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The protein kinase Akt plays a central role in a number of key biological functions including protein synthesis, glucose homeostasis, and the regulation of cell survival or death. The mechanism by which tyrosine kinase growth factor receptors stimulate Akt has been recently defined. In contrast, the mechanism of activation of Akt by other cell surface receptors is much less understood. For G protein-coupled receptors (GPCRs), conflicting data suggest that these receptors stimulate Akt in a cell type-specific manner by a yet to be fully elucidated mechanism. Here, we took advantage of the availability of cells, where Akt activity could not be enhanced by agonists acting on this large family of cell surface receptors, such as NIH 3T3 cells, to investigate the pathway linking GPCRs to Akt. We present evidence that expression of phosphatidylinositol 3-kinase (PI3K) β is necessary and sufficient to transmit signals from G proteins to Akt in these murine fibroblasts and that the activation of PI3Kβ may represent the most likely mechanism whereby GPCRs stimulate Akt, as the vast majority of cells do not express PI3Kγ, a known G protein-sensitive PI3K isoform. Furthermore, available evidence indicates that GPCRs activate Akt by a pathway distinct from that utilized by growth factor receptors, as it involves the tyrosine phosphorylation-independent activation of PI3Kβ by G protein βγ dimers.
G protein-coupled receptor(s)
β-adrenergic receptor kinase
muscarinic acetylcholine receptors
enhanced green fluorescent protein
extracellular signal-regulated kinase 2
mitogen-activated protein kinase
platelet-derived growth factor
green fluorescent protein
Akt homology domain
Many important intracellular downstream targets for phosphatidylinositol 3-kinases (PI3Ks)1 have been recently identified (
). Among them, the serine-threonine kinase, Akt-protein kinase B, has received special attention because of its central role in the regulation of cell survival or death in a large number of cellular systems (
). However, how the large family of cell surface receptors that transmit signals through heterotrimeric G proteins regulates the activity of Akt is still poorly understood. In this regard, seemingly conflicting data suggest that the ability of G protein-coupled receptors (GPCRs) to stimulate Akt is cell type-specific. For example, initial reports indicated that lysophosphatidic acid (LPA), which stimulates Gi-coupled receptors, does not enhance the activity of Akt in NIH 3T3 cells (
). The latter includes COS-7 cells, in which ectopically expressed Gq and Gi-coupled receptors, m1 and m2, muscarinic acetylcholine receptors (mAChRs), respectively, were shown to activate Akt effectively when stimulated by carbachol, a cholinergic agonist (
In the present study, we took advantage of the failure of GPCRs to stimulate Akt in NIH 3T3 cell lines to investigate the nature of the signaling molecules linking GPCRs to Akt. Here, we show that PI3Kβ is necessary and sufficient to stimulate Akt by the large family of GPCRs in the vast majority of cells, which do not normally express the G protein-regulated PI3Kγ isoform. Furthermore, we present evidence that GPCRs stimulate PI3Kβ by a pathway distinct from that utilized by tyrosine kinase growth factor receptors, as it does not require the phosphorylation of the p85 subunit in tyrosine residues or its recruitment to phosphotyrosine-containing complexes, but instead involves the activation of this PI3K isoform by the Gβγsubunits of heterotrimeric G proteins.
RESULTS AND DISCUSSION
To begin exploring the molecular basis for GPCR activation of Akt in each cellular setting, we first re-examined the effect of LPA and carbachol on the enzymatic activity of Akt in COS-7 and NIH 3T3 cells expressing endogenous and transfected LPA and m1 receptors, respectively. As shown in Fig.1A, these treatments stimulated Akt in COS-7 cells but not in NIH 3T3 cells, although these agonists enhanced the activity of MAPK in both cell types, which served as a control. Interestingly, serum stimulated Akt in both COS-7 and NIH 3T3 cells, thus suggesting that NIH 3T3 cells may be defective in one or more key molecular components involved in the transmission of signals from GPCRs to Akt.
As PI3Ks are central elements of intracellular signaling pathways linking cell surface receptors to Akt (
). These PI3K subunits form heterodimers with non-catalytic subunits of the p85 family and are stimulated by receptor tyrosine kinases upon their recruitment to the plasma membrane by the assembly of phosphotyrosine-containing multimolecular complexes (
). To explore which PI3K isoforms are expressed in NIH 3T3 and COS-7 cells, cellular lysates were subjected to immunoprecipitation with antibodies specific for each p110 PI3K subunit, and their kinase activity was assessed using an in vitro lipid-kinase reaction. As shown in Fig.1B, endogenous PI3Kα activity was readily detectable both in NIH 3T3 and COS-7 cells. In contrast, PI3Kγ activity was either absent or at levels below its limit of detection in both cell types. However, this PI3K was recovered from PI3Kγ-transfected NIH 3T3 cells (Fig. 1B, second panel) even if its expression levels were not high enough to be detectable by standard immunoblotting techniques (data not shown). Using this enzymatic assay as a sensitive approach to explore the expression of PI3Ks, we also did not observe any endogenous PI3Kγ activity in COS-7 cells. This suggested that the presence of this G protein-regulated PI3K isoform cannot explain the ability of GPCRs to stimulate Akt in COS-7 cells and raised the possibility that another PI3K catalytic subunit may transduce the G protein-initiated signals in this cell type. In this regard, very high levels of endogenous PI3Kβ activity were found in COS-7 cells (Fig.1B). Interestingly, no PI3Kβ activity could be detected in NIH 3T3 cells, although it was readily demonstrable upon exogenous expression of p110β.
These findings prompted us to explore whether the expression of PI3Kβ in NIH 3T3 might confer the ability to activate Akt in response to agonists acting on GPCRs. As shown in Fig.2A, whereas overexpression of p110α did not change the pattern of Akt activation observed in control-transfected cells, as expected, the expression of a G protein-regulated isoform, PI3Kγ, was sufficient to support the activation of Akt by the stimulation of m1 and LPA receptors (Fig.2A). Remarkably, expression of the p110β subunit of PI3K was also sufficient to enable the activation of Akt by both GPCR agonists. A detailed time course analysis of the Akt stimulation by carbachol and LPA in PI3Kβ-transfected NIH 3T3 cells revealed that the expression levels of PI3Kβ achieved (Fig. 2B) were sufficient to support the rapid and potent activation of Akt by these GPCR agonists, which was demonstrable as soon as 5 min after ligand addition and remained above control levels for more than 1 h (Fig.2C). Furthermore, expression of this PI3K isoform also enabled the subcellular redistribution of Akt in response to GPCR agonists in NIH 3T3 cells. As shown in Fig. 2D, an Akt-EGFP chimera containing the membrane-targeting domain of Akt (AH) fused to EGFP (
) displays a cytoplasmic localization in control cells, either untreated or when stimulated with carbachol or LPA, but readily translocates to the plasma membrane upon PDGF treatment (Fig.2D, upper panel). In contrast, carbachol and LPA promoted the accumulation of EGFP-Akt at the level of the plasma membrane when cells coexpressed PI3Kβ (Fig. 2D,lower panel). Taken together, these results indicate that PI3Kβ can mediate the activation of Akt by GPCRs and that the expression of this PI3K isoform is necessary and sufficient to transmit signals from G proteins to Akt in these murine fibroblasts.
Because the p110β PI3K catalytic subunit forms complexes with regulatory subunits of the p85 family (
), we next investigated whether tyrosine phosphorylation of p85 or its association to phosphotyrosine-containing proteins is also involved in the activation of PI3K by GPCRs in NIH 3T3 cells. As an approach, we performed PI3K assays on anti-phosphotyrosine immunoprecipitates from p110β-transfected NIH 3T3 cells upon treatment with serum, LPA, and carbachol. As shown in Fig.3A, treatment with serum resulted in the recovery of PI3K activity in anti-phosphotyrosine immunoprecipitates. In contrast, LPA and carbachol treatments did not result in any demonstrable effect. Similarly, the amount of endogenous p85 protein recovered in anti-phosphotyrosine immunoprecipitates was only enhanced by serum addition (Fig. 3A). In agreement with these findings, pretreatment of p110β-transfected cells with tyrosine kinase inhibitors such as genistein (100 μm for 30 min) diminished the activation of Akt by serum and PDGF but not when elicited by LPA and carbachol (data not shown). In addition, a mutant p85 protein lacking the p110 interacting domain, Δp85, which acts as a dominant negative interfering molecule for p85-mediated pathways (
), caused a significant reduction in the activation of Akt by PDGF but did not affect the activation of Akt by LPA and carbachol (Fig.3B). Taken together, these results strongly suggest that the activation of PI3Kβ by GPCR stimulation does not require tyrosine phosphorylation-dependent events.
We next explored the nature of the G protein(s) implicated in the activation of Akt through PI3Kβ, using the expression of GTPase-deficient forms of representative members of each G protein α subunit family as an approach. As shown in Fig.4A, coexpression of the epitope-tagged Akt together with constitutively active mutants of Gαs, Gαi2, Gαq, Gα12, and Gα13 (
) did not result in the activation of Akt in wild-type NIH 3T3 or in PI3Kβ-transfected cells, although Ras stimulated Akt potently in both cases. Thus, because of the failure of activated Gα subunits to stimulate Akt, we asked whether βγ dimers could induce the PI3Kβ-dependent activation of Akt. As shown in Fig.4A, overexpression in control NIH 3T3 cells of β1γ2 subunits either together or individually did not cause any significant change in the Akt activity. In contrast, overexpression of β1γ2 dimers induced a remarkable increase in the activity of Akt in PI3Kβ-expressing cells. This activation was not observed when β1 or γ2 were expressed alone or when HA-Akt was coexpressed with β1 and γ2*, a mutant γ2 that lacks the γ-isoprenylation signal and therefore fails to associate with the plasma membrane (
). These results indicated that βγ but not Gα subunits promote the PI3Kβ-dependent activation of Akt and that functional, membrane-bound βγ dimers are required for this effect.
To investigate whether βγ dimers participate in signaling from GPCRs to Akt, we expressed a chimeric molecule containing the extracellular and transmembrane domains of CD8 fused to the carboxyl-terminal domain of βARK, which includes the high affinity βγ binding region of this kinase and thus acts as a Gβγ scavenger. As shown in Fig. 4B, transfection of this chimeric molecule in NIH 3T3 cells expressing PI3Kβ dramatically reduced the activation of Akt in response to carbachol and LPA, whereas the stimulation of Akt by tyrosine kinase receptors, such as PDGF, was not affected. Taken together, these findings strongly suggest that βγ subunits of heterotrimeric G proteins play a key role in signaling from GPCRs to Akt by acting on the p110β isoform of PI3K.
Accumulated evidence indicates that PI3Kγ can link GPCRs to a number of signaling pathways (
). Indeed, we did not observe detectable levels of PI3Kγ protein or its lipid kinase activity in NIH 3T3 and COS-7 cells. Instead, this study and recently available evidence provide support for a novel role for PI3Kβ in signaling by GPCRs in the vast majority of cells, which do not express PI3Kγ. For example, it has been recently shown that this PI3K isozyme can be stimulated in vitro by purified G protein βγ subunits in synergism with p-Tyr peptides (
). Thus, these results and the present findings that PI3Kβ expression is necessary and sufficient to activate PI3K-depedent pathways by GPCRs in cells lacking PI3Kγ, such as fibroblasts, demonstrate a central role for the PI3Kβ isoform in signaling by G protein-linked receptors.
Interestingly, although there is extensive sequence homology between the p110α and p110β PI3K catalytic subunits (
). In this regard, our current findings suggest that one such distinct feature is the ability of PI3Kβ to act downstream from heterotrimeric G proteins. The structural elements responsible for this distinct coupling specificity are still unknown and are under current investigation.
The emerging picture is that GPCRs stimulate PI3Kβ in most tissues by a mechanism distinct from those utilized by tyrosine kinase receptors, as it does not require the functional activity of the p85 non-catalytic subunit, but instead involves the activation of p110β by Gβγ subunits. We can also conclude that the distinct tissue distribution of each PI3K isoform may play an unexpected role in controlling the specificity in signal transmission, as it may govern the ability of GPCRs to stimulate a variety of intracellular signaling pathways that require PI3K function. In turn, the availability and level of expression of PI3Kβ and PI3Kγ may determine the nature of the biological responses elicited by the large family of G protein-linked cell surface receptors in each cell type.
We thank Dr. M. Kasuga for providing the pSRα-Δp85 expression vector and Drs. Tsichlis and Franke for Akt expression plasmids. We appreciate the gifts of PI3Kγ and p101 expression constructs from Dr. L. R. Stephens and of a p85 expression vector from Dr. Paolo di Fiore.