Agonist-modulated regulation of AMP-activated protein kinase (AMPK) in endothelial cells. Evidence for an AMPK -> Rac1 -> Akt -> endothelial nitric-oxide synthase pathway

The endothelial isoform of nitric-oxide synthase (eNOS), a key determinant of vascular homeostasis, is a calcium/calmodulin-dependent phosphoprotein regulated by diverse cell surface receptors. Vascular endothelial growth factor (VEGF) and sphingosine 1-phosphate (S1P) stimulate eNOS activity through Akt/phosphoinositide 3-kinase and calcium-dependent pathways. AMP-activated protein kinase (AMPK) also activates eNOS in endothelial cells; however, the molecular mechanisms linking agonist-mediated AMPK regulation with eNOS activation remain incompletely understood. We studied the role of AMPK in VEGF- and S1P-mediated eNOS activation and found that both agonists led to a striking increase in AMPK phosphorylation in pathways involving the calcium/calmodulin-dependent protein kinase kinase beta. Treatment with tyrosine kinase inhibitors or the phosphoinositide 3-kinase inhibitor wortmannin demonstrated differential effects of VEGF versus S1P. Small interfering RNA (siRNA)-mediated knockdown of AMPKalpha1or Akt1 impaired the stimulatory effects of both VEGF and S1P on eNOS activation. AMPKalpha1 knockdown impaired agonist-mediated Akt phosphorylation, whereas Akt1 knockdown did not affect AMPK activation, thus suggesting that AMPK lies upstream of Akt in the pathway leading from receptor activation to eNOS stimulation. Importantly, we found that siRNA-mediated knockdown of AMPKalpha1 abrogates agonist-mediated activation of the small GTPase Rac1. Conversely, siRNA-mediated knockdown of Rac1 decreased the agonist-mediated phosphorylation of AMPK substrates without affecting that of AMPK, implicating Rac1 as a molecular link between AMPK and Akt in agonist-mediated eNOS activation. Finally, siRNA-mediated knockdown of caveolin-1 significantly enhanced AMPK phosphorylation, suggesting that AMPK is negatively regulated by caveolin-1. Taken together, these results suggest that VEGF and S1P differentially regulate AMPK and establish a central role for an agonist-modulated AMPK --> Rac1 --> Akt axis in the control of eNOS in endothelial cells.     

Akt1 and Akt2: differentiating the aktion

Kinases of the Akt family are integral and essential components in growth factor signaling pathways activated downstream of the membrane bound phospho-inositol-3 kinase. In light of strong homologies in the primary amino acid sequence, the three Akt kinases were long surmised to play redundant and overlapping roles in insulin signaling across the spectra of cell and tissue types. Over the last 10 years, work using mouse knockout models, cell specific inactivation, and more recently targeted gene inactivation, has brought into question the redundancy within Akt kinase isoforms and instead pointed to isoform specific functions in different cellular events and diseases. Here we concentrate on the differential roles played by Akt1 and Akt2 in a variety of cellular processes and in particular during cancer biogenesis. In this overview, we illustrate that while Akt1 and 2 are often implicated in many aspects of cellular transformation, the two isoforms frequently act in a complementary opposing manner. Furthermore, Akt1 and Akt2 kinases interact differentially with modulating proteins and are necessary in relaying roles during the evolution of cancers from deregulated growth into malignant metastatic killers. These different actions of the two isoforms point to the importance of treatments targeting isoform specific events in the development of effective approaches involving Akt kinases in human disease.     

Sphingosine 1-phosphate and activation of endothelial nitric-oxide synthase. differential regulation of Akt and MAP kinase pathways by EDG and bradykinin receptors in vascular endothelial cells

Sphingosine 1-phosphate (S1P) is a platelet-derived sphingolipid that elicits numerous biological responses in endothelial cells mediated by a family of G protein-coupled EDG receptors. Stimulation of EDG receptors by S1P has been shown to activate the endothelial isoform of nitric-oxide synthase (eNOS) in heterologous expression systems (Igarashi, J., and Michel, T. (2000) J. Biol. Chem. 275, 32363-32370). However, the signaling pathways that modulate eNOS regulation by S1P/EDG in vascular endothelial cells remain less well understood. We now report that S1P treatment of bovine aortic endothelial cells (BAEC) acutely increases eNOS enzyme activity; the EC(50) for S1P activation of eNOS is approximately 10 nm. The magnitude of eNOS activation by S1P in BAEC is equivalent to that elicited by the agonist bradykinin. S1P treatment activates Akt, a protein kinase implicated in phosphorylation of eNOS. S1P treatment of BAEC leads to eNOS phosphorylation at Ser(1179), a residue phosphorylated by Akt; an eNOS mutant in which this Akt phosphorylation site is inactivated shows attenuated S1P-induced eNOS activation. S1P-induced activation both of Akt and of eNOS is inhibited by pertussis toxin, by the phosphoinositide 3-kinase inhibitor wortmannin, and by the intracellular calcium chelator BAPTA (1,2-bis(aminophenoxy)ethane-N,N,N',N'-tetraacetic acid). By contrast to S1P, activation of G protein-coupled bradykinin B2 receptors neither activates kinase Akt nor promotes Ser(1179) eNOS phosphorylation despite robustly activating eNOS enzyme activity. Understanding the differential regulation of protein kinase pathways by S1P and bradykinin may lead to the identification of new points for eNOS regulation in vascular endothelial cells.     

The Akt kinases: Isoform specificity in metabolism and cancer

The Akt (PKB) protein kinases are critical regulators of human physiology that control an impressive array of diverse cellular functions, including the modulation of growth, survival, proliferation and metabolism. The Akt kinase family is comprised of three highly homologous isoforms: Akt1 (PKBα), Akt2 (PKBβ) and Akt3 (PKBγ). Phenotypic analyses of Akt isoform knockout mice documented Akt isoform specific functions in the regulation of cellular growth, glucose homeostasis and neuronal development. Those studies establish that the functions of the different Akt kinases are not completely overlapping and that isoform-specific signaling contributes to the diversity of Akt activities. However, despite these important advances, a thorough understanding about the specific roles of Akt family members and the molecular mechanisms that determine Akt isoform functional specificity will be essential to elucidate the complexity of Akt regulated cellular processes and how Akt isoform-specific deregulation might contribute to disease states. Here, we summarize recent advances in understanding the roles of Akt isoforms in the regulation of metabolism and cancer, and possible mechanisms contributing to Akt isoform functional specificity.     

Akt substrate TBC1D1 regulates GLUT1 expression through the mTOR pathway in 3T3-L1 adipocytes

Multiple studies have suggested that the protein kinase Akt/PKB (protein kinase B) is required for insulin-stimulated glucose transport in skeletal muscle and adipose cells. In an attempt to understand links between Akt activation and glucose transport regulation, we applied mass spectrometry-based proteomics and bioinformatics approaches to identify potential Akt substrates containing the phospho-Akt substrate motif RXRXXpS/T. The present study describes the identification of the Rab GAP (GTPase-activating protein)-domain containing protein TBC1D1 [TBC (Tre-2/Bub2/Cdc16) domain family, member 1], which is closely related to TBC1D4 [TBC domain family, member 4, also denoted AS160 (Akt substrate of 160 kDa)], as an Akt substrate that is phosphorylated at Thr(590). RNAi (RNA interference)-mediated silencing of TBC1D1 elevated basal deoxyglucose uptake by approx. 61% in 3T3-L1 mouse embryo adipocytes, while the suppression of TBC1D4 and RapGAP220 under the same conditions had little effect on basal and insulin-stimulated deoxyglucose uptake. Silencing of TBC1D1 strongly increased expression of the GLUT1 glucose transporter but not GLUT4 in cultured adipocytes, whereas the decrease in TBC1D4 had no effect. Remarkably, loss of TBC1D1 in 3T3-L1 adipocytes activated the mTOR (mammalian target of rapamycin)-p70 S6 protein kinase pathway, and the increase in GLUT1 expression in the cells treated with TBC1D1 siRNA (small interfering RNA) was blocked by the mTOR inhibitor rapamycin. Furthermore, overexpression of the mutant TBC1D1-T590A, lacking the putative Akt/PKB phosphorylation site, inhibited insulin stimulation of p70 S6 kinase phosphorylation at Thr(389), a phosphorylation induced by mTOR. Taken together, our data suggest that TBC1D1 may be involved in controlling GLUT1 glucose transporter expression through the mTOR-p70 S6 kinase pathway.     

Rac1 modulates sphingosine 1-phosphate-mediated activation of phosphoinositide 3-kinase/Akt signaling pathways in vascular endothelial cells

Sphingosine 1-phosphate (S1P) is a platelet-derived sphingolipid that activates G protein-coupled S1P receptors and initiates a broad range of responses in vascular endothelial cells. The small GTPase Rac1 is implicated in diverse S1P-modulated cellular responses in endothelial cells, yet the molecular mechanisms involved in S1P-mediated Rac1 activation are incompletely understood. We studied the pathways involved in S1P-mediated Rac1 activation in bovine aortic endothelial cells (BAEC) and found that S1P-induced Rac1 activation is impaired following chelation of G protein betagamma subunits by transfection of betaARKct. Treatment with the Src tyrosine kinase inhibitor PP2 completely attenuated S1P-mediated Rac1 activation; however, pretreatment of BAEC with wortmannin, an inhibitor of phosphoinositide (PI) 3-kinase, had no effect on Rac1 activation while completely blocking S1P-induced Akt phosphorylation. We used Rac1-specific small interfering RNA (siRNA) duplexes to "knock down" endogenous Rac1 expression and found that siRNA-mediated Rac1 knockdown significantly impaired basal as well as S1P-induced phosphorylation of protein kinase Akt, as well as several downstream targets of Akt including endothelial nitric-oxide synthase and glycogen synthase kinase 3beta. By contrast, S1P-induced phosphorylation of the mitogen-activated protein kinases ERK1/2 was unperturbed by siRNA-mediated Rac1 knockdown. We found that overexpression of the Rac1 guanine nucleotide exchange factor (GEF) Tiam1 markedly enhanced Rac1 activity, whereas a dominant negative Tiam1 mutant significantly attenuated S1P-mediated Rac1 activation. Taken together, these studies identify G protein betagamma subunits, Src kinase and the GEF Tiam1 as upstream modulators of S1P-mediated Rac1 activation, and establish a central role for Rac1 in S1P-mediated activation of PI 3-kinase/Akt/endothelial nitric-oxide synthase signaling in vascular endothelial cells.     

Akt is a major angiogenic mediator downstream of the Ang1/Tie2 signaling pathway

Tie2 and VEGF receptors (VEGFRs) are tyrosine kinases that play essential roles in angiogenesis. Activation of both receptors leads to the activation of Akt, an important mediator of cell survival and cell motility. In this study, we compared the role of Akt in Tie2-mediated versus VEGF-mediated endothelial cell (EC) survival and EC sprouting. Our data show that Akt is required and sufficient to mediate Ang1-induced EC survival in response to growth factor depletion. Blocking Akt function abolishes angiopoietin 1 (Ang1), a ligand for Tie2, mediated EC survival, and activating Akt rescues a Tie2 blockade-induced EC apoptosis. In contrast, activating Akt rescues EC apoptosis induced by a VEGF blockade, but interestingly, blocking Akt function has no effects on VEGF-induced EC survival, demonstrating that Akt is sufficient but not required for VEGF-mediated EC survival. In addition, we show that both Ang1 and VEGF induce EC sprouting in a three-dimensional collagen gel, which depends on the activation of Akt. Blocking Akt action inhibited EC sprouting induced by Ang1 or VEGF. Therefore, the data show that Akt is the primary mediator of Ang1-induced EC survival while multiple pathways are involved downstream of VEGF responsible for EC survival. However, Akt is required and sufficient to mediate the EC sprouting induced by both Ang1 and VEGF.     

Dephosphorylation by default, a potential mechanism for regulation of insulin receptor substrate-1/2, Akt, and ERK1/2

Protein phosphorylation is an important mechanism that controls many cellular activities. Phosphorylation of a given protein is precisely controlled by two opposing biochemical reactions catalyzed by protein kinases and protein phosphatases. How these two opposing processes are coordinated to achieve regulation of protein phosphorylation is unresolved. We have developed a novel experimental approach to directly study protein dephosphorylation in cells. We determined the kinetics of dephosphorylation of insulin receptor substrate-1/2, Akt, and ERK1/2, phosphoproteins involved in insulin receptor signaling. We found that insulin-induced ERK1/2 and Akt kinase activities were completely abolished 10 min after inhibition of the corresponding upstream kinases with PD98059 and LY294002, respectively. In parallel experiments, insulin-induced phosphorylation of Akt, ERK1/2, and insulin receptor substrate-1/2 was decreased and followed similar kinetics. Our findings suggest that these proteins are dephosphorylated by a default mechanism, presumably via constitutively active phosphatases. However, dephosphorylation of these proteins is overcome by activation of protein kinases following stimulation of the insulin receptor. We propose that, during acute insulin stimulation, the kinetics of protein phosphorylation is determined by the interplay between upstream kinase activity and dephosphorylation by default.     

Glutathione transferase Omega 1-1 (GSTO1-1) modulates Akt and MEK1/2 signaling in human neuroblastoma cell SH-SY5Y

In the human neuroblastoma SH-SY5Y cell line, the glutathione transferase Omega 1-1 (GSTO1-1) appears to modulate Akt and MEK1/2 kinase activation. We observed a glutathionylation modification was involved in the activation of Akt but not MEK1/2. With the specific GSTO1-1 inhibitor ML175, we show the enzyme activity of GSTO1-1 is important for modulation as the inhibited GSTO1-1 allowed activation of both Akt and MEK1/2. The inhibition of GSTO1-1 showed a similar extent of activation of Akt and MEK1/2 as treatment by the endotoxin lipopolysaccharide. The GSTO1-1 also either directly interacts with Akt and MEK1/2 or interacts with a protein complexed with Akt and MEK1/2 as both kinases coimmunoprecipitated with GSTO1-1. The results suggest that GSTO1-1 enzyme activity inhibits the activation of these two kinases to maintain basal levels. The possible regulation by GSTO1-1 is of interest as both kinases have hundreds of potential downstream targets that are known to have contributions to various cellular processes including survival, growth, proliferation, and metabolism.     

Sphingosine 1-phosphate and isoform-specific activation of phosphoinositide 3-kinase beta. Evidence for divergence and convergence of receptor-regulated endothelial nitric-oxide synthase signaling pathways

Sphingosine 1-phosphate (S1P) is a platelet-derived sphingolipid that elicits diverse biological responses, including angiogenesis, via the activation of G protein-coupled EDG receptors. S1P activates the endothelial isoform of nitric-oxide synthase (eNOS), associated with eNOS phosphorylation at Ser-1179, a site phosphorylated by protein kinase Akt. We explored the proximal signaling pathways that mediate Akt activation and eNOS regulation by S1P/EDG receptors. Akt is regulated by the lipid kinase phosphoinositide 3-kinase (PI3-K). We found that bovine aortic endothelial cells (BAEC) express both alpha and beta isoforms of PI3-K, while lacking the gamma isoform. S1P treatment led to the rapid and isoform-specific activation of PI3-Kbeta in BAEC. PI3-Kbeta can be regulated by G protein betagamma subunits (Gbetagamma). The overexpression of a peptide inhibitor of Gbetagamma attenuated S1P-induced eNOS enzyme activation, as well as S1P-induced phosphorylation of eNOS and Akt. In contrast, bradykinin, a classical eNOS agonist, neither activated any PI3-K isoform nor induced eNOS phosphorylation at Ser-1179, despite activating eNOS in BAEC. Vascular endothelial growth factor activated both PI3-Kalpha and PI3-Kbeta via tyrosine kinase pathways and promoted eNOS phosphorylation that was unaffected by Gbetagamma inhibition. These findings indicate that PI3-Kbeta (regulated by Gbetagamma) may represent a novel molecular locus for eNOS activation by EDG receptors in vascular endothelial cells. These studies also indicate that different eNOS agonists activate distinct signaling pathways that diverge proximally following receptor activation but converge distally to activate eNOS.     

Transactivation of vascular endothelial growth factor (VEGF) receptor Flk-1/KDR is involved in sphingosine 1-phosphate-stimulated phosphorylation of Akt and endothelial nitric-oxide synthase (eNOS)

Sphingosine 1-phosphate (S1P) and vascular endothelial growth factor (VEGF) elicit numerous biological responses including cell survival, growth, migration, and differentiation in endothelial cells mediated by the endothelial differentiation gene, a family of G-protein-coupled receptors, and fetal liver kinase-1/kinase-insert domain-containing receptor (Flk-1/KDR), one of VEGF receptors, respectively. Recently, it was reported that S1P or VEGF treatment of endothelial cells leads to phosphorylation at Ser-1179 in bovine endothelial nitric oxide synthase (eNOS), and this phosphorylation is critical for eNOS activation. S1P stimulation of eNOS phosphorylation was shown to involve G(i) protein, phosphoinositide 3-kinase, and Akt. VEGF also activates eNOS through Flk-1/KDR, phosphoinositide 3-kinase, and Akt, which suggested that S1P and VEGF may share upstream signaling mediators. We now report that S1P treatment of bovine aortic endothelial cells acutely increases the tyrosine phosphorylation of Flk-1/KDR, similar to VEGF treatment. S1P-mediated phosphorylation of Flk-1/KDR, Akt, and eNOS were all inhibited by VEGF receptor tyrosine kinase inhibitors and by antisense Flk-1/KDR oligonucleotides. Our study suggests that S1P activation of eNOS involves G(i), calcium, and Src family kinase-dependent transactivation of Flk-1/KDR. These data are the first to establish a critical role of Flk-1/KDR in S1P-stimulated eNOS phosphorylation and activation.     

Regulation of the Akt/Glycogen synthase kinase-3 axis by insulin-like growth factor-II via activation of the human insulin receptor isoform-A

Insulin-like growth factor II (IGF-II) plays a key role in mitogenesis during development and tumorigenesis and is believed to exert its mitogenic functions mainly through the IGF-I receptor. Recently, we identified the insulin receptor isoform A (IR(A)) as an additional high affinity receptor for IGF-II in both fetal and cancer cells. Here we investigated the mitogenic signaling of IGF-II via the Akt/Glycogen synthase kinase 3 (Gsk3) axis employing R-IR(A) cells that are IGF-I receptor null mouse embryonic fibroblasts expressing the human IR(A). IGF-II induced activation of the proto-oncogenic serine kinase Akt, reaching maximal at 5-10 min. IGF-II also caused the rapid and sustained deactivation of glycogen synthase kinase 3-beta (Gsk3beta), reaching maximal at 1-3 min, shortly preceding, therefore, maximal activation of Akt. Under our conditions, IGF-II and insulin induced 70-80% inhibition of Gsk3betaactivity. In these cells IGF-II also deactivated Gsk3alpha although less effectively than Gsk3beta. In parallel experiments, we found that IGF-II induced transient activation of extracellular-signal-regulated kinases (Erk) reaching maximal at 5-10 min and decreasing thereafter. Time courses and potencies of regulation of both mitogenic pathways (Akt/Gsk3beta and Erk) by IGF-II via IR(A) were similar to those of insulin. Furthermore, IGF-II like insulin effectively stimulated cell cycle progression from the G0/G1 to the S and G2/M phases. Interestingly, AP-1-mediated gene expression, that was reported to be negatively regulated by Gsk3beta was only weakly increased after IGF-II stimulation. Our present data suggest that the coordinated activation or deactivation of Akt, Gsk3beta, and Erk may account for IGF-II mitogenic effects and support an active role for IR(A) in IGF-II action.     

Regulation of vascular endothelial growth factor expression by EMMPRIN via the PI3K-Akt signaling pathway

Extracellular matrix metalloproteinase (MMP) inducer (EMMPRIN) is a cell surface glycoprotein overexpressed in many solid tumors. In addition to its ability to stimulate stromal MMP expression, tumor-associated EMMPRIN also induces vascular endothelial growth factor (VEGF) expression. To explore the underlying signaling pathways used by EMMPRIN, we studied the involvement of phosphoinositide 3-kinase (PI3K)-Akt, mitogen-activated protein kinase (MAPK), JUN, and p38 kinases in EMMPRIN-mediated VEGF regulation. Overexpression of EMMPRIN in MDA-MB-231 breast cancer cells stimulated the phosphorylation of only Akt and MAPKs but not that of JUN and p38 kinases. Conversely, inhibition of EMMPRIN expression resulted in suppressed Akt and MAPK phosphorylation. Furthermore, the PI3K-specific inhibitor LY294002 inhibited VEGF production by EMMPRIN-overexpressing cells in a dose- and time-dependent manner. On the other hand, the MAPK inhibitor U0126 did not affect VEGF production. In vivo, EMMPRIN-overexpressing tumors with elevated VEGF expression had a high level of phosphorylation of Akt and MAPK. Finally, when fibroblast cells were treated with recombinant EMMPRIN, Akt kinase but not MAPK was phosphorylated concomitant with an increase in VEGF production. Both the activation of Akt kinase and the induction of VEGF were specifically inhibited with a neutralizing antibody to EMMPRIN. Our results show that in both tumor and fibroblast cells EMMPRIN regulates VEGF production via the PI3K-Akt pathway but not via the MAPK, JUN, or p38 kinase pathways.     

Effect of orthodontic force on the expression of PI3K,Akt, and P70S6 K in the human periodontal ligament during orthodontic loading

The mammalian target of rapamycin (mTOR) is an atypical serine/threonine protein kinases involved in the regulation of cell growth, proliferation, and differentiation through the PI3K/Akt/mTOR/P70S6 K signalling pathway. P70S6 K as a downstream molecule of mTOR is activated by phosphorylation and subsequently promotes the synthesis of ribosomal and translational proteins. In this study, we investigated the role of PI3K, Akt, and P70S6 K in human periodontal tissue remodelling during orthodontic loading. The prepared tissue specimens taken from 4 extracted premolars were processed for immunolabelling. The changes in the expression of PI3K, Akt, and P70S6 K in the periodontal tissues were detected by real‐time quantitative‐polymerase chain reaction and Western blot analysis. The results from real‐time quantitative‐polymerase chain reaction and Western blot both showed that the expression of PI3K, Akt, and P70S6 K in the experimental group began to increase at 3 days and increased significantly at 10 days, then decreased approaching the control group level at 28 days. Our findings showed that the expression of PI3K, Akt, and P70S6 K in human periodontal ligament demonstrated a variability during the orthodontic loading, which suggested that the PI3K/Akt/mTOR/P70S6 K signal pathway was involved in orthodontic tooth movement and played a role in the process of periodontium remodelling.     

Flt-1, but not Flk-1 mediates hyperpermeability through activation of the PI3-K/Akt pathway

Vascular endothelial growth factor (VEGF), a potent mediator of endothelial proliferation and migration, has an important role also in brain edema formation during hypoxia and ischemia. VEGF binds to the tyrosine kinase receptors Flt-1 and Flk-1. Yet, their relative importance for hypoxia-induced hyperpermeability is not well understood. We used an in vitro blood-brain barrier (BBB) model consisting of porcine brain microvascular endothelial cells (BMEC) to determine the role of Flt-1 in VEGF-induced endothelial cell (EC) barrier dysfunction. Soluble Flt-1 abolished hypoxia/VEGF-induced hyperpermeability. Furthermore, selective antisense oligonucleotides to Flt-1, but not to Flk-1, inhibited hypoxia-induced permeability changes. Consistent with these data, addition of the receptor-specific homolog placenta-derived growth factor, which binds Flt-1 but not Flk-1, increased endothelial permeability to the same extent as VEGF, whereas adding VEGF-E, a viral VEGF molecule from the orf virus family activating Flk-1 and neuropilin-1, but not Flt-1, did not show any effect. Using the carcinoma submandibular gland cell line (CSG), only expressing Flt-1, it was demonstrated that activation of Flt-1 is sufficient to induce hyperpermeability by hypoxia and VEGF. Hyperpermeability, induced by hypoxia/VEGF, depends on activation of phosphatidylinositol 3-kinase/Akt (PI3-K/Akt), nitric oxide synthase (NOS) and protein kinase G (PKG). The activation of the PI3-K/Akt pathway by hypoxia was confirmed using an in vivo mice hypoxia model. These results demonstrate that hypoxia/VEGF-induced hyperpermeability can be mediated by activation of Flt-1 independently on the presence of Flk-1 and indicate a central role for activation of the PI3-K/Akt pathway, followed by induction of NOS and PKG activity.     

The docking protein Gab1 is an essential component of an indirect mechanism for fibroblast growth factor stimulation of the phosphatidylinositol 3-kinase/Akt antiapoptotic pathway

The docking protein Gab1 has been implicated as a mediator of multiple signaling pathways that are activated by a variety of receptor tyrosine kinases and cytokines. We have previously proposed that fibroblast growth factor 1 (FGF1) stimulation of tyrosine phosphorylation of Gab1 and recruitment of phosphatidylinositol (PI) 3-kinase are mediated by an indirect mechanism in which the docking protein fibroblast receptor substrate 2alpha (FRS2alpha) plays a critical role. In this report, we explore the role of Gab1 in FGF1 signaling by using mouse embryo fibroblasts (MEFs) derived from Gab1(-/-) or FRS2alpha(-/-) mice. We demonstrate that Gab1 is essential for FGF1 stimulation of both PI 3-kinase and the antiapoptotic protein kinase Akt, while FGF1-induced mitogen-activated protein kinase (MAPK) stimulation is not affected by Gab1 deficiency. To test the indirect mechanism for FGF1 stimulation of PI 3-kinase and Akt, we use a chimeric docking protein composed of the membrane targeting signal and the phosphotyrosine-binding domain of FRS2alpha fused to the C-terminal portion of Gab1, the region including the binding sites for the complement of signaling proteins that are recruited by Gab1. We demonstrate that expression of the chimeric docking protein in Gab1(-/-) MEFs rescues PI 3-kinase and the Akt responses, while expression of the chimeric docking protein in FRS2alpha(-/-) MEFs rescues stimulation of both Akt and MAPK. These experiments underscore the essential role of Gab1 in FGF1 stimulation of the PI 3-kinase/Akt signaling pathway and provide further support for the indirect mechanism for FGF1 stimulation of PI 3-kinase involving regulated assembly of a multiprotein complex.