The tyrosine phosphatase CD148 interacts with the p85 regulatory subunit of phosphoinositide 3-kinase

2008 ◽  
Vol 413 (1) ◽  
pp. 193-200 ◽  
Author(s):  
Nobuo Tsuboi ◽  
Tadahiko Utsunomiya ◽  
Richard L. Roberts ◽  
Hideyuki Ito ◽  
Keiko Takahashi ◽  
...  
Keyword(s):  
Tyrosine Phosphatase ◽  
Growth Factor Receptor ◽  
Regulatory Subunit ◽  
Mutant Form ◽  
Dependent Manner ◽  
Screening Assay ◽  
Intact Cells ◽  
Pi3k Activity ◽  
Phosphoinositide 3 Kinase

CD148 is a transmembrane tyrosine phosphatase that has been implicated in the regulation of cell growth and transformation. However, the signalling mechanisms of CD148 are incompletely understood. To identify the specific intracellular molecules involved in CD148 signalling, we carried out a modified yeast two-hybrid screening assay. Using the substrate-trapping mutant form of CD148 (CD148 D/A) as bait, we recovered the p85 regulatory subunit of PI3K (phosphoinositide 3-kinase). CD148 D/A, but not catalytically active CD148, interacted with p85 in a phosphorylation-dependent manner in vitro and in intact cells. Growth factor receptor and PI3K activity were also trapped by CD148 D/A via p85 from pervanadate-treated cell lysates. CD148 prominently and specifically dephosphorylated p85 in vitro. Co-expression of CD148 reduced p85 phosphorylation induced by active Src, and attenuated the increases in PI3K activity, yet CD148 did not alter the basal PI3K activity. Finally, CD148 knock-down by siRNA (short interfering RNA) increased PI3K activity on serum stimulation. Taken together, these results demonstrate that CD148 may interact with and dephosphorylate p85 when it is phosphorylated and modulate the magnitude of PI3K activity.

scholarly journals Interaction of Ras with phosphoinositide 3-kinase γ

1997 ◽  
Vol 326 (3) ◽  
pp. 891-895 ◽  
Author(s):  
Ignacio RUBIO ◽  
Pablo RODRIGUEZ-VICIANA ◽  
Julian DOWNWARD ◽  
Reinhard WETZKER
Keyword(s):  
Binding Site ◽  
G Proteins ◽  
Terminal Region ◽  
Regulatory Subunit ◽  
Heterotrimeric G Proteins ◽  
Modest Increase ◽  
Pi3k Activity ◽  
Phosphoinositide 3 Kinase ◽  
Stimulation Of

Phosphoinositide 3-kinase γ (PI3Kγ) can be activated in vitro by both α and βγ subunits of heterotrimeric G-proteins and does not interact with p85, the regulatory subunit of PI3Kα. Here we demonstrate the binding of Ras to PI3Kγ in vitro. An N-terminal region of PI3Kγ was identified as a binding site for Ras. After co-expression with PI3Kγ in COS-7 cells, Ras induced only a modest increase in PI3K activity compared with the stimulation of PI3Kα by Ras in the same cells.

scholarly journals Differential regulation of class IA phosphoinositide 3-kinase catalytic subunits p110α and β by protease-activated receptor 2 and β-arrestins

2007 ◽  
Vol 408 (2) ◽  
pp. 221-230 ◽  
Author(s):  
Ping Wang ◽  
Puneet Kumar ◽  
Chang Wang ◽  
Kathryn A. DeFea
Keyword(s):  
G Protein ◽  
Small Gtpase ◽  
Catalytic Subunit ◽  
Regulatory Subunit ◽  
Catalytic Subunits ◽  
Pi3k Activity ◽  
Phosphoinositide 3 Kinase ◽  
Protease Activated Receptor 2

PAR-2 (protease-activated receptor 2) is a GPCR (G-protein-coupled receptor) that can elicit both G-protein-dependent and -independent signals. We have shown previously that PAR-2 simultaneously promotes Gαq/Ca2+-dependent activation and β-arrestin-1-dependent inhibition of class IA PI3K (phosphoinositide 3-kinase), and we sought to characterize further the role of β-arrestins in the regulation of PI3K activity. Whereas the ability of β-arrestin-1 to inhibit p110α (PI3K catalytic subunit α) has been demonstrated, the role of β-arrestin-2 in PI3K regulation and possible differences in the regulation of the two catalytic subunits (p110α and p110β) associated with p85α (PI3K regulatory subunit) have not been examined. In the present study we have demonstrated that: (i) PAR-2 increases p110α- and p110β-associated lipid kinase activities, and both p110α and p110β are inhibited by over-expression of either β-arrestin-1 or -2; (ii) both β-arrestin-1 and -2 directly inhibit the p110α catalytic subunit in vitro, whereas only β-arrestin-2 directly inhibited p110β; (iii) examination of upstream pathways revealed that PAR-2-induced PI3K activity required the small GTPase Cdc (cell-division cycle)42, but not tyrosine phosphorylation of p85; and (iv) β-arrestins inhibit PAR-2-induced Cdc42 activation. Taken together, these results indicated that β-arrestins could inhibit PAR-2-stimulated PI3K activity, both directly and through interference with upstream pathways, and that the two β-arrestins differ in their ability to inhibit the p110α and p110β catalytic subunits. These results are particularly important in light of the growing interest in PAR-2 as a pharmacological target, as commonly used biochemical assays that monitor G-protein coupling would not screen for β-arrestin-dependent signalling events.

A cascade involving p85, Cdc42 and septin 2 regulates cytokinesis

2007 ◽  
Vol 35 (2) ◽  
pp. 222-224 ◽  
Author(s):  
V. Silió ◽  
M. Marqués ◽  
I. Cortés ◽  
S. Zuluaga ◽  
A.C. Carrera
Keyword(s):  
Cell Separation ◽  
Nuclear Division ◽  
Growth Factor Receptor ◽  
Regulatory Subunit ◽  
Final Phase ◽  
Receptor Stimulation ◽  
Pi3k Activity ◽  
Phosphoinositide 3 Kinase ◽  
The Impact ◽  
Early Mitosis

Mitosis, the final phase of cell division, includes the processes of nuclear division and cytosolic division (cytokinesis). Cytokinesis occurs when DNA separation terminates, and involves a number of proteins that induce furrowing at the region of cell separation, formation of new membrane, and abscission. This process is remarkably complex, and the list of proteins that regulate it is long. Our understanding is limited as to how these players are organized in space and time to ensure that the cytosol divides equally, and only after nuclear division. Class IA PI3K (phosphoinositide 3-kinase) is an enzyme activated by growth factor receptor stimulation, but it is re-activated in early mitosis and regulates mitosis entry. By the end of mitosis, PI3K activity is low; at this point, the class IA PI3K regulatory subunit p85 contributes to co-ordination of the cytoskeletal changes required for cytokinesis. The impact of these observations on current models of cytokinesis execution is discussed here.

A novel signaling pathway for β-adrenergic receptor-mediated activation of phosphoinositide 3-kinase in H9c2 cardiomyocytes

2007 ◽  
Vol 293 (1) ◽  
pp. H385-H393 ◽  
Author(s):  
Naohiro Yano ◽  
Vlad Ianus ◽  
Ting C. Zhao ◽  
Andy Tseng ◽  
James F. Padbury ◽  
...  
Keyword(s):  
Growth Factor ◽  
Protein Kinase ◽  
Intracellular Signaling ◽  
Growth Factor Receptor ◽  
Mouse Heart ◽  
Pi3k Activity ◽  
H9c2 Cardiomyocytes ◽  
Phosphoinositide 3 Kinase

Stimulation of cardiac β-adrenergic receptors (β-AR) activates both the Gs- and Gi-coupled signaling cascades, including the phosphoinositide 3 kinase (PI3K) pathway, that have important physiological implications. Multiple isoforms of PI3K exist in the heart. The goals of this study were to examine the intracellular signaling pathways linking β-AR to PI3K and to identify the PI3K isoform mediating this transactivation in a cardiac context. Acute β-AR stimulation with isoproterenol resulted in increased tyrosine kinase-associated PI3K activity and phosphorylation of Akt and p70S6K in H9c2 cardiomyocytes. Cotreatment with ICI-118,551, but not CGP-20712, abolished the increase in PI3K activity, suggesting a β2-AR-mediated event. PI3K activation was also abrogated by cotreatment with pertussis toxin, 4-amino-5-(4-chlorophenyl)-7-( t-butyl)pyrazolol[3,4-d]pyrimidine (PP2, a selective Src-family tyrosine kinases inhibitor), or AG-1296 [selective platelet-derived growth factor receptor (PDGFR) inhibitor] but not with an inhibitor for protein kinase A, protein kinase C, Ras, adenylyl cyclase, epidermal growth factor receptor, or insulin-like growth factor-1 receptor. β-AR stimulation induced an increase in tyrosine phosphorylation of PDGFR, which was abolished by inhibition of Src either by PP2 or small interfering RNA. Moreover, H9c2 cardiomyocytes stably transfected with a vector expressing a Gβγ sequestrant peptide derived from the COOH-terminus of β-AR kinase-1 failed to activate PI3K after β-AR stimulation, suggesting Gβγ is required for the transactivation. Furthermore, acute β-AR stimulation in vivo resulted in increases in PDGFR-associated PI3K and PI3Kα isoform activities but not the activities of other isoforms (PI3Kβ, -δ, -γ) in adult mouse heart. Taken together, these data provide in vitro and in vivo evidence for a novel mechanism of β-AR-mediated transactivation of cardiac PI3Kα via sequential involvement of Gαi/Gβγ, Src, and PDGFR.

scholarly journals p56lck, LFA-1 and PI3K but not SHP-2 interact with GM1- or GM3-enriched microdomains in a CD4–p56lck association-dependent manner

2007 ◽  
Vol 402 (3) ◽  
pp. 471-481 ◽  
Author(s):  
Christiane Barbat ◽  
Maylis Trucy ◽  
Maurizio Sorice ◽  
Tina Garofalo ◽  
Valeria Manganelli ◽  
...  
Keyword(s):  
Tyrosine Phosphatase ◽  
Structural Diversity ◽  
Mutant Form ◽  
Lymphocyte Function ◽  
Dependent Manner ◽  
Down Regulation ◽  
Src Homology 2 ◽  
Src Homology ◽  
Src Homology 2 Domain ◽  
Phosphoinositide 3 Kinase

We previously showed that the association of CD4 and GM3 ganglioside induced by CD4 ligand binding was required for the down-regulation of adhesion and that aggregation of ganglioside-enriched domains was accompanied by transient co-localization of LFA-1 (lymphocyte function-associated antigen-1), PI3K (phosphoinositide 3-kinase) and CD4. We also showed that these proteins co-localized with the GM1 ganglioside that partially co-localized with GM3 in these domains. In the present study, we show that CD4–p56lck association in CD4 signalling is required for the redistribution of p56lck, PI3K and LFA-1 in ganglioside-enriched domains, since ganglioside aggregation and recruitment of these proteins were not observed in a T-cell line (A201) expressing the mutant form of CD4 that does not bind p56lck. In addition, we show that although these proteins associated in different ways with GM1 and GM3, all of the associations were dependent on CD4–p56lck association. Gangliosides could associate with these proteins that differ in affinity binding and could be modified following CD4 signalling. Our results suggest that through these associations, gangliosides transiently sequestrate these proteins and consequently inhibit LFA-1-dependent adhesion. Furthermore, while structural diversity of gangliosides may allow association with distinct proteins, we show that the tyrosine phosphatase SHP-2 (Src homology 2 domain-containing protein tyrosine phosphatase 2), also required for the down-regulation of LFA-1-dependent adhesion, transiently and partially co-localized with PI3K and p56lck in detergent-insoluble membranes without association with GM1 or GM3. We propose that CD4 ligation and binding with p56lck and their interaction with GM3 and/or GM1 gangliosides induce recruitment of distinct proteins important for CD4 signalling to form a multimolecular signalling complex.

scholarly journals Activated STAT5 proteins induce activation of the PI 3-kinase/Akt and Ras/MAPK pathways via the Gab2 scaffolding adapter

2005 ◽  
Vol 390 (1) ◽  
pp. 359-366 ◽  
Author(s):  
Rémy Nyga ◽  
Christian Pecquet ◽  
Noria Harir ◽  
Haihua Gu ◽  
Isabelle Dhennin-Duthille ◽  
...  
Keyword(s):  
Cell Proliferation ◽  
Tyrosine Phosphatase ◽  
Growth Factor Receptor ◽  
Janus Kinase ◽  
Scaffolding Protein ◽  
Regulatory Subunit ◽  
Growth And Survival ◽  
Mapk Pathways ◽  
Mitogen Activated Protein ◽  
Phosphoinositide 3 Kinase

The active forms of STAT5A (signal transducer and activator of transcription 5A) and STAT5B are able to relieve the cytokine dependence of haematopoietic cells and to induce leukaemia in mice. We have demonstrated previously that activation of the PI3K (phosphoinositide 3-kinase) signalling cascade plays a major role in cell growth and survival induced by these proteins. Interaction between STAT5 and p85, the regulatory subunit of the PI3K, has been suggested to be required for this activation. We show in the present study that the scaffolding protein Gab2 [Grb2 (growth-factor-receptor-bound protein 2)-associated binder-2] is an essential component of this interaction. Gab2 is persistently tyrosine-phosphorylated in Ba/F3 cells expressing caSTAT5 (constitutively activated STAT5), independent of JAK2 (Janus kinase 2) activation where it interacts with STAT5, p85 and Grb2, but not with Shp2 [SH2 (Src homology 2)-domain-containing tyrosine phosphatase] proteins. Interaction of STAT5 with Gab2 was also observed in Ba/F3 cells stimulated with interleukin-3 or expressing the oncogenic fusion protein Tel–JAK2. The MAPKs (mitogen-activated protein kinases) ERK1 (extracellular-signal-regulated kinase 1) and ERK2 were constitutively activated in the caSTAT5-expressing cells and were found to be required for caSTAT5-induced cell proliferation. Overexpression of Gab2-3YF, a mutant of Gab2 incapable of binding PI3K, inhibited the proliferation and survival of caSTAT5-expressing cells as well as ERK1/2 and Akt/protein kinase B phosphorylation. Taken together, our results indicate that Gab2 is required for caSTAT5-induced cell proliferation by regulating both the PI3K/Akt and the Ras/MAPK pathways.

scholarly journals Calcium-sensitivity of inositol 1,4,5-trisphosphate metabolism in exocrine cells from the avian salt gland

1992 ◽  
Vol 282 (3) ◽  
pp. 703-710 ◽  
Author(s):  
J P Hildebrandt ◽  
T J Shuttleworth
Keyword(s):  
Substrate Concentration ◽  
Inositol Phosphates ◽  
Receptor Activation ◽  
Dependent Manner ◽  
Salt Glands ◽  
Concentration Dependent Manner ◽  
Intact Cells ◽  
Exocrine Cells ◽  
Tissue Homogenates

The generation of inositol phosphates upon muscarinic-receptor activation was studied in [3H]inositol-loaded exocrine cells from the nasal salt glands of the duck Anas platyrhynchos, and the metabolism of different inositol phosphates in vitro was studied in tissue homogenates, with particular reference to the possible interaction of changes in intracellular [Ca2+] ([Ca2+]i) with the metabolic processes. In intact cells, there was a rapid (within 15 s) generation of Ins(1,4,5)P3 and Ins(1,3,4,5)P4, followed by an accumulation of their breakdown products, Ins(1,3,4)P3 and inositol bis- and monophosphates. Ca(2+)-sensitivity of the Ins(1,4,5)P3 3-kinase was demonstrated in tissue homogenates, with the rate of phosphorylation increasing 2-fold at free Ca2+ concentrations greater than 1 microM. However, addition of calmodulin or the presence of the calmodulin inhibitor W-7 (up to 100 microM) had no effect. 3-Kinase activity increased proportionally with the initial Ins(1,4,5)P3 concentration up to 1 microM, but a 10-fold higher substrate concentration produced only a doubling in the phosphorylation rate. Ins(1,3,4,5)P4 was dephosphorylated to Ins(1,3,4)P3, which accumulated in the homogenate assays as well as in intact cells. Depending on its concentration, Ins(1,3,4)P3 was phosphorylated [in part to Ins(1,3,4,6)P4] or dephosphorylated. To investigate the Ca(2+)-sensitivity of the 3-kinase in intact cells, excess quin2 was used to buffer the receptor-mediated transient changes in [Ca2+]i in [3H]inositol-loaded cells. These experiments revealed that increasing [Ca2+]i from less than 100 to approx. 400 nM (i.e. within the physiological range) has no effect on the partitioning of Ins(1,4,5)P3 metabolism (phosphorylation versus dephosphorylation) and on the accumulation of Ins(1,4,5)P3 and Ins(1,3,4,5)P4. This indicates that activation of the 3-kinase by physiologically relevant Ca2+ concentrations may not play a major role in the generation of Ins(1,3,4,5)P4 signals upon receptor activation in these cells. The latter are mainly achieved by the receptor-mediated increase in Ins(1,4,5)P3 in the cell and its phosphorylation by the 3-kinase in a substrate-concentration-dependent manner.

Determination of the enhancing action of HSP90 on neuronal nitric oxide synthase by EPR spectroscopy

2001 ◽  
Vol 281 (6) ◽  
pp. C1819-C1824 ◽  
Author(s):  
Yao Song ◽  
Jay L. Zweier ◽  
Yong Xia
Keyword(s):  
Nitric Oxide ◽  
Electron Paramagnetic Resonance Spectroscopy ◽  
Heat Shock Protein 90 ◽  
Tryptophan Fluorescence ◽  
Hsp90 Inhibitor ◽  
Resonance Spectroscopy ◽  
No Production ◽  
Dependent Manner ◽  
Intact Cells

Recent studies showed that heat shock protein 90 (HSP90) enhances nitric oxide (NO) synthesis from endothelial and neuronal NO synthase (eNOS and nNOS, respectively). However, these findings were based on indirect NO measurements. Moreover, although our previous studies showed that the action of HSP90 involves increased Ca2+/calmodulin (Ca2+/CaM) binding, quantitative measurements of the effect of HSP90 on CaM binding to nNOS have been lacking. With electron paramagnetic resonance spectroscopy, we directly measured NO signals from purified nNOS. HSP90 augmented NO formation from nNOS in a dose-dependent manner. Tryptophan fluorescence-quenching measurements revealed that HSP90 markedly reduced the K d of CaM to nNOS (0.5 ± 0.1 nM vs. 9.4 ± 1.8 nM in the presence and absence of HSP90, P < 0.01). Ca2+ ionophore triggered strong NO production from nNOS-transfected cells, and this was significantly reduced by the HSP90 inhibitor geldanamycin. Thus these studies provide direct evidence demonstrating that HSP90 enhances nNOS catalytic function in vitro and in intact cells. The effect of HSP90 is mediated by the enhancement of CaM binding to nNOS.

scholarly journals The Receptor for Parathyroid Hormone and Parathyroid Hormone-Related Peptide Is Hydrolyzed and Its Signaling Properties Are Altered by Directly Binding the Calpain Small Subunit

Endocrinology ◽  
2005 ◽  
Vol 146 (5) ◽  
pp. 2336-2344 ◽  
Author(s):  
Masako Shimada ◽  
Matthew J. Mahon ◽  
Peter A. Greer ◽  
Gino V. Segre
Keyword(s):  
Parathyroid Hormone ◽  
Fluorescent Protein ◽  
Yellow Fluorescent Protein ◽  
Small Subunit ◽  
Hek293 Cells ◽  
Calpain Inhibitor ◽  
Dependent Manner ◽  
Intact Cells ◽  
Calcium Dependent

Abstract We show calcium-dependent, direct binding between the N-terminal portion of the PTH/PTHrP receptor (PTH1R) C-terminal intracellular tail and the calpain small subunit. Binding requires, but may not be limited to, amino acids W474, S475, and W477. The wild-type, full-length rat (r) PTH1R, but not rPTH1R with W474A/W477A substitutions, copurifies with the endogenous calpain small subunit in HEK293 cells. Calpain hydrolyzes ΔNt-rPTH1R, a receptor with a 156-amino acid N-terminal deletion, in a calcium-dependent manner in vitro and in intact cells. Most importantly, PTH stimulation increases the cleavage of ΔNt-rPTH1R and rPTH1R-yellow fluorescent protein in HEK293 cells, and of talin in HEK293 cells expressing rPTH1R-yellow fluorescent protein and in ROS17/2.8 osteoblast-like cells that express rPTH1R endogenously. The absence of calpain in Capn4-null embryonic fibroblasts and the lowered calpain activity in MC3T3-E1 osteoblastic cells due to stable expression of the calpain inhibitor, calpastatin, reduce PTH-stimulated cAMP accumulation. The calpain small subunit is the second protein, in addition to the sodium-hydrogen exchanger regulatory factor, and the first enzyme that binds the PTH1R; PTH1R bound to both of these proteins results in altered PTH signaling.

scholarly journals Ligand-dependent and -independent activation of the transcription factor γRF-1 in a cell-free system

1995 ◽  
Vol 310 (2) ◽  
pp. 461-467 ◽  
Author(s):  
C A Feghali ◽  
T M Wright
Keyword(s):  
Transcription Factor ◽  
Tyrosine Phosphatase ◽  
Cell Fractionation ◽  
Dependent Manner ◽  
Sodium Orthovanadate ◽  
Ifn Gamma ◽  
Free System ◽  
Cell Free System ◽  
A Cell

gamma RF-1 is a recently identified transcription factor induced by interferon-gamma (IFN-gamma) which binds to a unique palindromic enhancer, gamma RE-1, in the promoter of the mig gene. This paper describes the ligand-dependent and ligand-independent activation of gamma RF-1 in a cell-free system. gamma RF-1 activity was induced by IFN-gamma in a time-dependent manner from 5 to 60 min in lysates prepared from the human monocytic leukaemia line THP-1 and the human epidermoid carcinoma line A431. The activation of gamma RF-1 in vitro required both ATP and an inhibitor of tyrosine phosphatases (sodium orthovanadate or pervanadate). In the presence of limiting concentrations (micromolar) of ATP, activation was also dependent upon stimulation with IFN-gamma, whereas at millimolar concentrations of ATP, gamma RF-1 was activated by either sodium orthovanadate or pervanadate in the absence of ligand. Based on cell fractionation studies, both membrane and cytosol components were essential for activation of gamma RF-1 in vitro. Consistent with a role for one or more tyrosine kinases in the activation of gamma RF-1, its DNA binding activity was blocked by monoclonal anti-phosphotyrosine antibodies and by the tyrosine kinase inhibitors genistein, lavendustin A and herbimycin A. A comparison with recently described pathways of IFN-mediated transcription factor regulation indicates that the in vitro activation of gamma RF-1 is unique, requiring both membrane and cytosol fractions and inhibition of endogenous tyrosine phosphatase activity.

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