Inhibition of phosphatidylinositol 3-kinase activity by association with 14-3-3 proteins in T cells.

The Ca2+-activated K+ channel KCa3.1 is required for Ca2+ influx and the subsequent activation of T-cells. We previously showed that nucleoside diphosphate kinase beta (NDPK-B), a mammalian histidine kinase, directly phosphorylates and activates KCa3.1 and is required for the activation of human CD4 T lymphocytes. We now show that the class II phosphatidylinositol 3 kinase C2β (PI3K-C2β) is activated by the T-cell receptor (TCR) and functions upstream of NDPK-B to activate KCa3.1 channel activity. Decreased expression of PI3K-C2β by siRNA in human CD4 T-cells resulted in inhibition of KCa3.1 channel activity. The inhibition was due to decreased phosphatidylinositol 3-phosphate [PI(3)P] because dialyzing PI3K-C2β siRNA-treated T-cells with PI(3)P rescued KCa3.1 channel activity. Moreover, overexpression of PI3K-C2β in KCa3.1-transfected Jurkat T-cells led to increased TCR-stimulated activation of KCa3.1 and Ca2+ influx, whereas silencing of PI3K-C2β inhibited both responses. Using total internal reflection fluorescence microscopy and planar lipid bilayers, we found that PI3K-C2β colocalized with Zap70 and the TCR in peripheral microclusters in the immunological synapse. This is the first demonstration that a class II PI3K plays a critical role in T-cell activation.

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Phosphatidylinositol 3-kinase activation is mediated by high-affinity interactions between distinct domains within the p110 and p85 subunits

Molecular and Cellular Biology ◽

10.1128/mcb.14.1.42-49.1994 ◽

1994 ◽

Vol 14 (1) ◽

pp. 42-49

Author(s):

K H Holt ◽

L Olson ◽

W S Moye-Rowley ◽

J E Pessin

Keyword(s):

Gene Fusion ◽

Kinase Activity ◽

Expression System ◽

Sh2 Domain ◽

Full Length ◽

Transactivation Domain ◽

Phosphatidylinositol 3 Kinase ◽

Sh2 Domains ◽

Amino Terminal ◽

Phosphatidylinositol 3

Domains of interaction between the p85 and p110 subunits of phosphatidylinositol 3-kinase (PI 3-kinase) were studied with the yeast two-hybrid expression system. A gene fusion between the GAL4 transactivation domain and p85 activated transcription from a GAL1-lacZ reporter gene when complemented with a gene fusion between the GAL4 DNA binding domain and p110. To define subdomains responsible for this interaction, a series of p85 deletion mutants were analyzed. A 192-amino-acid inter-SH2 (IS) fragment (residues 429 to 621) was the smallest determinant identified that specifically associated with p110. In analogous experiments, the subdomain within p110 responsible for interaction with p85 was localized to an EcoRI fragment encoding the amino-terminal 127 residues. Expression of these two subdomains [p85(IS) with p110RI] resulted in 100-fold greater reporter activity than that obtained with full-length p85 and p110. Although the p85(IS) domain conferred a strong interaction with the p110 catalytic subunit, this region was not sufficient to impart phosphotyrosine peptide stimulation of PI 3-kinase activity. In contrast, coexpression of the p110 subunit with full-length p85 or with constructs containing the IS sequences flanked by both SH2 domains of p85 [p85(n/cSH2)] or either of the individual SH2 domains [p85(nSH2+IS) or p85(IS+cSH2)] resulted in PI 3-kinase activity that was activated by a phosphotyrosine peptide. These data suggest that phosphotyrosine peptide binding to either SH2 domain generates an intramolecular signal propagated through the IS region to allosterically activate p110.

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Enhanced Phosphatidylinositol 3-Kinase Activity and High Phosphorylation State of Its Downstream Signalling Molecules Mediated by Ret with the MEN 2B Mutation

Biochemical and Biophysical Research Communications ◽

10.1006/bbrc.1999.1186 ◽

1999 ◽

Vol 262 (1) ◽

pp. 68-75 ◽

Cited By ~ 56

Author(s):

Hideki Murakami ◽

Toshihide Iwashita ◽

Naoya Asai ◽

Yohei Shimono ◽

Yosuke Iwata ◽

...

Keyword(s):

Kinase Activity ◽

Phosphatidylinositol 3 Kinase ◽

Phosphorylation State ◽

Signalling Molecules ◽

Men 2B ◽

Phosphatidylinositol 3

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Association of phosphatidylinositol 3-kinase with the insulin receptor: compartmentation in rat liver

AJP Endocrinology and Metabolism ◽

10.1152/ajpendo.2000.279.2.e266 ◽

2000 ◽

Vol 279 (2) ◽

pp. E266-E274 ◽

Cited By ~ 9

Author(s):

Paul G. Drake ◽

Alejandro Balbis ◽

Jiong Wu ◽

John J. M. Bergeron ◽

Barry I. Posner

Keyword(s):

Plasma Membrane ◽

Cell Surface ◽

Insulin Receptor ◽

Rat Liver ◽

Intracellular Signaling ◽

Kinase Activity ◽

Glycogen Synthase ◽

Metabolic Effects ◽

Phosphatidylinositol 3 Kinase ◽

Phosphatidylinositol 3

Phosphatidylinositol 3-kinase (PI 3-kinase) plays an important role in a variety of hormone and growth factor-mediated intracellular signaling cascades and has been implicated in the regulation of a number of metabolic effects of insulin, including glucose transport and glycogen synthase activation. In the present study we have examined 1) the association of PI 3-kinase with the insulin receptor kinase (IRK) in rat liver and 2) the subcellular distribution of PI 3-kinase-IRK interaction. Insulin treatment promoted a rapid and pronounced recruitment of PI 3-kinase to IRKs located at the plasma membrane, whereas no increase in association with endosomal IRKs was observed. In contrast to IRS-1-associated PI 3-kinase activity, association of PI 3-kinase with the plasma membrane IRK did not augment the specific activity of the lipid kinase. With use of the selective PI 3-kinase inhibitor wortmannin, our data suggest that the cell surface IRK β-subunit is not a substrate for the serine kinase activity of PI 3-kinase. The functional significance for the insulin-stimulated selective recruitment of PI 3-kinase to cell surface IRKs remains to be elucidated.

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