Phosphorylation of Merlin Regulates its Stability and Tumor Suppressive Activity

The neurofibromatosis-2 (NF2) tumor suppressor protein, merlin or schwannomin, inhibits cell proliferation by modulating the growth activities of its binding partners, including the cell surface glycoprotein CD44, membrane-cytoskeleton linker protein ezrin and PIKE (PI 3-kinase enhancer) GTPase, etc. Merlin exerts its growth suppressive activity through a folded conformation that is tightly controlled through phosphorylation by numerous protein kinases including PAK, PKA and Akt. Merlin inhibits PI 3-kinase activity through binding to PIKE-L. Now, we show that merlin is a physiological substrate of Akt, which phosphorylates merlin on both T230 and S315 residues. This phosphorylation abolishes the folded conformation of merlin and inhibits its association with PIKE-L, provoking merlin polyubiquitination and proteasome-mediated degradation. This finding demonstrates a negative feed-back loop from merlin/PIKE-L/PI 3-kinase to Akt in tumors. The proliferation repressive activity of merlin is also partially regulated by S518 phosphorylation. Thus, Akt-mediated merlin T230/S315 phosphorylation, combined with S518 phosphorylation by PAK and PKA, provides new insight into abrogating merlin function in the absence of merlin mutational inactivation.Key Words: Akt, merlin, neurofibromatosis, phosphorylation, cell invasion and migrationNeurofibromatosis 2 (NF2) is a dominantly inherited disorder characterized by bilateral occurrence of vestibular schwannomas and other brain tumors, including meningiomas and ependymomas.¹ The NF2 tumor suppressor protein merlin belongs to the band 4.1 family of cytoskeleton-associated proteins.^2,3 Merlin isoform I possesses a “closed” conformation via an NTD (N-terminal domain)/CTD (Carboxy terminal domain) intramolecular interaction. In contrast, the alternatively spliced merlin isoform II exists in an “open” conformation that cannot function as a negative growth regulator.⁴ Merlin with NF2 patient missense mutations in the NTD or CTD exhibit an “open” conformation and do not suppress cell growth.⁵ Merlin plays a key role in regulating cell proliferation and cell migration. Merlin exerts its growth suppressive activity through intramolecular folding that dictates its binding affinities to various cellular partners including HRS (hepatocyte growth factor regulated tyrosine kinase substrate), CD44 cell surface glycoprotein, schwannomin interacting protein-1 (SCHIP1), βII-spectrin or fodrin, PIKE-L GTPase and other ERM proteins.^6–10 For instance, CD44 preferentially associates with hypophosphorylated merlin, and relatively little phosphorylated merlin binds CD44. Interference with merlin binding to CD44 impairs merlin growth suppression in RT4 rat schwannoma cells.¹¹We have previously shown that the PIKE/PI 3-kinase signaling pathway is negatively regulated by protein 4.1N, a neuronal selective isoform of band 4.1 superfamily.¹² Recently, we show that PIKE-L is an important mediator of merlin growth suppression. We show that merlin blocks cell proliferation by inhibiting PI 3-kinase through binding to PIKE-L. Interestingly, wild-type merlin, but not patient-derived mutant (L64P), binds PIKE-L and inhibits PI 3-kinase activity. This suppression of PI 3-kinase activity results from merlin disrupting the binding of PIKE-L to PI 3-kinase. Mutation of PIKE-L with Proline 187 into Leucine disrupts its interaction with merlin. Accordingly, merlin suppression of PI 3-kinase activity as well as schwannoma cell growth is abrogated by a single PIKE-L point mutation (P187L).¹⁰Merlin is phosphorylated on S518 by members of the PAK family of kinases, including PAK1 and PAK2,^13–15 which mislocates merlin from the plasma membrane to the cytoplasm. A merlin mutant that mimics S518 phosphorylation (S518D) cannot suppress cell growth or motility in RT4 rat schwannoma cells, and leads to dramatic changes in cell morphology and actin cytoskeleton organization.¹⁶ S518 phosphorylation results in impaired merlin NTD/CTD folding as well as altered interactions with critical merlin associated proteins, including CD44 and HRS.¹⁷ Recently, Alfthan and colleagues demonstrated that Protein Kinase-A (PKA) induces merlin phosphorylation on both N-and C-terminal residues.¹⁸ In addition to S518 phosphorylation, PKA can phosphorylate merlin at S66 in the N-terminal domain (Fig. 1). When PAK activity is suppressed, merlin can still be phosphorylated by PKA in cells, indicating that these two kinases function independently. The N-terminus of ezrin strongly binds to a PKA-phosphorylated, but not unphosphorylated, merlin CTD. In contrast, PAK2-induced S518 phosphorylation has a minimal effect on the interaction between full-length merlin and full-length ezrin.¹⁷ Besides regulation of cell growth, merlin also mediates cell motility presumably through directly binding to actin cytoskeleton.¹⁹ Depletion of merlin in normal fibroblast results in enhanced cell invasion. Nevertheless, expression of merlin attenuates Y397 phosphorylation on FAK, an essential player in cell migration and invasion. This observation might provide a molecular mechanism accounting for merlin inhibitory activity in cell motility.²⁰Open in a separate windowFigure 1Merlin phosphorylation sites by various kinases.In addition to PAK and PKA, we show that Akt potently phosphorylates merlin at both T230 and S315 residues. Blocking one site phosphorylation abolishes the other site phosphorylation by Akt, indicating that these two phosphorylation sites are mutually regulated.²¹ The physiological significance of the tight control on merlin phosphorylation by Akt remains incompletely understood. Presumably, only when mitogenic signal or oncogenic stress is strongly enough to provoke cell proliferation or migration, does Akt simultaneously phosphorylate both sites. Akt phosphorylation of merlin attenuates the NTD/CTD interaction and inhibits its binding activity to PIKE-L, CD44 and ezrin. Further, phosphorylation mediates the biological activities of merlin, as expression of a phosphomimetic merlin mutant (T230DS315D) increases cell motility and proliferation in a rat schwannoma cell line (Fig. 2). By contrast, expression of a mutant (T230A/S315A) that was unable to undergo phosphorylation inhibited cell growth and motility. The F1 motif in FERM proteins including merlin exhibits an ubiquitin-like structure. This domain facilitates MDM2 degradation and stimulates the ubiquitination and degradation of TRBP, a double-stranded RNA binding protein. Surprisingly, inhibition of the proteasome does not affect total merlin protein levels in human glioblastoma cells, but leads to a marked increase of phospho-S315 merlin. Simultaneous treatment with MG132, which blocks proteasome-mediated degradation and PI 3-kinase inhibitor, wortmannin, which inhibits Akt phosphorylation of merlin, substantially enhances merlin levels. Coimmunoprecipitation studies demonstrate that Akt-phosphorylated merlin is rapidly ubiquitinated, presumably by spectrin, which binds to merlin and possesses ubiquitin-conjugating and ubiquitin E3 ligase function. However, S518 phosphorylation fails to trigger merlin ubiquitination, suggesting that Akt, but not PAK or PKA, phosphorylation selectively elicits merlin ubiquitination. Using a panel of human primary nervous system tumors, we found that merlin phosphorylation by Akt also mediates its degradation in primary tumors. Accordingly, tumors that possess high levels of phospho-Akt exhibited low levels of merlin. Therefore, our data suggest a novel role for Akt in promoting phosphorylation and subsequent degradation of merlin. Loss of merlin has been linked to schwannomas and other nervous system tumors, and these results indicate that inhibitors for PI 3-kinase/Akt pathway might restore merlin function in tumors.Open in a separate windowFigure 2The model for Akt interaction with merlin and its phosphorylation.     

Akt phosphorylation regulates the tumour-suppressor merlin through ubiquitination and degradation

The neurofibromatosis-2 (NF2) tumour-suppressor gene encodes an intracellular membrane-associated protein, called merlin, whose growth-suppressive function is dependent on its ability to form interactions through its intramolecular amino-terminal domain (NTD) and carboxy-terminal domain (CTD). Merlin phosphorylation plays a critical part in dictating merlin NTD/CTD interactions as well as in controlling binding to its effector proteins. Merlin is partially regulated by phosphorylation of Ser 518, such that hyperphosphorylated merlin is inactive and fails to form productive intramolecular and intermolecular interactions. Here, we show that the protein kinase Akt directly binds to and phosphorylates merlin on residues Thr 230 and Ser 315, which abolishes merlin NTD/CTD interactions and binding to merlin's effector protein PIKE-L and other binding partners. Furthermore, Akt-mediated phosphorylation leads to merlin degradation by ubiquitination. These studies demonstrate that Akt-mediated merlin phosphorylation regulates the function of merlin in the absence of an inactivating mutation.     

Cyclic AMP-dependent protein kinase phosphorylates merlin at serine 518 independently of p21-activated kinase and promotes merlin-ezrin heterodimerization

Mutations in the NF2 tumor suppressor gene encoding merlin induce the development of tumors of the nervous system. Merlin is highly homologous to the ERM (ezrin-radixin-moesin) family of membrane/cytoskeleton linker proteins. However, the mechanism for the tumor suppressing activity of merlin is not well understood. Previously, we characterized a novel role for merlin as a protein kinase A (PKA)-anchoring protein, which links merlin to the cAMP/PKA signaling pathway. In this study we show that merlin is also a target for PKA-induced phosphorylation. In vitro [gamma-(33)P]ATP labeling revealed that both the merlin N and C termini are phosphorylated by PKA. Furthermore, both in vitro and in vivo phosphorylation studies of the wild-type and mutated C termini demonstrated that PKA can phosphorylate merlin at serine 518, a site that is phosphorylated also by p21-activated kinases (PAKs). Merlin was phosphorylated by PKA in cells in which PAK activity was suppressed, indicating that the two kinases function independently. Both in vitro and in vivo interaction studies indicated that phosphorylation of serine 518 promotes heterodimerization between merlin and ezrin, an event suggested to convert merlin from a growth-suppressive to a growth-permissive state. This study provides further evidence on the connection between merlin and cAMP/PKA signaling and suggests a role for merlin in the cAMP/PKA transduction pathway.     

Molecular Conformation of the Full-Length Tumor Suppressor NF2/Merlin—A Small-Angle Neutron Scattering Study

The tumor suppressor protein Merlin inhibits cell proliferation upon establishing cell–cell contacts. Because Merlin has high level of sequence similarity to the Ezrin-Radixin-Moesin family of proteins, the structural model of Ezrin-Radixin-Moesin protein autoinhibition and cycling between closed/resting and open/active conformational states is often employed to explain Merlin function. However, recent biochemical studies suggest alternative molecular models of Merlin function. Here, we have determined the low-resolution molecular structure and binding activity of Merlin and a Merlin(S518D) mutant that mimics the inactivating phosphorylation at S518 using small-angle neutron scattering and binding experiments. Small-angle neutron scattering shows that, in solution, both Merlin and Merlin(S518D) adopt a closed conformation, but binding experiments indicate that a significant fraction of either Merlin or Merlin(S518D) is capable of binding to the target protein NHERF1. Upon binding to the phosphatidylinositol 4,5-bisphosphate lipid, the wild-type Merlin adopts a more open conformation than in solution, but Merlin(S518D) remains in a closed conformation. This study supports a rheostat model of Merlin in NHERF1 binding and contributes to resolving a controversy about the molecular conformation and binding activity of Merlin.     

Merlin phosphorylation by p21-activated kinase 2 and effects of phosphorylation on merlin localization

The Nf2 tumor suppressor gene product merlin is related to the membrane-cytoskeleton linker proteins of the band 4.1 superfamily, including ezrin, radixin, and moesin (ERMs). Merlin is regulated by phosphorylation in a Rac/cdc42-dependent fashion. We report that the phosphorylation of merlin at serine 518 is induced by the p21-activated kinase PAK2. This is demonstrated by biochemical fractionation, use of active and dominant-negative mutants of PAK2, and immunodepletion. By using wild-type and mutated forms of merlin and phospho-directed antibodies, we show that phosphorylation of merlin at serine 518 leads to dramatic protein relocalization.     

The merlin interacting proteins reveal multiple targets for NF2 therapy

The neurofibromatosis 2 (NF2) tumor suppressor protein merlin is commonly mutated in human benign brain tumors. The gene altered in NF2 was located on human chromosome 22q12 in 1993 and the encoded protein named merlin and schwannomin. Merlin has homology to ERM family proteins, ezrin, radixin, and moesin, within the protein 4.1 superfamily. In efforts to determine merlin function several groups have discovered 34 merlin interacting proteins, including ezrin, radixin, moesin, CD44, layilin, paxillin, actin, N-WASP, betaII-spectrin, microtubules, TRBP, eIF3c, PIKE, NHERF, MAP, RalGDS, RhoGDI, EG1/magicin, HEI10, HRS, syntenin, caspr/paranodin, DCC, NGB, CRM1/exportin, SCHIP1, MYPT-1-PP1delta, RIbeta, PKA, PAK (three types), calpain and Drosophila expanded. Many of the proteins that interact with the merlin N-terminal domain also bind ezrin, while other merlin interacting proteins do not bind other members of the ERM family. Merlin also interacts with itself. This review describes these proteins, their possible roles in NF2, and the resultant hypothesized merlin functions. Review of all of the merlin interacting proteins and functional consequences of losses of these interactions reveals multiple merlin actions in PI3-kinase, MAP kinase and small GTPase signaling pathways that might be targeted to inhibit the proliferation of NF2 tumors.     

The NF2 tumor suppressor gene product, merlin, inhibits cell proliferation and cell cycle progression by repressing cyclin D1 expression

Inactivation of the NF2 tumor suppressor gene has been observed in certain benign and malignant tumors. Recent studies have demonstrated that merlin, the product of the NF2 gene, is regulated by Rac/PAK signaling. However, the mechanism by which merlin acts as a tumor suppressor has remained obscure. In this report, we show that adenovirus-mediated expression of merlin in NF2-deficient tumor cells inhibits cell proliferation and arrests cells at G1 phase, concomitant with decreased expression of cyclin D1, inhibition of CDK4 activity, and dephosphorylation of pRB. The effect of merlin on cell cycle progression was partially overridden by ectopic expression of cyclin D1. RNA interference experiments showed that silencing of the endogenous NF2 gene results in upregulation of cyclin D1 and S-phase entry. Furthermore, PAK1-stimulated cyclin D1 promoter activity was repressed by cotransfection of NF2, and PAK activity was inhibited by expression of merlin. Interestingly, the S518A mutant form of merlin, which is refractory to phosphorylation by PAK, was more efficient than the wild-type protein in inhibiting cell cycle progression and in repressing cyclin D1 promoter activity. Collectively, our data indicate that merlin exerts its antiproliferative effect, at least in part, via repression of PAK-induced cyclin D1 expression, suggesting a unifying mechanism by which merlin inactivation might contribute to the overgrowth seen in both noninvasive and malignant tumors.     

Activation of p21-activated kinase 2 by human immunodeficiency virus type 1 Nef induces merlin phosphorylation

The accessory human immunodeficiency virus type 1 (HIV-1) protein Nef activates the autophosphorylation activity of p21-activated kinase 2 (PAK2). Merlin, a cellular substrate of PAK2, is homologous to the ezrin-radixin-moesin family and plays a critical role in Rac signaling. To assess the possible impact on host cell metabolism of Nef-induced PAK2 activation, we investigated the phosphorylation of merlin in Nef expressing cells. Here we report that Nef induces merlin phosphorylation in multiple cell lines independently of protein kinase A. This intracellular phosphorylation of merlin directly correlates with in vitro assay of the autophosphorylation activity of Nef-activated PAK2. Importantly, merlin phosphorylation induced by Nef was also observed in human primary T cells. The finding that Nef induces phosphorylation of the key signaling molecule merlin suggests several possible roles for PAK2 activation in HIV pathogenesis.     

Regulation of merlin by protein phosphatase 1-TIMAP and EBP50 in endothelial cells

Merlin (moesin-ezrin-radixin like protein), the product of neurofibromatosis type 2 gene, was primarily recognized as a tumor suppressor, but it also functions as a membrane-cytoskeletal linker and regulator of multiple signaling pathways. The activity and localization of merlin is regulated by head to tail folding that is controlled by phosphorylation of the Ser518 side chain. Merlin localizes in the nucleus when the Ser518 side chain is not phosphorylated, while the phosphorylated form is present in the cytoplasm and the plasma membrane. In this work interactions and their impact on the subcellular localization and phosphorylation state of the Ser518 side chain of merlin were investigated in endothelial cells. It is shown that merlin (dephospho-Ser518 form) interacts in the nucleus of endothelial cells with the scaffolding protein EBP50, a member of the Na⁺/H⁺exchanger regulatory factor family. Upon EBP50 depletion, merlin translocated from the nucleus, suggesting that binding of merlin to EBP50 is critical in the nuclear localization of merlin. Along with the translocation, the phosphorylation level of phospho-Ser518-merlin was increased in EBP50 depleted cells. TIMAP (TGFβ-inhibited membrane-associated protein), a type 1 protein phosphatase (PP1) regulatory subunit, was newly recognized as an interacting partner for merlin. Domain mapping using truncated mutant forms in GST pull down revealed that the N-terminal half of TIMAP (aa 1-290) and the FERM domain of merlin are the regions responsible for the interaction.The catalytic subunit of PP1 (PP1c) was present in all merlin-TIMAP pull down or immunoprecipitation samples demonstrating that merlin actually interacts with the PP1c-TIMAP holoenzyme. On the other hand, from TIMAP depleted cells, without its targeting protein, PP1c could not bind to merlin. Also, when the phosphatase activity of PP1c-TIMAP was inhibited either with depletion of TIMAP or by treatment of the cells with specific PP1 inhibitor, there was an increase in the amount of phospho-Ser518 form of merlin in the membrane of the cells. These data strongly suggest that the PP1c-TIMAP- complex dephosphorylates phospho-Ser518-merlin. ECIS measurements indicate that phospho-merlin accelerates in vitro wound healing of the endothelial monolayer.In conclusion, in endothelial cells, EBP50 is required for the nuclear localization of merlin and the PP1c-TIMAP holoenzyme plays an important role in the dephosphorylation of merlin on its Ser518 side chain, which influence cell migration and proliferation.     

PIKE GTPase signaling and function

PIKE (PI 3-Kinase Enhancer) is a recently identified brain specific nuclear GTPase, which binds PI 3-kinase and stimulates its lipid kinase activity. Nerve growth factor treatment leads to PIKE activation by triggering the nuclear translocation of phospholipase C-gamma1 (PLC-gamma1), which acts as a physiologic guanine nucleotide exchange factor (GEF) for PIKE through its SH3 domain. To date, three forms of PIKE have been characterized: PIKE-S, PIKE-L and PIKE-A. PIKE-S is initially identified shorter isoform. PIKE-L, a longer isoform of PIKE gene, differs from PIKE-S by C-terminal extension containing Arf-GAP (ADP ribosylation factor-GTPase Activating Protein) and two ankyrin repeats domains. In contrast to the exclusive nuclear localization of PIKE-S, PIKE-L occurs in both the nucleus and the cytoplasm. PIKE-L physiologically associates with Homer 1, an mGluR I binding adaptor protein. The Homer/PIKE-L complex couples PI 3-kinase to mGluR I and regulates a major action of group I mGluRs, prevention of neuronal apoptosis. More recently, a third PIKE isoform, PIKE-A was identified in human glioblastoma multiforme brain cancers. Unlike the brain specific PIKE-L and -S isoforms, PIKE-A distributes in various tissues. PIKE-A contains the same domains present in PIKE-L but lacks N-terminal proline-rich domain (PRD), which binds PI 3-kinase and PLC-gamma1. Instead, PIKE-A specifically binds to active Akt and upregulates its activity in a GTP-dependent manner, mediating human cancer cell invasion and preventing apoptosis. Thus, PIKE extends its roles from the nucleus to the cytoplasm, mediating cellular processes from cell invasion to programmed cell death.     

Protein Kinase A-Dependent and -Independent Signaling Pathways Contribute to Cyclic AMP-Stimulated Proliferation

The effects of cyclic AMP (cAMP) on cell proliferation are cell type specific. Although the growth-inhibitory effects of cAMP have been well studied, much less is known regarding how cAMP stimulates proliferation. We report that cAMP stimulates proliferation through both protein kinase A (PKA)-dependent and PKA-independent signaling pathways and that phosphatidylinositol 3-kinase (PI3K) is required for cAMP-stimulated mitogenesis. In cells where cAMP is a mitogen, cAMP-elevating agents stimulate membrane ruffling, Akt phosphorylation, and p70 ribosomal S6 protein kinase (p70s6k) activity. cAMP effects on ruffle formation and Akt were PKA independent but sensitive to wortmannin. In contrast, cAMP-stimulated p70s6k activity was repressed by PKA inhibitors but not by wortmannin or microinjection of the N-terminal SH2 domain of the p85 regulatory subunit of PI3K, indicating that p70s6k and Akt can be regulated independently. Microinjection of highly specific inhibitors of PI3K or Rac1, or treatment with the p70s6k inhibitor rapamycin, impaired cAMP-stimulated DNA synthesis, demonstrating that PKA-dependent and -independent pathways contribute to cAMP-mediated mitogenesis. Direct elevation of PI3K activity through microinjection of an antibody that stimulates PI3K activity or stable expression of membrane-localized p110 was sufficient to confer hormone-independent DNA synthesis when accompanied by elevations in p70s6k activity. These findings indicate that multiple pathways contribute to cAMP-stimulated mitogenesis, only some of which are PKA dependent. Furthermore, they demonstrate that the ability of cAMP to stimulate both p70s6k- and PI3K-dependent pathways is an important facet of cAMP-regulated cell cycle progression.     

Gbetagamma subunits stimulate p21-activated kinase 1 (PAK1) through activation of PI3-kinase and Akt but act independently of Rac1/Cdc42

The p21-activated kinase (PAK) family is homologous to the yeast sterile 20 (Ste20) and regulates a wide variety of cellular responses, including cell morphology, proliferation, and survival. In this study we examined the activation of PAK1 by Gbetagamma subunits. Co-transfection of COS7 cells with Gbeta1gamma2 or Gbeta1gamma5 was sufficient to induce agonist-independent activation of PAK1. Expression of dominant/negative Rac, Cdc42, or Ras did not inhibit this Gbetagamma-dependent activation. Wortmannin, which inhibits phosphoinositide 3-kinase (PI3-kinase) activity, and expression of a dominant/negative form of Akt were sufficient to abrogate the activation of PAK1 that was induced by Gbetagamma. These results reveal that stimulation of PAK1 by Gbetagamma can occur via a PI3-kinase and Akt pathway that does not require Rac1 or Cdc42.     

The phosphoinositol-3-kinase-protein kinase B/Akt pathway is critical for Pseudomonas aeruginosa strain PAK internalization

Several Pseudomonas aeruginosa strains are internalized by epithelial cells in vitro and in vivo, but the host pathways usurped by the bacteria to enter nonphagocytic cells are not clearly understood. Here, we report that internalization of strain PAK into epithelial cells triggers and requires activation of phosphatidylinositol 3-kinase (PI3K) and protein kinase B/Akt (Akt). Incubation of Madin-Darby canine kidney (MDCK) or HeLa cells with the PI3K inhibitors LY294002 (LY) or wortmannin abrogated PAK uptake. Addition of the PI3K product phosphatidylinositol 3,4,5-trisphosphate [PtdIns(3,4,5)P3] to polarized MDCK cells was sufficient to increase PAK internalization. PtdIns(3,4,5)P3 accumulated at the site of bacterial binding in an LY-dependent manner. Akt phosphorylation correlated with PAK invasion. The specific Akt phosphorylation inhibitor SH-5 inhibited PAK uptake; internalization also was inhibited by small interfering RNA-mediated depletion of Akt phosphorylation. Expression of constitutively active Akt was sufficient to restore invasion when PI3K signaling was inhibited. Together, these results demonstrate that the PI3K signaling pathway is necessary and sufficient for the P. aeruginosa entry and provide the first example of a bacterium that requires Akt for uptake into epithelial cells.     

5(S)-hydroxyeicosatetraenoic acid stimulates DNA synthesis in human microvascular endothelial cells via activation of Jak/STAT and phosphatidylinositol 3-kinase/Akt signaling,leading to induction of expression of basic fibroblast growth factor 2

To understand the role of eicosanoids in angiogenesis, we have studied the effect of lipoxygenase metabolites of arachidonic acid on human microvascular endothelial cell (HMVEC) DNA synthesis. Among the various lipoxygenase metabolites of arachidonic acid tested, 5(S)-hydroxyeicosatetraenoic acid (5(S)-HETE) induced DNA synthesis in HMVEC. 5(S)-HETE also stimulated Jak-2, STAT-1, and STAT-3 tyrosine phosphorylation and STAT-3-DNA binding activity. Tyrphostin AG490, a specific inhibitor of Jak-2, significantly reduced tyrosine phosphorylation and DNA binding activity of STAT-3 and DNA synthesis induced by 5(S)-HETE. In addition, 5(S)-HETE stimulated phosphatidylinositol 3-kinase (PI3-kinase) activity and phosphorylation of its downstream targets Akt, p70S6K, and 4E-BP1 and their effector molecules ribosomal protein S6 and eIF4E. LY294002 and rapamycin, potent inhibitors of PI3-kinase and mTOR, respectively, also blocked the DNA synthesis induced by 5(S)-HETE. Interestingly, AG490 attenuated 5(S)-HETE-induced PI3-kinase activity and phosphorylation of Akt, p70S6K, ribosomal protein S6, 4E-BP1, and eIF4E. 5(S)-HETE induced the expression of basic fibroblast growth factor 2 (bFGF-2) in a Jak-2- and PI3-kinase-dependent manner. In addition, a neutralizing anti-bFGF-2 antibody completely blocked 5(S)-HETE-induced DNA synthesis in HMVEC. Together these results suggest that 5(S)-HETE stimulates HMVEC growth via Jak-2- and PI3-kinase-dependent induction of expression of bFGF-2. These findings also reveal a cross-talk between Jak-2 and PI3-kinase in response to 5(S)-HETE in HMVEC.     

Akt2, a novel functional link between p38 mitogen-activated protein kinase and phosphatidylinositol 3-kinase pathways in myogenesis

Activation of either the phosphatidylinositol 3-kinase (PI 3-kinase)/Akt or the p38 mitogen-activated protein kinase (MAPK) signaling pathways accelerates myogenesis but only when the reciprocal pathway is functional. We therefore examined the hypothesis that cross-activation between these signaling cascades occurs to orchestrate myogenesis. We reveal a novel and reciprocal cross-talk and activation between the PI 3-kinase/Akt and p38 MAPK pathways that is essential for efficient myoblast differentiation. During myoblast differentiation, Akt kinase activity correlated with S473 but not T308 phosphorylation and occurred 24 h after p38 activation. Inhibition or activation of p38 with SB203580, dominant-negative p38, or MKK6EE regulated Akt kinase activity. Analysis of Akt isoforms revealed a specific increase in Akt2 protein levels that coincided with AktS473 phosphorylation during myogenesis and an enrichment of S473-phosphorylated Akt2. Akt2 promoter activity and protein levels were regulated by p38 activation, thus providing a mechanism for communication. Subsequent Akt activation by S473 phosphorylation was PI 3-kinase dependent and specific for Akt2 rather than Akt1. Complementary to p38-mediated transactivation of Akt, activation or inhibition of PI 3-kinase regulated p38 activity upstream of MKK6, demonstrating reciprocal communication and positive feedback characteristic of myogenic regulation. Our findings have identified novel communication between p38 MAPK and PI 3-kinase/Akt via Akt2.     

Somatostatin interferes with thyrotropin-induced G1-S transition mediated by cAMP-dependent protein kinase and phosphatidylinositol 3-kinase. Involvement of RhoA and cyclin E x cyclin-dependent kinase 2 complexes

cAMP-mediated cell proliferation is a complex process that involves multiple pathways. Using a cAMP-dependent cell system, FRTL-5 thyroid cells, we have previously demonstrated the existence of a precise autocrine loop in the control of cell proliferation that involves the positive effector thyrotropin (TSH) and the general inhibitor somatostatin. In search of the regulatory mechanisms responsible for the TSH and somatostatin control of cell proliferation, we analyzed the cell cycle regulatory proteins and the cellular pathways involved in the action of both signals. The results show that specific inhibition of cAMP-dependent protein kinase (PKA) and phosphatidylinositol (PI) 3-kinase blocks independently TSH-induced FRTL-5 cell proliferation and that somatostatin interferes with both signals. Each pathway activates different proteins required for G(1)/S progression. Thus, PKA is responsible for the TSH-induction of 3-hydroxy-3-methylglutaryl-CoA reductase mRNA levels, RhoA activation, and down-regulation of p27(kip1). These correlated events are necessary for FRTL-5 cell proliferation after TSH stimulation. Moreover, TSH through PKA pathway increases cyclin-dependent kinase 2 levels, whereas PI 3-kinase signaling increases cyclin E levels. Together, both pathways finally converge, increasing the formation and activation of cyclin E x cyclin-dependent kinase 2 complexes and the phosphorylation of the retinoblastoma protein, two important steps in the transition from G(1) to S phase in growth-stimulated cells. Somatostatin exerts its antiproliferative effect inhibiting more upstream the TSH stimulation of PKA and PI 3-kinase, interfering with the TSH-mediated increases of intracellular cAMP levels by inactivation of adenylyl cyclase activity. Together, these results suggest the existence of a PKA-dependent pathway and a new PKA-independent PI 3-kinase pathway in the TSH/cAMP-mediated proliferation of FRTL-5 thyroid cells.     

Merlin, the product of the Nf2 tumor suppressor gene, is an inhibitor of the p21-activated kinase, Pak1

The Nf2 tumor suppressor gene codes for merlin, a protein whose function has been elusive. We describe a novel interaction between merlin and p21-activated kinase 1 (Pak1), which is dynamic and facilitated upon increased cellular confluence. Merlin inhibits the activation of Pak1, as the loss of merlin expression results in the inappropriate activation of Pak1 under conditions associated with low basal activity. Conversely, the overexpression of merlin in cells that display a high basal activity of Pak1 resulted in the inhibition of Pak1 activation. This inhibitory function of merlin is mediated through its binding to the Pak1 PBD and by inhibiting Pak1 recruitment to focal adhesions. This link provides a possible mechanism for the effect of loss of merlin expression in tumorigenesis.