Heregulin-dependent activation of phosphoinositide 3-kinase and Akt via the ErbB2/ErbB3 co-receptor

The ErbB2/ErbB3 heregulin co-receptor has been shown to couple to phosphoinositide (PI) 3-kinase in a heregulin-dependent manner. The recruitment and activation of PI 3-kinase by this co-receptor is presumed to occur via its interaction with phosphorylated Tyr-Xaa-Xaa-Met (YXXM) motifs occurring in the ErbB3 C terminus. In this study, mutant ErbB3 receptor proteins expressed in COS7 cells were used to investigate PI 3-kinase-dependent signaling pathways activated by the ErbB2/ErbB3 co-receptor. We observed that a mutant ErbB3 protein with each of its six YXXM motifs containing a Tyr --> Phe substitution was unable to bind either the p85 regulatory or p110 catalytic subunit of PI 3-kinase. However, restoration of a single YXXM motif was sufficient to mediate association with the PI 3-kinase holoenzyme, although at a lower level than wild-type ErbB3. When ErbB3 YXXM motifs were restored in pairs, evidence for cooperativity between two, those incorporating Tyr-1273 and Tyr-1286, was observed. Interestingly, we have shown that an apparent association of PI 3-kinase activity with ErbB2/Neu was due to the residual presence of ErbB3 in ErbB2 immunoprecipitates. The necessity of ErbB3 association with PI 3-kinase for downstream signaling to the effector kinase Akt was also investigated. Here, the heregulin-dependent translocation of Akt to the plasma membrane and its subsequent activation was observed in intact NIH-3T3 fibroblasts. Recruitment of PI 3-kinase to ErbB3 was required for both activities, and it appeared that ErbB2 activation alone was not sufficient to activate PI 3-kinase signaling in these cells.     

PI3-Kinase Is Involved in Mitogenic Signaling by the Oncogenic Receptor Tyrosine Kinase Xiphophorus Melanoma Receptor Kinase in Fish Melanoma

Overexpression of the mutationally activated receptor tyrosine kinase Xiphophorus melanoma receptor kinase (Xmrk) initiates formation of hereditary malignant melanoma in the fish Xiphophorus. In melanoma as well as in a melanoma-derived cell line (PSM) this receptor is highly activated resulting in constitutive Xmrk-mediated mitogenic signaling. In order to analyze mitogenic signaling triggered by Xmrk a possible involvement of phosphatidylinositol 3 (PI3)-kinase in Xmrk signal transduction was examined. Constitutive binding of the p85 adapter subunit of PI3-kinase to the Xmrk receptor was detected in PSM melanoma cells. Further analyses in BHK cells expressing a Xmrk chimera (HER-mrk) showed that p85 association with the intracellular part of Xmrk was dependent on autophosphorylation of the receptor. In vitro binding studies revealed that the interaction is mediated mainly through the N-terminal SH2 domain of p85 which directly binds to a sequence motif around phosphorylated Tyr-983 in the Xmrk carboxy-terminus. In accordance with recruitment of p85 by Xmrk in PSM cells, the PI3-kinase downstream target Akt was found to be highly phosphorylated on Ser-473, indicating efficient PI3-kinase signaling in melanoma cells. PI3-kinase activation was also detected in Xiphophorus melanoma. Moreover, malignant melanomas exhibited an increased level of PI3-kinase activity which was about three times higher than that in benign pigmented lesions. Inhibition of PI3-kinase activity in PSM melanoma cells by both Wortmannin and LY294002 blocked entry into S-phase. Together these data demonstrate that PI3-kinase is a substrate of the oncogenic Xmrk receptor and plays a significant role in mitogenic signaling of melanoma cells and the formation of malignant melanoma in Xiphophorus.     

Hydrogen peroxide activates endothelial nitric-oxide synthase through coordinated phosphorylation and dephosphorylation via a phosphoinositide 3-kinase-dependent signaling pathway.

Endothelial nitric-oxide synthase (eNOS) is an important component of vascular homeostasis. During vascular disease, endothelial cells are exposed to excess reactive oxygen species that can alter cellular phenotype by inducing various signaling pathways. In the current study, we examined the implications of H(2)O(2)-induced signaling for eNOS phosphorylation status and activity in porcine aortic endothelial cells. We found that H(2)O(2) treatment enhanced eNOS activity and NO bioactivity as determined by the conversion of l-[(3)H]arginine to l-[(3)H]citrulline and cellular cGMP content. Concomitant with eNOS activation, H(2)O(2) also activated Akt, increased eNOS phosphorylation at Ser-1177, and decreased eNOS phosphorylation at Thr-495. H(2)O(2)-induced promotion of eNOS activity and modulation of the eNOS phosphorylation status at Ser-1177 and Thr-495 were significantly attenuated by selective inhibitors of Src kinase, the ErbB receptor family, and phosphoinositide 3-kinase (PI 3-K). We found that Akt activation, eNOS Ser-1177 phosphorylation, and eNOS activation by H(2)O(2) were calcium-dependent, whereas eNOS dephosphorylation at Thr-495 was not, suggesting a branch point in the signaling cascade downstream from PI 3-K. Consistent with this, overexpression of a dominant negative isoform of Akt inhibited H(2)O(2)-induced phosphorylation of eNOS at Ser-1177 but not dephosphorylation of eNOS at Thr-495. Together, these data indicate that H(2)O(2) promotes calcium-dependent eNOS activity through a coordinated change in the phosphorylation status of the enzyme mediated by Src- and ErbB receptor-dependent PI 3-K activation. In turn, PI 3-K mediates eNOS Ser-1177 phosphorylation via a calcium- and Akt-dependent pathway, whereas eNOS Thr-495 dephosphorylation does not involve calcium or Akt. This response may represent an attempt by endothelial cells to maintain NO bioactivity under conditions of enhanced oxidative stress.     

Protein kinase A-mediated gating of neuregulin-dependent ErbB2-ErbB3 activation underlies the synergistic action of cAMP on Schwann cell proliferation

In Schwann cells (SCs), cyclic adenosine monophosphate (cAMP) enhances the action of neuregulin, the most potent known mitogen for SCs, by synergistically increasing the activation of two crucial signaling pathways: ERK and Akt. However, the underlying mechanism of cross-talk between neuregulin and cAMP signaling remains mostly undefined. Here, we report that the activation of protein kinase A (PKA), but not that of exchange protein activated by cAMP (EPAC), enhances S-phase entry of SCs by synergistically enhancing the ligand-dependent tyrosine phosphorylation/activation of the neuregulin co-receptor, ErbB2-ErbB3. The role of PKA in neuregulin-ErbB signaling was confirmed using PKA inhibitors, pathway-selective cAMP analogs, and natural ligands stimulating PKA activity in SCs, such as adenosine and epinephrine. Two basic observations defined the synergistic action of PKA as "gating" for neuregulin-ErbB signaling: 1) the activation of PKA was not sufficient to induce S-phase entry or the activation of either ErbB2 or ErbB3; and 2) the presence of neuregulin was strictly required to ignite ErbB activation and thereby ERK and Akt signaling. However, PKA directly phosphorylated ErbB2 on Thr-686, a highly conserved intracellular regulatory site that was required for the PKA-mediated synergistic enhancement of neuregulin-induced ErbB2-ErbB3 activation and proliferation in SCs. The gating action of PKA on neuregulin-induced ErbB2-ErbB3 activation has important biological significance, because it insures signal amplification into the ERK and Akt pathways without compromising either the neuregulin dependence or the high specificity of ErbB signaling pathways.     

Regulation of MDR1 expression and drug resistance by a positive feedback loop involving hyaluronan, phosphoinositide 3-kinase, and ErbB2

Multidrug resistance is a potent barrier to effective, long term therapy in cancer patients. It is frequently attributed to enhanced expression of multidrug transporters or to the action of receptor kinases, such as ErbB2, and downstream anti-apoptotic signaling pathways, such as the phosphoinositide 3-kinase/Akt pathway. However, very few connections have been made between receptor kinases or anti-apoptotic pathways and multidrug transporter expression or function. Data presented herein show that constitutive interaction of the pericellular polysaccharide, hyaluronan, with its receptor, CD44, regulates assembly and activation of an ErbB2-containing signaling complex, which in turn stimulates phosphoinositide 3-kinase activity in multidrug-resistant MCF-7/Adr human breast carcinoma cells. Phosphoinositide 3-kinase activates Akt and downstream anti-apoptotic events, which contribute to drug resistance. However, hyaluronan and phosphoinositide 3-kinase stimulate expression of the multidrug transporter, MDR1 (P-glycoprotein), in an interdependent, but Akt-independent, manner. Furthermore, constitutively active phosphoinositide 3-kinase, but not Akt, stimulates hyaluronan production. These Akt-independent effects are dominant over the effects of Akt on doxorubicin resistance in MCF-7/Adr cells. Thus hyaluronan, phosphoinositide 3-kinase, and ErbB2 form a positive feedback loop that strongly amplifies MDR1 expression and regulates drug resistance in these cells. This pathway may also be important in progression of other malignant characteristics. These results illustrate the potential importance of hyaluronan as a therapeutic target in multidrug-resistant carcinomas.     

Differential regulation of the phosphatidylinositol 3-kinase/Akt and p70 S6 kinase pathways by the alpha(1A)-adrenergic receptor in rat-1 fibroblasts

Phosphatidylinositol (PI) 3-kinase and its downstream effector Akt are thought to be signaling intermediates that link cell surface receptors to p70 S6 kinase. We examined the effect of a G(q)-coupled receptor on PI 3-kinase/Akt signaling and p70 S6 kinase activation using Rat-1 fibroblasts stably expressing the human alpha(1A)-adrenergic receptor. Treatment of the cells with phenylephrine, a specific alpha(1)-adrenergic receptor agonist, activated p70 S6 kinase but did not activate PI 3-kinase or any of the three known isoforms of Akt. Furthermore, phenylephrine blocked the insulin-like growth factor-I (IGF-I)-induced activation of PI 3-kinase and the phosphorylation and activation of Akt-1. The effect of phenylephrine was not confined to signaling pathways that include insulin receptor substrate-1, as the alpha(1)-adrenergic receptor agonist also inhibited the platelet-derived growth factor-induced activation of PI 3-kinase and Akt-1. Although increasing the intracellular Ca(2+) concentration with the ionophore A23187 inhibited the activation of Akt-1 by IGF-I, Ca(2+) does not appear to play a role in the phenylephrine-mediated inhibition of the PI 3-kinase/Akt pathway. The differential ability of phenylephrine and IGF-I to activate Akt-1 resulted in a differential ability to protect cells from UV-induced apoptosis. These results demonstrate that activation of p70 S6 kinase by the alpha(1A)-adrenergic receptor in Rat-1 fibroblasts occurs in the absence of PI 3-kinase/Akt signaling. Furthermore, this receptor negatively regulates the PI 3-kinase/Akt pathway, resulting in enhanced cell death following apoptotic insult.     

Conjugated linoleic acid inhibits cell proliferation and ErbB3 signaling in HT-29 human colon cell line

Conjugated linoleic acid (CLA) has chemoprotective properties in experimental cancer models, and in vitro studies have shown that CLA inhibits HT-29 colon cancer cell growth. ErbB2 and ErbB3 have been implicated in the development of colon cancer, and both proteins are expressed at high levels in the HT-29 cell line. Activation of ErbB2/ErbB3 heterodimers is regulated by the ErbB3 ligand heregulin. To examine CLA regulation of HT-29 cell proliferation and apoptosis and the influence of CLA on the ErbB3 signaling pathway, HT-29 cells were cultured in the presence of CLA and/or heregulin. CLA inhibited DNA synthesis and induced apoptosis of HT-29 cells. Although the addition of heregulin-alpha led to an increase in cell number, it was not able to counteract the negative growth regulatory effect of CLA. Immunoprecipitation/Western blot studies revealed that CLA inhibited heregulin-alpha-stimulated phosphorylation of ErbB2 and ErbB3, recruitment of the p85 subunit of phosphoinositide 3-kinase (PI3-kinase) to the ErbB3 receptor, ErbB3-associated PI3-kinase activities, and phosphorylation of Akt. CLA decreased ErbB2 and ErbB3 mRNA and protein levels in a dose-dependent manner. In conclusion, we demonstrate that CLA inhibits cell proliferation and stimulates apoptosis in HT-29 cells and that this may be mediated by its ability to downregulate ErbB3 signaling and the PI3-kinase/Akt pathway.     

Targeting of solid tumors and blood malignancies by antibody-based therapies — EGFR-pathway as an example

A well-coordinated interaction between extracellular signals and intracellular response forms the basis of life within multicellular organisms, with growth factors playing a crucial role in these interactions. Discoveries in recent years have shown that components of the Epidermal Growth Factor (EGF) signaling system have frequently been used by cancer cells to autonomously provide survival and proliferation signals. The main focus of this review is the ErbB epidermal growth factor receptor (EGFR) family of receptor tyrosine kinases including ErbB1/EGFR, ErbB2/HER2/neu, ErbB3/HER3, and ErbB4/HER4 as therapeutic targets. Since the ErbB receptor family regulates cell proliferation through the Ras-mitogen-activated protein kinase (RAS/MAPK) pathway, and cell survival and transformation through the phosphatidylinositol 3-kinase (PI3K/AKT) pathway, pharmacological targeting of these pathways is also discussed. We will also address the clinical studies that have been conducted to evaluate antibody-based therapies mostly on solid tumors and hematologic malignancies.     

PKC-ι promotes glioblastoma cell survival by phosphorylating and inhibiting BAD through a phosphatidylinositol 3-kinase pathway

The focus of this research was to investigate the role of protein kinase C-iota (PKC-ι) in regulation of Bad, a pro-apoptotic BH3-only molecule of the Bcl-2 family in glioblastoma. Robust expression of PKC-ι is a hallmark of human glioma and benign and malignant meningiomas. The results were obtained from the two human glial tumor derived cell lines, T98G and U87MG. In these cells, PKC-ι co-localized and directly associated with Bad, as shown by immunofluorescence, immunoprecipitation, and Western blotting. Furthermore, in-vitro kinase activity assay showed that PKC-ι directly phosphorylated Bad at phospho specific residues, Ser-112, Ser-136 and Ser-155 which in turn induced inactivation of Bad and disruption of Bad/Bcl-XL dimer. Knockdown of PKC-ι by siRNA exhibited a corresponding reduction in Bad phosphorylation suggesting that PKC-ι may be a Bad kinase. PKC-ι knockdown also induced apoptosis in both the cell lines. Since, PKC-ι is an essential downstream mediator of the PI (3)-kinase, we hypothesize that glioma cell survival is mediated via a PI (3)-kinase/PDK1/PKC-ι/Bad pathway. Treatment with PI (3)-kinase inhibitors Wortmannin and LY294002, as well as PDK1 siRNA, inhibited PKC-ι activity and subsequent phosphorylation of Bad suggesting that PKC-ι regulates the activity of Bad in a PI (3)-kinase dependent manner. Thus, our data suggest that glioma cell survival occurs through a novel PI (3)-kinase/PDK1/PKC-ι/BAD mediated pathway.     

A natural ErbB4 isoform that does not activate phosphoinositide 3-kinase mediates proliferation but not survival or chemotaxis

ErbB4 is a member of the epidermal growth factor receptor (ErbB) family that mediates cellular responses activated by neuregulins (NRG) and other epidermal growth factor-like growth factors. Two naturally occurring ErbB4 isoforms, ErbB4 CYT-1 and ErbB4 CYT-2, have previously been identified. Unlike ErbB4 CYT-1, ErbB4 CYT-2 lacks a phosphoinositide 3-kinase (PI3-K)-binding site and is incapable of activating PI3-K. We have now examined the consequences of the inability of this isoform to activate PI3-K on cell proliferation, survival, and chemotaxis in response to NRG-1beta: (i) NRG-1beta stimulated proliferation of cells expressing either ErbB4 CYT-1 or ErbB4 CYT-2. Consistent with the mitogenic responsiveness, analysis of downstream signaling showed that Shc and MAPK were phosphorylated after stimulating either isoform with NRG-1beta. (ii) NRG-1beta protected cells expressing ErbB4 CYT-1 but not cells expressing ErbB4 CYT-2 from starvation-induced apoptosis as measured by effects on cell number and 4', 6-diamidino-2-phenylindole staining. Furthermore, in cells expressing ErbB4 CYT-2, Akt, a protein kinase that mediates cell survival, was not phosphorylated. (iii) NRG-1beta stimulated chemotaxis and membrane ruffling in cells expressing ErbB4 CYT-1 but not in cells expressing ErbB4 CYT-2. In summary, ErbB4 CYT-2 can mediate proliferation but not chemotaxis or survival. These results suggest a novel mechanism by which cellular responses such as chemotaxis and survival may be regulated by the expression of alternative receptor-tyrosine kinase isoforms that differ in their coupling to PI3-K signaling.     

Estradiol rapidly activates Akt via the ErbB2 signaling pathway

Previously, we have demonstrated that the two mitogenic growth factors epidermal growth factor and IGF-I can activate Akt and estrogen receptor-alpha (ERalpha) in the hormone-dependent breast cancer cell line, MCF-7. In this report we now show that estradiol can also rapidly activate phosphatidylinositol 3-kinase (PI 3-K)/Akt and that this effect is mediated by the ErbB2 signaling pathway. Treatment of cells with estradiol resulted in phosphorylation of Akt and a 9-fold increase in Akt activity in 10 min. Akt activation was blocked by wortmannin and LY 294,002, two inhibitors of PI 3-K; by genistein, a protein tyrosine kinase inhibitor and an ER agonist; by AG825, a selective ErbB2 inhibitor; and by the antiestrogens ICI 182,780 and 4-hydroxy-tamoxifen; but not by rapamycin, an inhibitor of the ribosomal protein kinase p70S6K; nor by AG30, a selective epidermal growth factor receptor inhibitor. Akt activation by estradiol was abrogated by an arginine-to-cysteine mutation in the pleckstrin homology domain of Akt (R25C). Growth factors also activated Akt in the ER-negative variant of MCF-7, MCF-7/ADR, but estradiol did not induce Akt activity in these cells. Transient transfection of ERalpha into these cells restored Akt activation by estradiol, suggesting that estradiol activation of Akt requires the ERalpha. Estradiol did not activate Akt in MCF-7 cells stably transfected with an anti-ErbB2-targeted ribozyme, further confirming a role for ErbB2. In vitro kinase assays using immunoprecipitation and anti-Akt1, -Akt2, and -Akt3-specific antibodies demonstrated that Akt1 is activated by estradiol in MCF-7 cells whereas Akt3 is the activated isoform in ER-negative MDA-MB231 cells, implying that selective activation of Akt subtypes plays a role in the actions of estradiol. Taken together, our data suggest that estradiol, bound to membrane ERalpha, interacts with and activates an ErbB dimer containing ErbB2, inducing activation of PI 3-K/Akt.     

Survival signaling in type II pneumocytes activated by surfactant protein-A

Surfactant-associated protein-A (SP-A) is a component of pulmonary surfactant that acts as a cytokine through interaction with a cell-surface receptor (SPAR) on lung epithelial cells. SP-A regulates important physiological processes including surfactant secretion, gene expression, and protection against apoptosis. Tyrosine kinase and PI3K inhibitors block effects of SP-A, suggesting that SPAR may be a receptor tyrosine kinase and activate the PI3K-PKB/Akt pathway. Here we report that SP-A treatment leads to rapid tyrosine-specific phosphorylation of several important proteins in lung epithelial cells including insulin receptor substrate-1 (IRS-1), an upstream activator of PI3K. Analysis of anti-apoptotic signaling species downstream of IRS-1 showed activation of PKB/Akt but not of MAPK. Phosphorylation of IkappaB was minimally affected by SP-A as was NFkappaB gel shift activity. However, FKHR was rapidly phosphorylated in response to SP-A and its DNA-binding activity was significantly reduced. Since FKHR is pro-apoptotic, this may play an important role in signaling the anti-apoptotic effects of SP-A. Therefore, we have characterized survival-enhancing signaling activated by SP-A leading from SPAR through IRS-1, PI3K, PKB/Akt, and FKHR. The activity of this pathway may explain, in part, the resilience of type II cells to lung injury and their survival to repopulate alveolar epithelium after peripheral lung damage.     

High density lipoprotein-induced endothelial nitric-oxide synthase activation is mediated by Akt and MAP kinases

High density lipoprotein (HDL) activates endothelial nitric-oxide synthase (eNOS), leading to increased production of the antiatherogenic molecule NO. A variety of stimuli regulate eNOS activity through signaling pathways involving Akt kinase and/or mitogen-activated protein (MAP) kinase. In the present study, we investigated the role of kinase cascades in HDL-induced eNOS stimulation in cultured endothelial cells and COS M6 cells transfected with eNOS and the HDL receptor, scavenger receptor B-I. HDL (10-50 microg/ml, 20 min) caused eNOS phosphorylation at Ser-1179, and dominant negative Akt inhibited both HDL-mediated phosphorylation and activation of the enzyme. Phosphoinositide 3-kinase (PI3 kinase) inhibition or dominant negative PI3 kinase also blocked the phosphorylation and activation of eNOS by HDL. Studies with genistein and PP2 showed that the nonreceptor tyrosine kinase, Src, is an upstream stimulator of the PI3 kinase-Akt pathway in this paradigm. In addition, HDL activated MAP kinase through PI3 kinase, and mitogen-activated protein kinase/extracellular signal-regulated kinase kinase inhibition fully attenuated eNOS stimulation by HDL without affecting Akt or eNOS Ser-1179 phosphorylation. Conversely, dominant negative Akt did not alter HDL-induced MAP kinase activation. These results indicate that HDL stimulates eNOS through common upstream, Src-mediated signaling, which leads to parallel activation of Akt and MAP kinases and their resultant independent modulation of the enzyme.     

The mTOR (mammalian target of rapamycin) kinase maintains integrity of mTOR complex 2

In higher eukaryotes, growth factors promote anabolic processes and stimulate cell growth, proliferation, and survival by activation of the phosphoinositide 3-kinase (PI3K)/Akt pathway. Deregulation of PI3K/Akt signaling is linked to human diseases, including cancer and metabolic disorders. The PI3K-dependent signaling kinase complex mTORC2 (mammalian target of rapamycin complex 2) has been defined as the regulatory Ser-473 kinase of Akt. The regulation of mTORC2 remains very poorly characterized. We have reconstituted mTORC2 by its assembly in vitro or by co-expression its four essential components (rictor, SIN1, mTOR, mLST8). We show that the functional mTOR kinase domain is required for the mTORC2 activity as the Ser-473 kinase of Akt. We also found that mTOR by phosphorylation of SIN1 prevents its lysosomal degradation. Thus, the kinase domain of mTOR is required for the functional activity of mTORC2, and it controls integrity of mTORC2 by maintaining the protein stability of SIN1.     

The T-cell receptor regulates Akt (protein kinase B) via a pathway involving Rac1 and phosphatidylinositide 3-kinase

The serine/threonine kinase Akt (also known as protein kinase B) (Akt/PKB) is activated upon T-cell antigen receptor (TCR) engagement or upon expression of an active form of phosphatidylinositide (PI) 3-kinase in T lymphocytes. Here we report that the small GTPase Rac1 is implicated in this pathway, connecting the receptor with the lipid kinase. We show that in Jurkat cells, activated forms of Rac1 or Cdc42, but not Rho, stimulate an increase in Akt/PKB activity. TCR-induced Akt/PKB activation is inhibited either by PI 3-kinase inhibitors (LY294002 and wortmannin) or by overexpression of a dominant negative mutant of Rac1 but not Cdc42. Accordingly, triggering of the TCR rapidly stimulates a transient increase in GTP-Rac content in these cells. Similar to TCR stimulation, L61Rac-induced Akt/PKB kinase activity is also LY294002 and wortmannin sensitive. However, induction of Akt/PKB activity by constitutive active PI 3-kinase is unaffected when dominant negative Rac1 is coexpressed, placing Rac1 upstream of PI 3-kinase in the signaling pathway. When analyzing the signaling hierarchy in the pathway leading to cytoskeleton rearrangements, we found that Rac1 acts downstream of PI 3-kinase, a finding that is in accordance with numerous studies in fibroblasts. Our results reveal a previously unrecognized role of the GTPase Rac1, acting upstream of PI 3-kinase in linking the TCR to Akt/PKB. This is the first report of a membrane receptor employing Rac1 as a downstream transducer for Akt/PKB activation.