordingly, that these AFP+clones were more responsive to exogenous insulin in terms of protein tyrosine phosphorylation. Finally, growth rate in cells of AFP+ clones was higher than that measured in cells of AFP− clones, and inhibition of phosphatidylinositol-3-kinase by LY294002 and Wortmannin blocked insulin- and serum-stimulated DNA synthesis only in cells of AFP+ clones. These studies provide evidences in support of the hypothesis that signaling via insulin prevents hepatocyte differentiation by promoting fetal hepatocyte growth.

Download Full-text

Regulation of class IA PI3Ks

Biochemical Society Transactions ◽

10.1042/bst0350242 ◽

2007 ◽

Vol 35 (2) ◽

pp. 242-244 ◽

Cited By ~ 33

Author(s):

H. Wu ◽

Y. Yan ◽

J.M. Backer

Keyword(s):

Tyrosine Kinases ◽

Regulatory Subunit ◽

Sh2 Domains ◽

Src Homology 2 ◽

Pi3k Activity ◽

Wide Range ◽

Rho Family Gtpases ◽

Src Homology ◽

Rho Family

Class IA PI3Ks (phosphoinositide 3-kinases) regulate a wide range of cellular responses through the production of PI(3,4,5)P3 (phosphatidylinositol 3,4,5-trisphosphate) in cellular membranes. They are activated by receptor tyrosine kinases, by Ras and Rho family GTPases, and in some cases by Gβγ subunits from trimeric G-proteins. Crystallographic studies on the related class IB PI3Kγ, and biochemical and structural studies on the class IA PI3Ks, have led to new insights into how these critical enzymes are regulated in normal cells and how mutations can lead to their constitutive activation in transformed cells. The present paper will discuss recent studies on the regulation of class I (p85/p110) PI3Ks, with a focus on the role of SH2 domains (Src homology 2 domains) in the p85 regulatory subunit in modulating PI3K activity.

Download Full-text