scholarly journals PI3Kβ—A Versatile Transducer for GPCR, RTK, and Small GTPase Signaling

Endocrinology ◽  
2019 ◽  
Vol 160 (3) ◽  
pp. 536-555 ◽  
Author(s):  
Anne R Bresnick ◽  
Jonathan M Backer
Keyword(s):  
G Proteins ◽  
Tyrosine Kinases ◽  
Small Gtpases ◽  
Small Gtpase ◽  
Catalytic Subunits ◽  
Regulatory Subunits ◽  
Phosphoinositide 3 Kinase ◽  
G Protein Coupled

AbstractThe phosphoinositide 3-kinase (PI3K) family includes eight distinct catalytic subunits and seven regulatory subunits. Only two PI3Ks are directly regulated downstream from G protein–coupled receptors (GPCRs): the class I enzymes PI3Kβ and PI3Kγ. Both enzymes produce phosphatidylinositol 3,4,5-trisposphate in vivo and are regulated by both heterotrimeric G proteins and small GTPases from the Ras or Rho families. However, PI3Kβ is also regulated by direct interactions with receptor tyrosine kinases (RTKs) and their tyrosine phosphorylated substrates, and similar to the class II and III PI3Ks, it binds activated Rab5. The unusually complex regulation of PI3Kβ by small and trimeric G proteins and RTKs leads to a rich landscape of signaling responses at the cellular and organismic levels. This review focuses first on the regulation of PI3Kβ activity in vitro and in cells, and then summarizes the biology of PI3Kβ signaling in distinct tissues and in human disease.

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 Evidence for Two CRIB Domains in Phospholipase D2 (PLD2) That the Enzyme Uses to Specifically Bind to the Small GTPase Rac2

2011 ◽  
Vol 286 (18) ◽  
pp. 16308-16320 ◽  
Author(s):  
Hong-Juan Peng ◽  
Karen M. Henkels ◽  
Madhu Mahankali ◽  
Mary C. Dinauer ◽  
Julian Gomez-Cambronero
Keyword(s):  
Specific Binding ◽  
Strong Association ◽  
Living Cells ◽  
Small Gtpases ◽  
Small Gtpase ◽  
Deletion Mutants ◽  
Ph Domain ◽  
Phospholipase D2

Phospholipase D (PLD) and small GTPases are vital to cell signaling. We report that the Rac2 and the PLD2 isoforms exist in the cell as a lipase-GTPase complex that enables the two proteins to elicit their respective functionalities. A strong association between the two molecules was demonstrated by co-immunoprecipitation and was confirmed in living cells by FRET with CFP-Rac2 and YFP-PLD2 fluorescent chimeras. We have identified the amino acids in PLD2 that define a specific binding site to Rac2. This site is composed of two CRIB (Cdc42-and Rac-interactive binding) motifs that we have named “CRIB-1” and “CRIB-2” in and around the PH domain in PLD2. Deletion mutants PLD2-ΔCRIB-1/2 negate co-immunoprecipitation with Rac2 and diminish the FRET signal in living cells. The PLD2-Rac2 association was further confirmed in vitro using affinity-purified recombinant proteins. Binding was saturable with an apparent Kd of 3 nm and was diminished with PLD2-ΔCRIB mutants. Furthermore, PLD2 bound more efficiently to Rac2-GTP than to Rac2-GDP or to a GDP-constitutive Rac2-N17 mutant. Increasing concentrations of recombinant Rac2 in vitro and in vivo during cell adhesion inhibit PLD2. Conversely, Rac2 activity is increased in the presence of PLD2-WT but not in PLD2-ΔCRIB. We propose that in activated cells PLD2 affects Rac2 in an initial positive feedback, but as Rac2-GTP accumulates in the cell, this constitutes a “termination signal” leading to PLD2 inactivation.

scholarly journals Phosphoinositide 3-kinase activates Rac by entering in a complex with Eps8, Abi1, and Sos-1

2003 ◽  
Vol 160 (1) ◽  
pp. 17-23 ◽  
Author(s):  
Metello Innocenti ◽  
Emanuela Frittoli ◽  
Isabella Ponzanelli ◽  
John R. Falck ◽  
Saskia M. Brachmann ◽  
...  
Keyword(s):  
Tyrosine Kinases ◽  
Physical Interaction ◽  
Nucleotide Exchange ◽  
Downstream Target ◽  
Signaling Complex ◽  
Guanine Nucleotide Exchange ◽  
Nucleotide Exchange Factor ◽  
Phosphoinositide 3 Kinase

Class I phosphoinositide 3-kinases (PI3Ks) are implicated in many cellular responses controlled by receptor tyrosine kinases (RTKs), including actin cytoskeletal remodeling. Within this pathway, Rac is a key downstream target/effector of PI3K. However, how the signal is routed from PI3K to Rac is unclear. One possible candidate for this function is the Rac-activating complex Eps8–Abi1–Sos-1, which possesses Rac-specific guanine nucleotide exchange factor (GEF) activity. Here, we show that Abi1 (also known as E3b1) recruits PI3K, via p85, into a multimolecular signaling complex that includes Eps8 and Sos-1. The recruitment of p85 to the Eps8–Abi1–Sos-1 complex and phosphatidylinositol 3, 4, 5 phosphate (PIP3), the catalytic product of PI3K, concur to unmask its Rac-GEF activity in vitro. Moreover, they are indispensable for the activation of Rac and Rac-dependent actin remodeling in vivo. On growth factor stimulation, endogenous p85 and Abi1 consistently colocalize into membrane ruffles, and cells lacking p85 fail to support Abi1-dependent Rac activation. Our results define a mechanism whereby propagation of signals, originating from RTKs or Ras and leading to actin reorganization, is controlled by direct physical interaction between PI3K and a Rac-specific GEF complex.

BCR activation of PI3K is Vav-independent in murine B cells

2004 ◽  
Vol 32 (5) ◽  
pp. 781-784 ◽  
Author(s):  
E. Vigorito ◽  
E. Clayton ◽  
M. Turner
Keyword(s):  
B Cells ◽  
Tyrosine Kinases ◽  
Adaptor Proteins ◽  
Pleckstrin Homology Domain ◽  
Pi3k Activation ◽  
Lipid Product ◽  
Lipid Kinases ◽  
Phosphoinositide 3 Kinase

BCR (B-cell antigen receptor)-induced Ca2+ signalling is initiated by activation of tyrosine kinases, which in concert with adaptor proteins and lipid kinases regulate PLC (phospholipase C) γ2 activation. Vav and PI3K (phosphoinositide 3-kinase) are required for optimal Ca2+ responses, although it has not been established, in primary B-cells, if both proteins are components of the same pathway. In vitro evidence suggests that binding of the PI3K lipid product PIP3 to Vav pleckstrin homology domain contributes to Vav activation. However, pharmacological inhibition of PI3K by wortmannin or deletion of the p110δ catalytic subunit has no effect on Vav activation in response to BCR engagement, suggesting that this mechanism does not operate in vivo. We also show that PI3K recruitment to phosphorylated-tyrosine-containing complexes is Vav-independent. Taken together with our previous observation that protein kinase B phosphorylation is normal in Vav-deficient B-cells, we suggest that PI3K activation is Vav-independent in response to strong signals delivered by multivalent cross-linking.

scholarly journals Structure of the phosphoinositide 3-kinase p110γ-p101 complex reveals molecular mechanism of GPCR activation

2021 ◽  
Author(s):  
Manoj K Rathinaswamy ◽  
Udit Dalwadi ◽  
Kaelin D Fleming ◽  
Carson Adams ◽  
Jordan TB Stariha ◽  
...  
Keyword(s):  
Cell Function ◽  
Immune Cell ◽  
Inflammatory Diseases ◽  
Regulatory Subunit ◽  
Regulatory Subunits ◽  
Gpcr Activation ◽  
Oncogenic Mutations ◽  
Phosphoinositide 3 Kinase ◽  
G Protein Coupled

The class IB phosphoinositide 3-kinase (PI3K), PI3Kγ, is a master regulator of immune cell function, and a promising drug target for both cancer and inflammatory diseases. Critical to PI3Kγ function is the association of the p110γ catalytic subunit to either a p101 or p84 regulatory subunit, which mediates activation by G-protein coupled receptors (GPCRs). Here, we report the cryo-EM structure of a hetero-dimeric PI3Kγ complex, p110γ-p101. This structure reveals a unique assembly of catalytic and regulatory subunits that is distinct from other class I PI3K complexes. p101 mediates activation through its Gβγ binding domain, recruiting the hetero-dimer to the membrane and allowing for engagement of a secondary Gβγ binding site in p110γ. Multiple oncogenic mutations mapped to these novel interfaces and enhanced Gβγ activation. A nanobody that specifically binds to the p101-Gβγ interface blocks activation providing a novel tool to study and target p110γ-p101-specific signaling events in vivo.

scholarly journals Mechanisms through which Sos-1 coordinates the activation of Ras and Rac

2002 ◽  
Vol 156 (1) ◽  
pp. 125-136 ◽  
Author(s):  
Metello Innocenti ◽  
Pierluigi Tenca ◽  
Emanuela Frittoli ◽  
Mario Faretta ◽  
Arianna Tocchetti ◽  
...  
Keyword(s):  
Tyrosine Kinases ◽  
Adaptor Protein ◽  
Small Gtpases ◽  
Nucleotide Exchange ◽  
Guanine Nucleotide Exchange ◽  
Nucleotide Exchange Factor ◽  
Son Of Sevenless ◽  
Sequential Activation

Signaling from receptor tyrosine kinases (RTKs)* requires the sequential activation of the small GTPases Ras and Rac. Son of sevenless (Sos-1), a bifunctional guanine nucleotide exchange factor (GEF), activates Ras in vivo and displays Rac-GEF activity in vitro, when engaged in a tricomplex with Eps8 and E3b1–Abi-1, a RTK substrate and an adaptor protein, respectively. A mechanistic understanding of how Sos-1 coordinates Ras and Rac activity is, however, still missing. Here, we demonstrate that (a) Sos-1, E3b1, and Eps8 assemble into a tricomplex in vivo under physiological conditions; (b) Grb2 and E3b1 bind through their SH3 domains to the same binding site on Sos-1, thus determining the formation of either a Sos-1–Grb2 (S/G) or a Sos-1–E3b1–Eps8 (S/E/E8) complex, endowed with Ras- and Rac-specific GEF activities, respectively; (c) the Sos-1–Grb2 complex is disrupted upon RTKs activation, whereas the S/E/E8 complex is not; and (d) in keeping with the previous result, the activation of Ras by growth factors is short-lived, whereas the activation of Rac is sustained. Thus, the involvement of Sos-1 at two distinct and differentially regulated steps of the signaling cascade allows for coordinated activation of Ras and Rac and different duration of their signaling within the cell.

scholarly journals Lymphocyte cell motility: the twisting, turning tale of phosphoinositide 3-kinase

2007 ◽  
Vol 35 (5) ◽  
pp. 1109-1113 ◽  
Author(s):  
J.S. Oak ◽  
M.P. Matheu ◽  
I. Parker ◽  
M.D. Cahalan ◽  
D.A. Fruman
Keyword(s):  
Cell Motility ◽  
Cell Types ◽  
Lymphocyte Migration ◽  
Regulatory Subunits ◽  
Lipid Kinases ◽  
Cell Type Specific ◽  
Phosphoinositide 3 Kinase ◽  
Lymphocyte Motility

The PI3K (phosphoinositide 3-kinase) family of lipid kinases regulate cell motility in diverse organisms and cell types. In mammals, the main PI3K enzyme activated by chemokine receptor signalling is the class IB isoform, p110γ. Studies of p110γ-knockout mice have shown an essential function for this isoform in chemotaxis of neutrophils and macrophages both in vitro and in vivo. However, the roles of p110γ and other PI3K enzymes and regulatory subunits in lymphocyte motility have been more difficult to discern. Recent studies of adoptively transferred, fluorescently labelled lymphocytes have revealed complex and unexpected functions for PI3K in lymphocyte migration in vivo. In this review we highlight cell-type-specific roles for PI3K catalytic and regulatory subunits in the homing and basal motility of lymphocytes in the intact lymph node.

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.

Simvastatin induces the apoptosis of normal vascular smooth muscle through the disruption of actin integrity via the impairment of RhoA/ Rac-1 activity

2016 ◽  
Vol 116 (09) ◽  
pp. 496-505 ◽  
Author(s):  
Seojin Kang ◽  
Keunyoung Kim ◽  
Ji-Yoon Noh ◽  
Yeryeon Jung ◽  
Ok-Nam Bae ◽  
...  
Keyword(s):  
Smooth Muscle ◽  
Vascular Smooth Muscle ◽  
Time Course ◽  
Heart Diseases ◽  
Small Gtpases ◽  
Small Gtpase ◽  
Lipid Lowering ◽  
Contractile Dysfunction

SummaryStatins, lipid-lowering agents for the prevention of atherosclerosis and fatal coronary heart diseases, have pleiotropic modalities on the function and physiology of vascular smooth muscle that include anti-contractile and pro-apoptotic effects. These effects were suggested to stem from the inhibition of small GTPase Rho A, but they are largely regarded as distinct and unrelated. Recently, we discovered that simvastatin causes both contractile dysfunction and apoptosis of vascular smooth muscle cells (VSMCs), reflecting that they may be closely related, yet their connecting link remains unexplained. Here, we elaborated the mechanism underlying simvastatin-induced apoptosis of normal VSMCs in connection with contractile dysfunction. Repeated oral administration of simvastatin to rats in vivo resulted in contractile dysfunction and apoptosis of vascular smooth muscle, of which pattern was well reproduced in rat VSMCs in vitro. Of note, contractile dysfunction and apoptosis occurred in concerted manners both in vivo and in vitro in the aspects of time course and dose of exposure. In rat VSMCs, simvastatin impaired the activation of small GTPases, RhoA along with Rac-1, which resulted in the disruption of actin integrity, a pivotal factor both for the generation of contractile force and survival of VSMCs. In line with the disruption of actin integrity, Bmf, a pro-apoptotic factor bound to intact actin, dissociated and translocated into mitochondria, which corresponded well with the dissipation of mitochondrial membrane potential, caspase-3 activation and ultimately apoptosis. These events were all rescued by an actin stabilisation agent, jasplakinolide as well as geranylgeraniol, indicating that damages of the actin integrity from disrupted activation of RhoA/ Rac-1 lies at the center of simvastatin-induced contractile dysfunction and apoptosis in vascular smooth muscle.Supplementary Material to this article is available online at www.thrombosis-online.com.

Zoledronate Exerts Direct Antiproliferative and Apoptotic Effects on Human Myeloma Cells In Vitro and In Vivo.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 3387-3387
Author(s):  
Andreas Guenther ◽  
Sharon Gordon ◽  
Frank Bakker ◽  
Renate Burger ◽  
Markus Tiemann ◽  
...  
Keyword(s):  
Molecular Mechanisms ◽  
Specific Effect ◽  
Small Gtpases ◽  
Small Gtpase ◽  
Control Group ◽  
Dependent Manner ◽  
Myeloma Cells ◽  
Osteolytic Bone Disease

Abstract Zoledronate (ZOL) is the most potent nitrogen-containing bisphosphonate and is effective at preventing osteolytic bone disease in patients with multiple myeloma (MM) and solid tumors. ZOL inhibits the enzyme farnesylpyrophosphate synthase and thus blocks the prenylation of small GTPases. In vitro studies have demonstrated that ZOL can also directly affect the growth and viability of myeloma cells, however, the molecular mechanisms underlying this activity have not been fully elucidated. The goal of our study was to investigate direct antimyeloma effects of ZOL in vitro and in vivo. In five myeloma cell lines (RPMI8226, L363, U266, JK-6L, and the IL-6 dependent INA-6), growth was inhibited and apoptosis induced by ZOL in a dose-dependent manner (IC50’s between 30 μM and 285 μM). Similar results were obtained in the presence of bone marrow stromal cells, IL-6 (20 ng/mL), IGF-1 (200 ng/mL), or a combination of both cytokines. The potential antitumor effect of ZOL on myeloma cells in vivo was studied in the INA-6 SCID model, in which mice are injected intraperitoneally with INA-6 cells and subsequently develop plasmacytomas. Mice treated with ZOL had reduced tumor burden and a significant survival benefit compared to the control group (p=0.002). Histological examination of plasmacytomas explanted 72 hours after a single injection of 8 μg ZOL revealed extensive apoptotic/necrotic areas while no such areas were found in tumors of untreated animals. Induction of apoptosis was confirmed by Western blot analysis of tumor lysates, which revealed increased levels of cleaved poly (ADP-ribose) polymerase (PARP) in tumors of ZOL treated vs. untreated animals. This correlated with an accumulation of the unprenylated form of the small GTPase Rap1A, which was virtually absent in tumors of untreated mice. Our findings demonstrate a direct and specific effect of ZOL in plasmacytomas in vitro and in vivo and point to a therapeutic potential in MM beyond the prevention of osteolytic lesions.

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