Characterization of the bovine brain cytosolic phosphatidylinositol 3-kinase complex

Ruiz-Larrea, Fernanda; Vicendo, Patricia; Yaish, Pnina; End, Peter; Panayotou, George; Fry, Michael; Morgan, Sarah J.; Thompson, Andrew R.; Parker, Peter J.; Waterfield, Michael D.

doi:10.1042/bj2900609

Cited by 32 publications

(16 citation statements)

References 26 publications

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“…Three sites of tyrosine phosphorylation have been mapped to residues 368, 580, and 607 of p85␣ (53,54). Consistent with the hypothesis that tyrosine phosphorylation of PI 3-kinase leads to an increase in its enzymatic activity is the report that a decrease in lipid kinase activity follows treatment of the purified bovine brain enzyme with a phosphotyrosyl protein phosphatase (55). Mutagenesis of these sites of tyrosine phosphorylation sites will be required, however, to precisely define their role in the activation of PI 3-kinase activity.…”

Section: P85␣-p110␣ Pi 3-kinase Complex and Pdgf Receptor Bindingsupporting

confidence: 50%

Binding to the Platelet-derived Growth Factor Receptor Transiently Activates the p85α-p110α Phosphoinositide 3-Kinase Complex in Vivo

Domin

Dhand

Waterfield

1996

Journal of Biological Chemistry

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Ligand stimulation of the platelet-derived growth factor (PDGF) receptor results in its association with phosphoinositide 3-kinase activity and a corresponding synthesis of 3 -phosphorylated lipids. Early studies that examined this interaction in vivo employed anti-phosphotyrosine antiserum or antiserum against the PDGF receptor. The recent identification of multiple isoforms of both the regulatory and the catalytic subunit of the enzyme have led us to utilize antisera against p85␣ and p110␣ to characterize the association of this particular phosphoinositide 3-kinase complex with the PDGF receptor following ligand stimulation of murine fibroblasts. Both the p85␣ and p110␣ subunits rapidly associated with the ligand-activated receptor resulting in a transient, 2-fold increase in the total pool of p110␣ lipid kinase activity. This association was stable for 15 min after initial stimulation. Subsequently, both subunits began to dissociate from the receptor with similar kinetics. By 60 min this process was complete, demonstrating that p85␣ and p110␣ both associate with the receptor and dissociate from the receptor as a dimeric complex. At this time, marked PDGF receptor down-regulation was observed. Immunoprecipitation from metabolically labeled cells revealed that p85␣ is constitutively phosphorylated on serine residues in quiescent cultures. Upon PDGF stimulation, this phosphorylation upon serine residues was maintained in addition to tyrosine phosphorylation of this subunit. No phosphorylation of the p110␣ subunit was detected in either quiescent or PDGF-stimulated cells. Quantitation of Western blot analysis demonstrated that only 5% of the total pool of p85␣ associated with the PDGF receptor upon ligand stimulation. The 2-fold increase in the lipid kinase activity measured in immunoprecipitates using either anti-p85␣ or anti-p110␣ antiserum therefore reflects a far greater increase in the specific activity of the enzyme upon its association with the PDGF receptor.Platelet derived growth factor (PDGF) 1 potently stimulates the proliferation of cells of mesenchymal origin. It has been reported to play a role in the physiology of early development, wound healing, and inflammation in addition to the etiology of atherosclerosis, rheumatoid arthritis, and oncogenesis, (1, 2). PDGF consists of a disulfide-linked dimer of two related polypeptide chains assembled either as homodimers or as a heterodimer. The binding of PDGF to its cell surface receptors causes receptor dimerization and transphosphorylation of specific residues along the PDGF receptor polypeptide chain (1, 3, 4). These phosphorylated tyrosine residues then serve as specific attachment sites for many cytoplasmic effector proteins (5). Substrates for the PDGF receptor include phospholipase C␥, ras GAP, members of the pp60 src family of protein tyrosine kinases, Nck, Grb2, and the phosphotyrosine phosphatase Syp (6 -9). Once bound to the receptor, many of these substrates are themselves phosphorylated on tyrosine residues by the intrinsic receptor kinase ac...

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Section: P85␣-p110␣ Pi 3-kinase Complex and Pdgf Receptor Bindingsupporting

confidence: 50%

Binding to the Platelet-derived Growth Factor Receptor Transiently Activates the p85α-p110α Phosphoinositide 3-Kinase Complex in Vivo

Domin

Dhand

Waterfield

1996

Journal of Biological Chemistry

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“…After insulin stimulation, PIP2 and PIP3 bind with Akt and translocate to the plasma membrane where Akt is phosphorylated by phosphatidylinositoldependent kinase (PDK1) and PDK2 on Thr 308 and Ser 473 respectively (Stokoe et al 1997, Stephens et al 1998, Andjelkovic et al 1999. The activation of PI 3-kinase by insulin requires phosphorylation of tyrosine residues on both p85 and p110 (Hayashi et al 1992, Ruiz-Larrea et al 1993. Former studies showed that activation of PI 3-kinase not only increases serine phosphorylation and activates Akt kinase but also induces the translocation of Akt to the plasma membrane, where Akt is activated (Wijkander et al 1997, Goransson et al 1998.…”

Section: Discussionmentioning

confidence: 99%

Decreased Akt kinase activity and insulin resistance in C57BL/KsJ-Leprdb/db mice

Shao¹,

Yamashita²,

Qiao³

et al. 2000

Journal of Endocrinology

142

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Recent studies suggest that the serine/threonine kinase protein kinase B (PKB or Akt) is involved in the pathway for insulin-stimulated glucose transporter 4 (GLUT4) translocation and glucose uptake. In this study we examined the components of the Akt signaling pathway in skeletal muscle and adipose tissue in vivo from C57BL/ KsJ-Lepr db/db mice (db/db), a model of obesity, insulin resistance, and type II diabetes. There were no changes in the protein levels of GLUT4, p85 , or Akt in tissues from db/db mice compared with non-diabetic littermate controls (+/+). In response to acute insulin administration, GLUT4 recruitment to the plasma membrane increased twofold in muscle and adipose tissue from +/+ mice, but was significantly reduced by 42-43% (P<0·05) in both tissues from db/db mice. Insulin increased Akt-Ser 473 phosphorylation by two-to fivefold in muscle and adipose tissue from all mice. However, in db/db mice, maximal Akt-Ser 473 phosphorylation was decreased by 32% (P<0·05) and 69% (P<0·05) in muscle and adipose tissue respectively. This decreased phosphorylation in db/db mice corresponded with a significant decrease in maximal Akt kinase activity using a glycogen synthase kinase-3 fusion protein as a substrate (P<0·05). The level of insulin-stimulated tyrosine phosphorylation of p85 from phosphatidylinositol 3 (PI 3)-kinase, which is upstream of Akt, was also reduced in muscle and adipose tissue from db/db mice (P<0·05); however, there was no change in extracellular signal-regulated kinase-1 or -2 phosphorylation. These data implicate decreased insulin-stimulated Akt kinase activity as an important component underlying impaired GLUT4 translocation and insulin resistance in tissues from db/db mice. However, impaired insulin signal transduction appears to be specific for the PI 3-kinase pathway of insulin signaling, while the MAP kinase pathway remained intact.

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“…In addition, the activated growth factor receptor tyrosine kinases also phosphorylate both catalytic and regulatory subunits of PI 3-kinase on tyrosine. This tyrosine phosphorylation increased the PI 3-kinase activity (33)(34)(35)(36). Thus, the activated PI 3-kinase could be immunoprecipitated with antiphosphotyrosine antibody.…”

Section: Involvement Of Pi 3-kinase-pkb/akt Signal Cascade In 3t3-l1 mentioning

confidence: 99%

Protein Kinase B/AKT 1 Plays a Pivotal Role in Insulin-like Growth Factor-1 Receptor Signaling Induced 3T3-L1 Adipocyte Differentiation

Liao

2004

Journal of Biological Chemistry

191

168

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During 3T3-L1 preadipocyte differentiation induction, the insulin-stimulated insulin-like growth factor-1 (IGF-1) receptor signal is responsible for the induction of adipocyte differentiation. Treatment with inhibitors of phosphatidylinositol 3-kinase, LY294002 or wortmannin, leads to the complete blockade of adipocyte differentiation in 3T3-L1 preadipocytes. Of the three factors (1-methyl-3-isobutylxanthine, dexamethasone, and insulin) inducing 3T3-L1 preadipocyte differentiation, only insulin was able to activate the phosphatidylinositol 3-kinase-protein kinase B/Akt signal cascade. In 3T3-L1 preadipocytes, protein kinase B/Akt 1 RNA interference not only suppressed the expression of protein kinase B/Akt 1 but also blocked hormone-induced adipocyte differentiation. In these protein kinase B/Akt 1 RNA interference cells, the signal molecules upstream of protein kinase B/Akt 1, such as IGF-1 receptor and insulin receptor substrate-1, were normally activated by insulin stimulation, whereas insulin-stimulated phosphorylation of forkhead transcription factor (FKHR), which is a downstream molecule of PKB/Akt 1, was blocked. Thus, protein kinase B/Akt 1 is an important signal mediator in IGF-1 receptor signal cascade for inducing adipocyte differentiation.Most of our current understandings on the cellular mechanisms of adipocyte differentiation regulation have come from the studies on established in vitro preadipocyte cell lines (1-3). These in vitro preadipocyte cell lines, e.g. 3T3-L1, 3T3-F442A, and TA1, can be induced to differentiate into adipocytes by insulin or in combination with other hormones. Subsequent studies on 3T3-L1 adipocyte differentiation induction indicate that the authentic hormone to induce the adipocyte differentiation is insulin-like growth factor-1 (IGF-1), 1 and insulin acts through the IGF-1 receptor on the cell membrane to induce the differentiation (4). In post-confluent 3T3-L1 preadipocytes, IGF-1 receptor signal initiates the adipocyte differentiation process. Immediately after the hormonal stimulation, postconfluent G 0 3T3-L1 preadipocytes reenter the cell cycle and start the adipocyte differentiation program. It is clear that the IGF-1 receptor signal plays an irreplaceable role in inducing the adipocyte differentiation process in 3T3-L1 cells.The signaling pathways or networks involved in mediating IGF-1 receptor signal for adipocyte differentiation are not fully understood. Two kinase systems, MAP kinases and phosphatidylinositol 3-kinase-protein kinase B/Akt, in 3T3-L1 cells can be activated by IGF-1 receptor signaling. The function of the MAP kinase system in IGF-1 receptor signal-induced 3T3-L1 cell adipogenesis is not clear. It is further complicated by the different effects exhibited by p38 MAP kinase and ERK1/2 on 3T3-L1 cell adipogenesis (5-12). More molecular studies are required in order to understand the relationship between MAP kinase system and 3T3-L1 cell adipogenesis.PI 3-kinase-PKB/Akt as an important intracellular signal cascade has been involved in the regulation o...

show abstract

Characterization of the bovine brain cytosolic phosphatidylinositol 3-kinase complex

Cited by 32 publications

References 26 publications

Binding to the Platelet-derived Growth Factor Receptor Transiently Activates the p85α-p110α Phosphoinositide 3-Kinase Complex in Vivo

Binding to the Platelet-derived Growth Factor Receptor Transiently Activates the p85α-p110α Phosphoinositide 3-Kinase Complex in Vivo

Decreased Akt kinase activity and insulin resistance in C57BL/KsJ-Leprdb/db mice

Protein Kinase B/AKT 1 Plays a Pivotal Role in Insulin-like Growth Factor-1 Receptor Signaling Induced 3T3-L1 Adipocyte Differentiation

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