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.

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 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.

scholarly journals RGS3 interacts with 14-3-3 via the N-terminal region distinct from the RGS (regulator of G-protein signalling) domain

2002 ◽  
Vol 365 (3) ◽  
pp. 677-684 ◽  
Author(s):  
Jiaxin NIU ◽  
Astrid SCHESCHONKA ◽  
Kirk M. DRUEY ◽  
Amanda DAVIS ◽  
Eleanor REED ◽  
...  
Keyword(s):  
Binding Site ◽  
G Protein ◽  
G Proteins ◽  
Rgs Proteins ◽  
Heterotrimeric G Proteins ◽  
G Protein Signalling ◽  
Vitro Binding ◽  
Rgs Domain ◽  
Hybrid Screening

RGS3 belongs to a family of the regulators of G-protein signalling (RGS), which bind and inhibit the Gα subunits of heterotrimeric G-proteins via a homologous RGS domain. Increasing evidence suggests that RGS proteins can also interact with targets other than G-proteins. Employing yeast two-hybrid screening of a cDNA library, we identified an interaction between RGS3 and the phosphoserine-binding protein 14-3-3. This interaction was confirmed by in vitro binding and co-immunoprecipitation experiments. RGS3-deletion analysis revealed the presence of a single 14-3-3-binding site located outside of the RGS domain. Ser264 was then identified as the 14-3-3-binding site of RGS3. The S264A mutation resulted in the loss of RGS3 binding to 14-3-3, without affecting its ability to bind Gαq. Signalling studies showed that the S264A mutant was more potent than the wild-type RGS3 in inhibition of G-protein-mediated signalling. Binding experiments revealed that RGS3 exists in two separate pools, either 14-3-3-bound or G-protein-bound, and that the 14-3-3-bound RGS3 is unable to interact with G-proteins. These data are consistent with the model wherein 14-3-3 serves as a scavenger of RGS3, regulating the amounts of RGS3 available for binding G-proteins. This study describes a new level in the regulation of G-protein signalling, in which the inhibitors of G-proteins, RGS proteins, can themselves be regulated by phosphorylation and binding 14-3-3.

Phosphoinositide 3-kinase is activated by MUC1 but not responsible for MUC1-induced suppression of Toll-like receptor 5 signaling

2007 ◽  
Vol 293 (3) ◽  
pp. L686-L692 ◽  
Author(s):  
Kosuke Kato ◽  
Wenju Lu ◽  
Hirofumi Kai ◽  
K. Chul Kim
Keyword(s):  
Cross Talk ◽  
Negative Regulator ◽  
Regulatory Subunit ◽  
Binding Motif ◽  
Toll Like Receptor ◽  
Akt Phosphorylation ◽  
Phosphoinositide 3 Kinase ◽  
Toll Like Receptor 5

MUC1 is a membrane-tethered mucin-like glycoprotein expressed on the surface of various mucosal epithelial cells as well as hematopoietic cells. Recently, we showed that MUC1 suppresses flagellin-induced Toll-like receptor (TLR) 5 signaling both in vivo and in vitro through cross talk with TLR5. In this study, we determined whether phosphoinositide 3-kinase (PI3K), a negative regulator of TLR5 signaling, is involved in the cross talk between MUC1 and TLR5 using various genetically modified epithelial cell lines. Our results showed 1) activation of MUC1 induced recruitment of the PI3K regulatory subunit p85 to the MUC1 cytoplasmic tail (CT) as well as Akt phosphorylation, 2) MUC1-induced Akt phosphorylation required the presence of Tyr20 within the PI3K binding motif of the MUC1 CT, and 3) mutation of Tyr20 or pharmacological inhibition of PI3K activation failed to block MUC1-induced suppression of TLR5 signaling. We conclude that whereas PI3K is downstream of MUC1 activation and negatively regulates TLR5 signaling, it is not responsible for MUC1-induced suppression of TLR5 signaling.

scholarly journals Identification of Tetratricopeptide Repeat 1 as an Adaptor Protein That Interacts with Heterotrimeric G Proteins and the Small GTPase Ras

2003 ◽  
Vol 23 (11) ◽  
pp. 3847-3858 ◽  
Author(s):  
Caroline Marty ◽  
Darren D. Browning ◽  
Richard D. Ye
Keyword(s):  
G Proteins ◽  
Mammalian Cells ◽  
Active Form ◽  
Adaptor Protein ◽  
Small Gtpases ◽  
Small Gtpase ◽  
Tetratricopeptide Repeat ◽  
Heterotrimeric G Proteins ◽  
Interacting Protein

ABSTRACT The biological functions of heterotrimeric G proteins and small GTPases are modulated by both extracellular stimuli and intracellular regulatory proteins. Using Saccharomyces cerevisiae two-hybrid screening, we identified tetratricopeptide repeat 1 (TPR1), a 292-amino-acid protein with three TPR motifs, as a Gα16-binding protein. The interaction was confirmed both in vitro and in transfected mammalian cells, where TPR1 also binds to several other Gα proteins. TPR1 was found to interact with Ha-Ras preferentially in its active form. Overexpression of TPR1 promotes accumulation of active Ras. TPR1 was found to compete with the Ras-binding domain (RBD) of Raf-1 for binding to the active Ras, suggesting that it may also compete with Ras GTPase-activating protein, thus contributing to the accumulation of GTP-bound Ras. Expression of Gα16 strongly enhances the interaction between TPR1 and Ras. Removal of the TPR1 N-terminal 112 residues abolishes potentiation by Gα16 while maintaining the interaction with Gα16 and the ability to discriminate active Ras from wild-type Ras. We have also observed that LGN, a Gαi-interacting protein with seven TPR motifs, binds Ha-Ras. Thus, TPR1 is a novel adaptor protein for Ras and selected Gα proteins that may be involved in protein-protein interaction relating to G-protein signaling.

scholarly journals Complementary biosensors reveal different G-protein signaling modes triggered by GPCRs and non-receptor activators

eLife ◽  
2021 ◽  
Vol 10 ◽  
Author(s):  
Mikel Garcia-Marcos
Keyword(s):  
G Protein ◽  
G Proteins ◽  
Protein Signaling ◽  
Heterotrimeric G Proteins ◽  
Nucleotide Exchange ◽  
Guanine Nucleotide Exchange ◽  
Cytoplasmic Proteins ◽  
Nucleotide Exchange Factor ◽  
In Cells

It has become evident that activation of heterotrimeric G-proteins by cytoplasmic proteins that are not G-protein-coupled receptors (GPCRs) plays a role in physiology and disease. Despite sharing the same biochemical guanine nucleotide exchange factor (GEF) activity as GPCRs in vitro, the mechanisms by which these cytoplasmic proteins trigger G-protein-dependent signaling in cells have not been elucidated. Heterotrimeric G-proteins can give rise to two active signaling species, Gα-GTP and dissociated Gβγ, with different downstream effectors, but how non-receptor GEFs affect the levels of these two species in cells is not known. Here, a systematic comparison of GPCRs and three unrelated non-receptor proteins with GEF activity in vitro (GIV/Girdin, AGS1/Dexras1, and Ric-8A) revealed high divergence in their contribution to generating Gα-GTP and free Gβγ in cells directly measured with live-cell biosensors. These findings demonstrate fundamental differences in how receptor and non-receptor G-protein activators promote signaling in cells despite sharing similar biochemical activities in vitro.

scholarly journals Latrophilins are essential for endothelial junctional fluid shear stress mechanotransduction

2020 ◽  
Author(s):  
Keiichiro Tanaka ◽  
Andrew Prendergast ◽  
Jared Hintzen ◽  
Abhishek Kumar ◽  
Minhwan Chung ◽  
...  
Keyword(s):  
Shear Stress ◽  
G Proteins ◽  
Fluid Shear Stress ◽  
Affinity Purification ◽  
Cell Junctions ◽  
Heterotrimeric G Proteins ◽  
Fluid Shear ◽  
Vascular Morphogenesis ◽  
Flow Activation

AbstractEndothelial cell (EC) responses to fluid shear stress (FSS) are crucial for vascular development, adult physiology and disease. PECAM1 is an important transducer but earlier events remain poorly understood. We therefore investigated heterotrimeric G proteins in FSS sensing. Knockdown (KD) in ECs of single Gα proteins had little effect but combined depletion of Gαi and Gαq/11 blocked all known PECAM1-dependent responses. Re-expression of Gαi2 and Gαq but not Gαi1 and Gαi3 rescued these effects. Sequence alignment and mutational studies identified that K307 in Gαi2 and Gq/11 (Q306 in Gαi1/3), determines participation in flow signaling. We developed pull-down assays for measuring Gα activation and found that this residue, localized to the GPCR interface, determines activation by FSS. We developed a protocol for affinity purification of GPCRs on activated Gα’s, which identified latrophilins (ADGRLs) as specific upstream interactors for Gαi2 and Gq/11. Depletion of latrophilin-2 blocked EC activation of Gαi2 and Gαq, downstream events in vitro, and flow-dependent vascular morphogenesis in zebrafish embryos. Surprisingly, latrophilin-2 depletion also blocked flow activation of two additional pathways activated at cell-cell junctions, Smad1/5 and Notch1, independently of Gα proteins. Latrophilins are thus central mediators of junctional shear stress mechanotransduction via Gα protein-dependent and -independent mechanisms.

Characterisation of the paxillin-binding site and the C-terminal focal adhesion targeting sequence in vinculin

1994 ◽  
Vol 107 (2) ◽  
pp. 709-717 ◽  
Author(s):  
C.K. Wood ◽  
C.E. Turner ◽  
P. Jackson ◽  
D.R. Critchley
Keyword(s):  
Amino Acids ◽  
Fusion Protein ◽  
Binding Site ◽  
Focal Adhesion ◽  
Focal Adhesions ◽  
Cytoskeletal Proteins ◽  
Terminal Region ◽  
Nih3t3 Cells ◽  
Cell Biol

Paxillin and vinculin are cytoskeletal proteins that colocalise to focal adhesions, specialised regions of the cell involved in attachment to the extracellular matrix. These two molecules form part of a complex of proteins that link the actin network to the plasma membrane. Paxillin has been shown to bind directly in vitro to the C-terminal region of vinculin (Turner et al. (1990). J. Cell Biol. 111, 1059–1068), which also contains a focal adhesion targeting sequence (Bendori et al. (1989). J. Cell Biol. 108, 2383–2393). In the present study, we have used a series of vinculin deletion mutants to map more precisely the sites in vinculin responsible for paxillin binding and focal adhesion localisation. A glutathione-S-transferase fusion protein spanning vinculin residues 881–1000 was sufficient to support 125I-paxillin binding in a gel-blot assay while no detectable binding was observed to a fusion protein spanning residues 881–978. Transfection experiments using cDNAs encoding chick vinculin residues 398–1066 and 398–1028 demonstrated that amino acids C-terminal to residue 1028 were not necessary for targeting to focal adhesions. In contrast, a vinculin polypeptide expressed from a cDNA encoding residues 398–1000 failed to localise to focal adhesions in stably transfected NIH3T3 cells. We have therefore identified a region of 50 amino acids (residues 979–1028) within the C-terminal region of vinculin that contains both the paxillin-binding site and the focal adhesion targeting sequence. This region is highly conserved in human and chicken vinculin and is likely to be important in regulation of the assembly of focal adhesions.

scholarly journals Glucocorticoid receptor-glucocorticoid response element binding stimulates nucleosome disruption by the SWI/SNF complex.

1997 ◽  
Vol 17 (2) ◽  
pp. 895-905 ◽  
Author(s):  
A K Ostlund Farrants ◽  
P Blomquist ◽  
H Kwon ◽  
O Wrange
Keyword(s):  
Glucocorticoid Receptor ◽  
Binding Site ◽  
Glucocorticoid Response Element ◽  
Response Element ◽  
Nucleosome Remodeling ◽  
Synthetic Dna ◽  
Glucocorticoid Response ◽  
Bending Sequence ◽  
Stimulation Of

The organization of DNA in chromatin is involved in repressing basal transcription of a number of inducible genes. Biochemically defined multiprotein complexes such as SWI/SNF (J. Côté, J. Quinn, J. L. Workman, and C. L. Peterson, Science 265:53-60, 1994) and nucleosome remodeling factor (T. Tsukiyama and C. Wu, Cell 83:1011-1020, 1995) disrupt nucleosomes in vitro and are thus candidates for complexes which cause chromatin decondensation during gene induction. In this study we show that the glucocorticoid receptor (GR), a hormone-inducible transcription factor, stimulates the nucleosome-disrupting activity of the SWI/SNF complex partially purified either from HeLa cells or from rat liver tissue. This GR-mediated stimulation of SWI/SNF nucleosome disruption depended on the presence of a glucocorticoid response element. The in vitro-reconstituted nucleosome probes used in these experiments harbored 95 bp of synthetic DNA-bending sequence in order to rotationally position the DNA. The GR-dependent stimulation of SWI/SNF-mediated nucleosome disruption, as evaluated by DNase I footprinting, was 2.7- to 3.8-fold for the human SWI/SNF complex and 2.5- to 3.2-fold for the rat SWI/SNF complex. When nuclear factor 1 (NF1) was used instead of GR, there was no stimulation of SWI/SNF activity in the presence of a mononucleosome containing an NF1 binding site. On the other hand, the SWI/SNF nucleosome disruption activity increased the access of NF1 for its nucleosomal binding site. No such effect was seen on binding of GR to its response element. Our results suggest that GR, but not NF1, is able to target the nucleosome-disrupting activity of the SWI/SNF complex.

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