Different inhibition of Gβγ-stimulated class IB phosphoinositide 3-kinase (PI3K) variants by a monoclonal antibody. Specific function of p101 as a Gβγ-dependent regulator of PI3Kγ enzymatic activity

Shymanets, Aliaksei; Prajwal,; Vadas, Oscar; Czupalla, Cornelia; LoPiccolo, Jaclyn; Brenowitz, Michael; Ghigo, Alessandra; Hirsch, Emilio; Krause, Eberhard; Wetzker, Reinhard; Williams, Roger L.; Harteneck, Christian; Nürnberg, Bernd

doi:10.1042/bj20150099

Cited by 18 publications

(26 citation statements)

References 67 publications

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“…The cleaved material was eluted, brought to 10% glycerol, and stored at −80°C. Purification of heterologously expressed Gβ 1 γ 2 in Sf9 cells following infection with recombinant baculovirus has been recently described [18]. Proteins were stored at −80°C until use.…”

Section: Methodsmentioning

confidence: 99%

Rac1-stimulated macropinocytosis enhances Gβγ activation of PI3Kβ

Erami

Khalil

Salloum

et al. 2017

Biochemical Journal

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Phosphoinositide 3-kinases (PI 3-kinases) are regulated by a diverse range of upstream activators, including receptor tyrosine kinases (RTKs), G-protein-coupled receptors (GPCRs), and small GTPases from the Ras, Rho and Rab families. For the Class IA PI 3-kinase PI3Kβ, two mechanisms for GPCR-mediated regulation have been described: direct binding of Gβγ subunits to the C2-helical domain linker of p110β, and Dock180/Elmo1-mediated activation of Rac1, which binds to the Ras-Binding Domain of p110β. We now show that the integration of these dual pathways is unexpectedly complex. In breast cancer cells, expression of constitutively activated Rac1 (CA-Rac1) along with either GPCR stimulation or expression of Gβγ led to an additive PI3Kβ-dependent activation of Akt. Whereas CA-Rac1-mediated activation of Akt was blocked in cells expressing a mutated PI3Kβ that cannot bind Gβγ, Gβγ and GPCR-mediated activation of Akt was preserved when Rac1 binding to PI3Kβ was blocked. Surprisingly, PI3Kβ-dependent CA-Rac1 signaling to Akt was still seen in cells expressing a mutant p110β that cannot bind Rac1. Instead of directly binding to PI3Kβ, CA-Rac1 acts by enhancing Gβγ coupling to PI3Kβ, as CA-Rac1-mediated Akt activation was blocked by inhibitors of Gβγ. Cells expressing CA-Rac1 exhibited a robust induction of macropinocytosis, and inhibitors of macropinocytosis blocked the activation of Akt by CA-Rac1 or lysophosphatidic acid. Our data suggest that Rac1 can potentiate the activation of PI3Kβ by GPCRs through an indirect mechanism, by driving the formation of macropinosomes that serve as signaling platforms for Gβγ coupling to PI3Kβ.

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Section: Methodsmentioning

confidence: 99%

Rac1-stimulated macropinocytosis enhances Gβγ activation of PI3Kβ

Erami

Khalil

Salloum

et al. 2017

Biochemical Journal

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“…Although class 1A PI3Ks (PI3Kα, -β, and -δ) are downstream of tyrosine kinase receptors, the class 1B isoform PI3Kγ plays a critical role in coupling signals downstream of activated GPCRs (39,45). Classically, Gβγ subunits recruit PI3Kγ to the GPCR complex, wherein its kinase lipid function generates membrane PtdIns 3,4,5 P 3 (PIP 3 ) from phosphatidylinositol 4,5-bis-phosphate (PtdIns 4,5 P 2 ;PIP 2 ), resulting in activation of downstream signaling, including Akt (46,47). Our findings demonstrate a unique mechanism of PI3Kγ recruitment to the plasma membrane through formation of an intracellular complex with TRPV4.…”

Section: Discussionmentioning

confidence: 99%

Translocation of TRPV4-PI3Kγ complexes to the plasma membrane drives myofibroblast transdifferentiation

et al. 2019

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Myofibroblasts are key contributors to pathological fibrotic conditions of several major organs. The transdifferentiation of fibroblasts into myofibroblasts requires both a mechanical signal and transforming growth factor–β (TGF-β) signaling. The cation channel transient receptor potential vanilloid 4 (TRPV4) is a critical mediator of myofibroblast transdifferentiation and in vivo fibrosis through its mechanosensitivity to extracellular matrix stiffness. Here, we showed that TRPV4 promoted the transdifferentiation of human and mouse lung fibroblasts through its interaction with phosphoinositide 3-kinase γ (PI3Kγ), forming nanomolar-affinity, intracellular TRPV4-PI3Kγ complexes. TGF-β induced the recruitment of TRPV4-PI3Kγ complexes to the plasma membrane and increased the activities of both TRPV4 and PI3Kγ. Using gain- and loss-of-function approaches, we showed that both TRPV4 and PI3Kγ were required for myofibroblast transdifferentiation as assessed by the increased production of α-smooth muscle actin and its incorporation into stress fibers, cytoskeletal changes, collagen-1 production, and contractile force. Expression of various mutant forms of the PI3Kγ catalytic subunit (p110γ) in cells lacking PI3Kγ revealed that only the noncatalytic, amino-terminal domain of p110γ was necessary and sufficient for TGF-β–induced TRPV4 plasma membrane recruitment and myofibroblast transdifferentiation. These data suggest that TGF-β stimulates a noncanonical scaffolding action of PI3Kγ, which recruits TRPV4-PI3Kγ complexes to the plasma membrane, thereby increasing myofibroblast transdifferentiation. Given that both TRPV4 and PI3Kγ have pleiotropic actions, targeting the interaction between them could provide a specific therapeutic approach for inhibiting myofibroblast transdifferentiation.

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“…The lysates were clarified, and GST-Rac1 was purified as described above using wash buffer (50 mM Tris-HCl, pH 7.6, 50 mM NaCl, 5 mM MgCl 2 , 1 mM DTT) and elution buffer (50 mM Tris-HCl, pH 7.6, 150 mM NaCl, 1 mM DTT, 25 mM reduced L-GSH (Sigma-Aldrich)). Purification of lipidated G␤ 1 ␥ 2 protein was performed as described previously (33).…”

Section: Protein Expression and Purification For Binding Assaysmentioning

confidence: 99%

“…Lipid vesicles (37 l) were incubated on ice for 30 min with 1.5 M purified G␤ 1 ␥ 2 protein or an equal volume of G␤ 1 ␥ 2 buffer (33). The eluted p110␤-StrepII(ϫ3)/p85␣-FLAG(ϫ3) heterodimer (9 l) was incubated with the lipid vesicles with or…”

Section: Lipid Kinase Assaymentioning

confidence: 99%

A single discrete Rab5-binding site in phosphoinositide 3-kinase β is required for tumor cell invasion

Heitz

Hamelin

Hoffmann

et al. 2019

Journal of Biological Chemistry

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Phosphoinositide 3-kinase ␤ (PI3K␤) is regulated by receptor tyrosine kinases (RTKs), G protein-coupled receptors (GPCRs), and small GTPases such as Rac1 and Rab5. Our lab previously identified two residues (Gln 596 and Ile 597) in the helical domain of the catalytic subunit (p110␤) of PI3K␤ whose mutation disrupts binding to Rab5. To better define the Rab5-p110␤ interface, we performed alanine-scanning mutagenesis and analyzed Rab5 binding with an in vitro pulldown assay with GST-Rab5 GTP. Of the 35 p110␤ helical domain mutants assayed, 11 disrupted binding to Rab5 without affecting Rac1 binding, basal lipid kinase activity, or G␤␥-stimulated kinase activity. These mutants defined the Rab5-binding interface within p110␤ as consisting of two perpendicular ␣-helices in the helical domain that are adjacent to the initially identified Gln 596 and Ile 597 residues. Analysis of the Rab5-PI3K␤ interaction by hydrogendeuterium exchange MS identified p110␤ peptides that overlap with these helices; no interactions were detected between Rab5 and other regions of p110␤ or p85␣. Similarly, the binding of Rab5 to isolated p85␣ could not be detected, and mutations in the Ras-binding domain (RBD) of p110␤ had no effect on Rab5 binding. Whereas soluble Rab5 did not affect PI3K␤ activity in vitro, the interaction of these two proteins was critical for chemotaxis, invasion, and gelatin degradation by breast cancer cells. Our results define a single, discrete Rab5-binding site in the p110␤ helical domain, which may be useful for generating inhibitors to better define the physiological role of Rab5-PI3K␤ coupling in vivo. Class I phosphoinositide 3-kinases (PI3Ks) 8 are lipid kinases that regulate cell motility, growth, and survival. Class IA PI3Ks are obligate heterodimers that contain a regulatory subunit (p85␣/␤, p55␣/␥, or p50␣) and a catalytic (p110␣,-␤, or-␦) subunit (1). The full-length regulatory p85 subunit consists of an N-terminal SH3 domain, two proline-rich domains (nPRD and cPRD) that flank a BCR homology domain (BCR), and two SH2 domains (N-terminal (nSH2) and C-terminal (cSH2)) linked by the inter-SH2 (iSH2) domain (1). The p110 catalytic subunit contains an N-terminal adaptor-binding domain (ABD), a Ras-binding domain (RBD), a C2 domain, a helical domain, and a C-terminal kinase domain (Fig. 1A) (2). PI3K␤ can be activated by both receptor tyrosine kinase binding to the p85 SH2 domains (3) and by G protein-coupled receptors (GPCRs), which stimulate G␤␥ binding to the C2-helical linker region of p110␤ (the G␤␥-binding loop) (Fig. 1A) (4, 5). In contrast to the other Class IA PI3Ks, which bind Ras, p110␤ binds the small GTPases Rac1 and Cdc42 via the RBD (6). Rab5 was first identified as a p110␤-binding partner in a screen for Rab5 effectors (7). Rab5 localizes to early endosomes and other vesicular structures (8, 9), and PI3K␤ has been implicated in endocytic trafficking of cell-surface receptors (10, 11). Rab5 binding to p110␤ is required for macroautophagy induced by growth factor limitation (12).

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Different inhibition of Gβγ-stimulated class IB phosphoinositide 3-kinase (PI3K) variants by a monoclonal antibody. Specific function of p101 as a Gβγ-dependent regulator of PI3Kγ enzymatic activity

Cited by 18 publications

References 67 publications

Rac1-stimulated macropinocytosis enhances Gβγ activation of PI3Kβ

Rac1-stimulated macropinocytosis enhances Gβγ activation of PI3Kβ

Translocation of TRPV4-PI3Kγ complexes to the plasma membrane drives myofibroblast transdifferentiation

A single discrete Rab5-binding site in phosphoinositide 3-kinase β is required for tumor cell invasion

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