Activation of Phosphatidylinositol-3′ Kinase by Src-Family Kinase SH3 Binding to the p85 Subunit

Pleiman, Christopher M.; Hertz, WM; Jc, Cambier

doi:10.1126/science.8128248

Cited by 421 publications

(316 citation statements)

References 44 publications

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“…Sitedirected mutagenesis of PDGFb receptor YXXM motifs and in vitro tyrosyl phosphorylated peptide studies have con®rmed this mechanism of activation (Valius and Kazlauskas, 1993). A role for SH3 domain as a second mechanism of activation has also been suggested through the use of GST ± SH3 binding of p85 (Pleiman et al, 1994;.…”

Section: Discussionmentioning

confidence: 99%

“…GST constructs of Lyn have been found to associate with and activate PI 3-kinase ( Pleiman et al, 1994). Lyn and other Srcrelated PTK have been found to associate with Cbl (Cory et al, 1995;Fukazawa et al, 1995).…”

Section: Discussionmentioning

confidence: 99%

“…Receptor-encoded tyrosine kinases become tyrosyl phosphorylated, and these tyrosyl phosphorylated residues may serve as a docking site for the tandem SH2 domains of the p85 subunit of PI 3-kinase. Src-related protein tyrosine kinases have been found to contain p85 in antibody or a nity reagent co-precipitations, and thus, it has been postulated that the tyrosyl phosphorylated kinase is recognized by p85's SH2 domains and the kinase's SH3 domain recognizes the proline-rich domain of p85 (Pleiman et al, 1994). The adaptor protein Cbl, when expressed as an oncoprotein, results in B cell and myeloid leukemias (Langdon et al, 1989).…”

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confidence: 99%

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Yeast two-hybrid in vivo association of the Src kinase Lyn with the proto-oncogene product Cbl but not with the p85 subunit of PI 3-kinase

Dombrosky-Ferlan

Corey

1997

Oncogene

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Ligand binding of multi-chain antigen receptors and hematopoietin/cytokine receptors results in rapid activation of protein tyrosine kinase (PTK)-dependent signalling molecules such as phosphatidylinositol 3-kinase (PI 3-kinase). Co-precipitation studies have shown that Srcrelated PTK, such as Lyn, associates with the p85 regulatory subunit of PI 3-kinase via SH2 and SH3 domain binding with their cognate ligands. More recent studies have shown that the proto-oncogene product Cbl co-precipitates with p85 following engagement of cytokine and antigen receptors. As opposed to in vitro co-precipitation studies, the yeast two-hybrid screen reveals in vivo protein ± protein interactions. Using the yeast two-hybrid screen, we demonstrate an in vivo association of Lyn's SH3 and SH2 domains with the proline-rich domain of Cbl. Lyn's SH3 and SH2 domains do not interact with p85 in the yeast two-hybrid screen, as would be predicted from glutathione-S-transferase (GST) fusion protein pull-down or co-immunoprecipitation studies from whole cell lysates. However, the SH3 domain of p85 interacts with the proline-rich domain of Cbl. When yeast were transformed with catalytic Lyn, an interaction between p85's SH2 domain and Cbl occurred. From the data, we propose the following three step process of PI 3-kinase activation: (1) complexes of Lyn ± Cbl and Cbl ± p85 exist without ligand stimulation, (2) upon ligand binding, Lyn becomes active and phosphorylates Cbl, and (3) Cbl's tyrosine phosphorylated residue serves as a docking site for the SH2 domains of p85 ± thereby stabilizing the complex and activating PI 3-kinase. The yeast two-hybrid system can be used to dissect the precise mechanisms of in vivo protein ± protein interactions, including those between phosphotyrosine and SH2-containing proteins.

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Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

mentioning

confidence: 99%

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Yeast two-hybrid in vivo association of the Src kinase Lyn with the proto-oncogene product Cbl but not with the p85 subunit of PI 3-kinase

Dombrosky-Ferlan

Corey

1997

Oncogene

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“…Localization of PI 3-K by p85 to the occupied receptor has already been shown to be regulated by exposure of certain cells to platelet-derived growth factor [17], since p85 binds via its two SH2 domains to the tyrosinephosphorylated receptor. Other possible targets may involve exposure of proline-rich domains of p85 to the SH3 domain of src-family proteins [18]. Neither the thrombin receptor nor the LPA receptor is a tyrosine-autophosphorylating kinase, although tyrosine kinases are activated in response to occupancy of these receptors.…”

Section: Discussionmentioning

confidence: 99%

Thrombin stimulates wortmannin-inhibitable phosphoinositide 3-kinase and membrane blebbing in CHRF-288 cells

Vemuri

Zhang

Huang

et al. 1996

Biochemical Journal

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We have investigated thrombin-stimulated morphological changes and the activation of phosphoinositide 3-kinase (PI 3-K), as manifested by the accumulation of PtdIns(3,4)P2 and PtdIns(3,4,5)P3 (labelled with 32P or myo-[3H]inositol), in CHRF-288 cells, a leukaemic cell line derived from a platelet progenitor cell. We report that these cells, when exposed to thrombin or SFLLRN (the peptide Ser-Phe-Leu-Leu-Arg-Asn, a thrombin-receptor ligand) rapidly change shape, forming membrane ‘blebs’, detectable by differential interference contrast or confocal microscopy, as well as labelled 3-phosphorylated phosphoinositides. The ‘blebs’ are distinguishable from ‘ruffles’ or lamellae, since they do not contain phalloidin-detectable actin. Studies with permeabilized cells indicate that PI 3-K is activated synergistically by thrombin+guanosine 5´-[γ-thio]triphosphate. Two forms of PI 3-K, i.e. PI 3-K(γ) and p85/PI 3-K, regulated by Gβγ subunits of heterotrimeric G-protein and the small G-protein Rho, respectively, are present in these cells, as is true for platelets. Wortmannin, a known potent and specific inhibitor of PI 3-K activities, inhibits thrombin-stimulated accumulation of 3-phosphorylated phosphoinositides in a dose-dependent manner (IC50 ~ 10nM), without affecting phospholipase C activation. Pretreatment of CHRF-288 cells with either wortmannin (100 nM) or an unrelated synthetic PI 3-K inhibitor, LY294002 (50 μM), abolishes thrombin-receptor-stimulated blebbing. These results suggest that thrombin-stimulated accumulation of 3-phosphorylated phosphoinositide(s) is required for the shape-change response in CHRF-288 cells.

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“…The binding of tyrosine-phosphorylated proteins to the nSH2 domain of p85 inhibits PI3-K and is most frequently responsible for kinase activation, however, alternate mechanisms have been reported. For example, the conformational switch between p85 and p110 holoenzyme can also occur upon the interaction of signaling mediators with the SH3 or breakpoint cluster region homology domain (Liu et al, 1993;Prasad et al, 1993;Pleiman et al, 1994;Zheng et al, 1994). To investigate the apoptin interaction site of the p85 subunit, we cotransfected PC-3 cells with a vector encoding full-length apoptin, together with full-length p85 or various deletion mutants.…”

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confidence: 99%

Interaction with PI3-kinase contributes to the cytotoxic activity of Apoptin

et al. 2007

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Apoptin, a small protein from the chicken anemia virus, has attracted attention because of its specificity in killing tumor cells. Localization of apoptin in the nucleus of tumor cells has been shown to be vital for proapoptotic activity, however, targeted expression of apoptin in the nucleus of normal cells does not harm the cells, indicating that nuclear localization of apoptin is insufficient for its cytotoxicity. Here, we demonstrate for the first time that apoptin interacts with the SH3 domain of p85, the regulatory subunit of phosphoinositide 3-kinase (PI3-K), through its proline-rich region. Apoptin derivatives devoid of this proline-rich region do not interact with p85, are unable to activate PI3-K, and show impaired apoptosis induction. Moreover, apoptin mutants containing the proline-rich domain are sufficient to elevate PI3-K activity and to induce apoptosis in cancer cells. Downregulation of p85 leads to nuclear exclusion of apoptin and impairs cell death induction, indicating that interaction with the p85 PI3-K subunit essentially contributes to the cytotoxic activity of apoptin.

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Activation of Phosphatidylinositol-3′ Kinase by Src-Family Kinase SH3 Binding to the p85 Subunit

Cited by 421 publications

References 44 publications

Yeast two-hybrid in vivo association of the Src kinase Lyn with the proto-oncogene product Cbl but not with the p85 subunit of PI 3-kinase

Yeast two-hybrid in vivo association of the Src kinase Lyn with the proto-oncogene product Cbl but not with the p85 subunit of PI 3-kinase

Thrombin stimulates wortmannin-inhibitable phosphoinositide 3-kinase and membrane blebbing in CHRF-288 cells

Interaction with PI3-kinase contributes to the cytotoxic activity of Apoptin

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