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 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 p56lck interacts via its src homology 2 domain with the ZAP-70 kinase.

1994 ◽  
Vol 179 (4) ◽  
pp. 1163-1172 ◽  
Author(s):  
P Duplay ◽  
M Thome ◽  
F Hervé ◽  
O Acuto
Keyword(s):  
Tyrosine Kinases ◽  
Intracellular Signaling ◽  
Specific Binding ◽  
Sh2 Domain ◽  
Jurkat Cells ◽  
Vitro Assay ◽  
Phosphorylated Proteins ◽  
Src Homology 2 ◽  
Src Homology

p56lck, a member of the src family of protein tyrosine kinases, is an essential component in T cell receptor (TCR) signal transduction. p56lck contains a src homology 2 (SH2) domain found in a number of proteins involved in intracellular signaling. SH2 domains have been implicated in protein-protein interactions by binding to sequences in target proteins containing phosphorylated tyrosine. Using an in vitro assay, we have studied specific binding of tyrosine-phosphorylated proteins to a recombinant p56lck SH2 domain. In nonactivated Jurkat cells, two tyrosine-phosphorylated proteins were detected. Stimulation with anti-CD3 monoclonal antibodies induced the binding of seven additional tyrosine-phosphorylated proteins to the SH2 domain of p56lck. We have identified the zeta-associated tyrosine kinase, ZAP-70, as one of these proteins. Evidence suggests that binding of ZAP-70 to p56lck SH2 is direct and not mediated by zeta. The significance of this interaction was further investigated in vivo. p56lck could be coprecipitated with the zeta/ZAP-70 complex and conversely, ZAP-70 was detected in p56lck immunoprecipitates of activated Jurkat cells. The physical association of p56lck and ZAP-70 during activation supports the recently proposed functional cooperation of these two tyrosine kinases in TCR signaling.

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.

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.

Triazolopyrimidinium salts: discovery of a new class of agents for cancer therapy

2020 ◽  
Vol 12 (5) ◽  
pp. 387-402
Author(s):  
Mariateresa Badolato ◽  
Fabrizio Manetti ◽  
Antonio Garofalo ◽  
Francesca Aiello
Keyword(s):  
Anticancer Agents ◽  
Docking Studies ◽  
In Vitro Models ◽  
Sh2 Domain ◽  
Activity Profile ◽  
Cancer Cell Growth ◽  
New Class ◽  
Src Homology 2 ◽  
Src Homology

Aim: The [1,2,4]triazolo[1,5- a]pyrimidine core is highly privileged in medicinal chemistry due to its versatile pharmacological activity profile. Recently, the search for novel anticancer agents has focused on [1,2,4]triazolo[1,5- a]pyrimidine derivatives. Results: Our hit functionalization has led to the discovery of new [1,2,4]triazolo[1,5- a]pyrimidinium salts with potential anticancer activity. Among a small library of molecules, compound 9 significantly inhibits cancer cell growth in a panel of in vitro models. Molecular docking studies and preliminary binding assay have displayed that 9 could directly bind the Src homology 2 (SH2) domain of STAT3 protein. Conclusion: Compound 9 is a novel promising lead compound that motivates additional evaluation of [1,2,4]triazolo[1,5- a]pyrimidinium salts as novel potential chemotherapeutics.

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

1994 ◽  
Vol 14 (9) ◽  
pp. 5929-5938 ◽  
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.

scholarly journals The common src homology region 2 domain of cytoplasmic signaling proteins is a positive effector of v-fps tyrosine kinase function.

1989 ◽  
Vol 9 (10) ◽  
pp. 4131-4140 ◽  
Author(s):  
C A Koch ◽  
M Moran ◽  
I Sadowski ◽  
T Pawson
Keyword(s):  
Tyrosine Kinase ◽  
Tyrosine Kinases ◽  
Kinase Activity ◽  
Specific Activity ◽  
Intramolecular Interaction ◽  
Kinase Domain ◽  
Sh2 Domain ◽  
Homology Region ◽  
Src Homology ◽  
Type V

A conserved noncatalytic domain SH2 (for src homology region 2) is located immediately N terminal to the kinase domains of all cytoplasmic protein-tyrosine kinases. We found that the wild-type v-fps SH2 domain stimulated the enzymatic activity of the adjacent kinase domain 10-fold and functioned as a powerful positive effector of catalytic and transforming activities within the v-fps oncoprotein (P130gag-fps). Partial proteolysis of P130gag-fps and supporting genetic data indicated that the v-fps SH2 domain exerts its effect on catalytic activity through an intramolecular interaction with the kinase domain. Amino acid alterations in the SH2 domain that impaired kinase function interfered with association of the SH2 domain with the kinase domain. Deletion of a conserved octapeptide motif converted the v-fps SH2 domain from an activator to an inhibitor of tyrosine kinase activity. This latent inhibitory activity of v-fps SH2 has functional implications for phospholipase C-gamma and p21ras GTPase-activating protein, both of which have two distinct SH2 domains suggestive of complex regulation. In addition to regulating the specific activity of the kinase domain, the SH2 domain of P130gag-fps was also found to be required for the tyrosine phosphorylation of specific cellular proteins, notably polypeptides of 124 and 62 kilodaltons. The SH2 domain therefore appears to play a dual role in regulation of kinase activity and recognition of cellular substrates.

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