Phosphatidylinositol 3-kinase: Inhibition of intrinsic protein-serine kinase activity by phosphoinositides, and of lipid kinase activity by Mn2+

The class III phosphatidylinositol 3-kinase complex I (PI3KC3-C1) that functions in early autophagy consists of the lipid kinase VPS34, the scaffolding protein VPS15, the tumor suppressor BECN1, and the autophagy-specific subunit ATG14. The structure of the ATG14-containing PI3KC3-C1 was determined by single-particle EM, revealing a V-shaped architecture. All of the ordered domains of VPS34, VPS15, and BECN1 were mapped by MBP tagging. The dynamics of the complex were defined using hydrogen–deuterium exchange, revealing a novel 20-residue ordered region C-terminal to the VPS34 C2 domain. VPS15 organizes the complex and serves as a bridge between VPS34 and the ATG14:BECN1 subcomplex. Dynamic transitions occur in which the lipid kinase domain is ejected from the complex and VPS15 pivots at the base of the V. The N-terminus of BECN1, the target for signaling inputs, resides near the pivot point. These observations provide a framework for understanding the allosteric regulation of lipid kinase activity.

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The p85 and p110 Subunits of Phosphatidylinositol 3-Kinase-α Are Substrates, In Vitro, for a Constitutively Associated Protein Tyrosine Kinase in Platelets

Blood ◽

10.1182/blood.v91.3.930.930_930_939 ◽

1998 ◽

Vol 91 (3) ◽

pp. 930-939 ◽

Cited By ~ 1

Author(s):

Norman R. Geltz ◽

James A. Augustine

Keyword(s):

Tyrosine Kinase ◽

Protein Tyrosine Kinase ◽

Kinase Activity ◽

Protein Kinase Activity ◽

Phosphatidylinositol 3 Kinase ◽

Lipid Kinase ◽

Protein Tyrosine ◽

Dependent Protein ◽

Phosphatidylinositol 3

Phosphatidylinositol 3-kinase (PI3K) is a heterodimer lipid kinase consisting of an 85-kD subunit bound to a 110-kD catalytic subunit that also possesses intrinsic, Mn2+-dependent protein serine kinase activity capable of phosphorylating the 85-kD subunit. Here, we examine the Mn2+-dependent protein kinase activity of PI3Kα immunoprecipitated from normal resting or thrombin-stimulated platelets, and characterize p85/p110 phosphorylation, in vitro. Phosphoamino acid analysis of phosphorylated PI3Kα showed p85 and p110 were phosphorylated on serine, but in contrast to previous results, were also phosphorylated on threonine and tyrosine. Wortmannin and LY294002 inhibited p85 phosphorylation; however, p110 phosphorylation was also inhibited suggesting p110 autophosphorylation on serine/threonine. The protein tyrosine kinase inhibitor, erbstatin analog, partially inhibited p85 and p110 phosphorylation but did not appear to affect PI3K lipid kinase activity. The in vitro phosphorylation of p85α or p110α derived from thrombin-stimulated platelets was no different than that of resting platelets, but we confirm that in thrombin receptor-stimulated platelets enhanced levels of p85α and PI3K lipid kinase activity were recovered in antiphosphotyrosine antibody immunoprecipitates. These results suggest PI3Kα can autophosphorylate on serine and threonine, and both p85α and p110α are substrates for a constitutively-associated protein tyrosine kinase in platelets.

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Insulin receptor substrate 1 is phosphorylated by the serine kinase activity of phosphatidylinositol 3-kinase

Biochemical Journal ◽

10.1042/bj3040017 ◽

1994 ◽

Vol 304 (1) ◽

pp. 17-21 ◽

Cited By ~ 64

Author(s):

J F Tanti ◽

T Grémeaux ◽

E Van Obberghen ◽

Y Le Marchand-Brustel

Keyword(s):

Insulin Receptor ◽

Kinase Activity ◽

Pi3 Kinase ◽

Serine Phosphorylation ◽

Kinase Assay ◽

Insulin Receptor Substrate ◽

Phosphatidylinositol 3 Kinase ◽

Serine Kinase ◽

Phosphatidylinositol 3

Insulin receptor substrate (IRS) 1, which is tyrosine phosphorylated in response to insulin, presents multiple serine/threonine phosphorylation sites. To search for a serine kinase activity towards IRS 1, immunoprecipitates from basal or stimulated 3T3-L1 adipocytes were used in an in vitro kinase assay. When IRS 1 was isolated from insulin-treated cells, serine phosphorylation of IRS 1 occurred, which we attribute to the kinase activity of the phosphatidylinositol 3-kinase (PI3-kinase). Importantly, in an in vitro reconstitution assay, an excess of the PI3-kinase subunit prevents this phosphorylation. Together, our results suggest that following insulin stimulation, PI3-kinase associates with IRS 1, allowing for its serine phosphorylation. This phosphorylation event could play a role in the modulation of insulin signalling.

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Phosphatidylinositol 3–Kinase Is Required for the Formation of Constitutive Transport Vesicles from the TGN

The Journal of Cell Biology ◽

10.1083/jcb.139.2.339 ◽

1997 ◽

Vol 139 (2) ◽

pp. 339-349 ◽

Cited By ~ 57

Author(s):

Steven M. Jones ◽

Kathryn E. Howell

Keyword(s):

Kinase Activity ◽

Pi3 Kinase ◽

Regulatory Subunit ◽

Vesicle Formation ◽

Phosphatidylinositol 3 Kinase ◽

Response Curves ◽

Direct Role ◽

Lipid Kinase ◽

Transport Vesicles ◽

Phosphatidylinositol 3

An 85-kD cytosolic complex (p62cplx), consisting of a 62-kD phosphoprotein (p62) and a 25-kD GTPase, has been shown to be essential for the cell-free reconstitution of polymeric IgA receptor (pIgA-R)-containing exocytic transport vesicle formation from the TGN (Jones, S.M., J.R. Crosby, J. Salamero, and K.E. Howell. 1993. J. Cell Biol. 122:775–788). Here the p62cplx is identified as a regulatory subunit of a novel phosphatidylinositol 3–kinase (PI3-kinase). This p62cplx-associated PI3-kinase activity is stimulated by activation of the p62cplx-associated GTPase, and is specific for phosphatidylinositol (PI) as substrate, and is sensitive to wortmannin at micromolar concentrations. The direct role of this p62cplx-associated PI3-kinase activity in TGN-derived vesicle formation is indicated by the finding that both lipid kinase activity and the formation of pIgA-R–containing exocytic vesicles from the TGN are inhibited by wortmannin with similar dose-response curves and 50% inhibitory concentrations (3.5 μM). These findings indicate that phosphatidylinositol-3-phosphate (PI[3]P) is required for the formation of TGN-derived exocytic transport vesicles, and that the p62cplx-associated PI3-kinase and an activated GTPase are the essential molecules that drive production of this PI(3)P.

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The p85 and p110 Subunits of Phosphatidylinositol 3-Kinase-α Are Substrates, In Vitro, for a Constitutively Associated Protein Tyrosine Kinase in Platelets

Blood ◽

10.1182/blood.v91.3.930 ◽

1998 ◽

Vol 91 (3) ◽

pp. 930-939 ◽

Cited By ~ 14

Author(s):

Norman R. Geltz ◽

James A. Augustine

Keyword(s):

Tyrosine Kinase ◽

Protein Tyrosine Kinase ◽

Kinase Activity ◽

Protein Kinase Activity ◽

Phosphatidylinositol 3 Kinase ◽

Lipid Kinase ◽

Protein Tyrosine ◽

Dependent Protein ◽

Phosphatidylinositol 3

Abstract Phosphatidylinositol 3-kinase (PI3K) is a heterodimer lipid kinase consisting of an 85-kD subunit bound to a 110-kD catalytic subunit that also possesses intrinsic, Mn2+-dependent protein serine kinase activity capable of phosphorylating the 85-kD subunit. Here, we examine the Mn2+-dependent protein kinase activity of PI3Kα immunoprecipitated from normal resting or thrombin-stimulated platelets, and characterize p85/p110 phosphorylation, in vitro. Phosphoamino acid analysis of phosphorylated PI3Kα showed p85 and p110 were phosphorylated on serine, but in contrast to previous results, were also phosphorylated on threonine and tyrosine. Wortmannin and LY294002 inhibited p85 phosphorylation; however, p110 phosphorylation was also inhibited suggesting p110 autophosphorylation on serine/threonine. The protein tyrosine kinase inhibitor, erbstatin analog, partially inhibited p85 and p110 phosphorylation but did not appear to affect PI3K lipid kinase activity. The in vitro phosphorylation of p85α or p110α derived from thrombin-stimulated platelets was no different than that of resting platelets, but we confirm that in thrombin receptor-stimulated platelets enhanced levels of p85α and PI3K lipid kinase activity were recovered in antiphosphotyrosine antibody immunoprecipitates. These results suggest PI3Kα can autophosphorylate on serine and threonine, and both p85α and p110α are substrates for a constitutively-associated protein tyrosine kinase in platelets.

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Phosphatidylinositol 3-kinase inhibition induces vasodilator effect of sevoflurane via reduction of Rho kinase activity

Life Sciences ◽

10.1016/j.lfs.2017.04.005 ◽

2017 ◽

Vol 177 ◽

pp. 20-26 ◽

Cited By ~ 2

Author(s):

Yasunori Miyamoto ◽

Guo-Gang Feng ◽

Shiho Satomi ◽

Katsuya Tanaka ◽

Yoshihiro Fujiwara ◽

...

Keyword(s):

Kinase Activity ◽

Rho Kinase ◽

Phosphatidylinositol 3 Kinase ◽

Kinase Inhibition ◽

Vasodilator Effect ◽

Phosphatidylinositol 3

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Regulation of the p85/p110 Phosphatidylinositol 3′-Kinase: Stabilization and Inhibition of the p110α Catalytic Subunit by the p85 Regulatory Subunit

Molecular and Cellular Biology ◽

10.1128/mcb.18.3.1379 ◽

1998 ◽

Vol 18 (3) ◽

pp. 1379-1387 ◽

Cited By ~ 336

Author(s):

Jinghua Yu ◽

Yitao Zhang ◽

James McIlroy ◽

Tamara Rordorf-Nikolic ◽

George A. Orr ◽

...

Keyword(s):

Mammalian Cells ◽

Kinase Activity ◽

Thermal Inactivation ◽

Insect Cells ◽

Catalytic Subunit ◽

Regulatory Subunit ◽

Phosphatidylinositol 3 Kinase ◽

Lipid Kinase ◽

Phosphatidylinositol 3

ABSTRACT We propose a novel model for the regulation of the p85/p110α phosphatidylinositol 3′-kinase. In insect cells, the p110α catalytic subunit is active as a monomer but its activity is decreased by coexpression with the p85 regulatory subunit. Similarly, the lipid kinase activity of recombinant glutathione S-transferase (GST)-p110α is reduced by 65 to 85% upon in vitro reconstitution with p85. Incubation of p110α/p85 dimers with phosphotyrosyl peptides restored activity, but only to the level of monomeric p110α. These data show that the binding of phosphoproteins to the SH2 domains of p85 activates the p85/p110α dimers by inducing a transition from an inhibited to a disinhibited state. In contrast, monomeric p110 had little activity in HEK 293T cells, and its activity was increased 15- to 20-fold by coexpression with p85. However, this apparent requirement for p85 was eliminated by the addition of a bulky tag to the N terminus of p110α or by the growth of the HEK 293T cells at 30°C. These nonspecific interventions mimicked the effects of p85 on p110α, suggesting that the regulatory subunit acts by stabilizing the overall conformation of the catalytic subunit rather than by inducing a specific activated conformation. This stabilization was directly demonstrated in metabolically labeled HEK 293T cells, in which p85 increased the half-life of p110. Furthermore, p85 protected p110 from thermal inactivation in vitro. Importantly, when we examined the effect of p85 on GST-p110α in mammalian cells at 30°C, culture conditions that stabilize the catalytic subunit and that are similar to the conditions used for insect cells, we found that p85 inhibited p110α. Thus, we have experimentally distinguished two effects of p85 on p110α: conformational stabilization of the catalytic subunit and inhibition of its lipid kinase activity. Our data reconcile the apparent conflict between previous studies of insect versus mammalian cells and show that p110α is both stabilized and inhibited by dimerization with p85.

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