Hdm2 nuclear export, regulated by insulin-like growth factor-I/MAPK/p90Rsk signaling, mediates the transformation of human cells

Insulin-like growth factor (IGF)-I receptor activation leads to enhanced proliferation and cell survival via the MAP kinase and phosphatidylinositol 3-kinase-signaling pathways. Upon stimulation by IGF-I, the Hdm2 oncoprotein is phosphorylated by AKT, leading to its rapid nuclear translocation and subsequent inhibition of p53. We now show that IGF-I stimulation regulates the nuclear export of Hdm2 and p53 via the MAP kinase pathway. Inhibition of p38 MAPK or MEK via pharmacological means or expression of dominant negative proteins inhibited the cytoplasmic accumulation of Hdm2 and increased Hdm2 and p53 protein levels, whereas constitutively active p90Rsk promoted the nuclear export of Hdm2. Expression of constitutively active p90Rsk with E1A, oncogenic H-Ras, and hTERT resulted in the anchorage-independent growth of normal human fibroblasts. Our findings link p90Rsk-mediated modulation of Hdm2 nuclear to cytoplasmic shuttling with the diminished ability of p53 to regulate cell cycle checkpoints that ultimately leads to transformation.     

Intrinsically unstructured domains of Arf and Hdm2 form bimolecular oligomeric structures in vitro and in vivo

Arf, Hdm2, and p53 regulate the tumor-suppressor pathway that is most frequently disrupted in human cancer. In the absence of tumorigenic stress, Hdm2 actively attenuates p53-dependent cell cycle arrest and apoptosis by mediating ubiquitination-dependent degradation of p53. Mitogenic stress activates Arf, which indirectly activates p53 by binding to and nullifying the anti-p53 activities of Hdm2. Small conserved domains within Arf and Hdm2 mediate their direct interaction. Individually, these domains are intrinsically unstructured and, when combined in vitro, cofold into bimolecular oligomeric structures that resemble amyloid fibrils in some features. Detailed structural characterization of Hdm2/Arf complexes has previously been hampered by their heterogeneity and large size. Here, we report that a nine-residue fragment of the N-terminus of mouse Arf (termed "A1-mini") cofolds specifically with the Arf-binding domain of Hdm2 to form bimolecular oligomers. We characterized these unprecedented structures using analytical ultracentrifugation and NMR spectroscopy, providing insights into their structural organization. The A1-mini peptide not only binds specifically to Hdm2 in vitro but also recapitulates the nucleolar localization features of full-length Arf in cells. Furthermore, larger fragments of Arf that contain the A1-mini segment have previously been shown to activate p53 in mouse and human cells. Our studies provide the first insights into the molecular basis through which Arf nullifies the p53-inhibiting activity of Hdm2, indirectly activating the tumor-suppressor function of p53 in mammalian cells.     

Identity of common phosphoprotein substrates stimulated by interleukin 2 and diacylglycerol suggests a role of protein kinase C for IL 2 signal transduction

Interleukin 2 (IL 2) is a polypeptide growth factor essential for the proliferation and differentiation of T lymphocytes, large granulocytic lymphocytes, and, potentially, cells of the antibody-producing lineage, B lymphocytes. Many of the biological properties of IL 2 may be mimicked or potentiated by a potent class of tumor promoters, phorbol esters. Phorbol esters have recently been shown to associate with and activate a unique phospholipid/Ca2+-dependent phosphotransferase, protein kinase C (PK-C). Utilizing two-dimensional gel electrophoresis, we have compared the IL 2 and diacylglycerol-induced protein phosphorylation patterns of several IL 2-dependent murine cell lines. Both IL 2 and synthetic diacylglycerol, 1-oleyl-2-acetylglycerol (OAG), stimulated phosphorylation of a number of protein substrates in intact cells compared to unstimulated controls. Three groups of substrates were identified; the first showed increased phosphorylation following stimulation with either IL 2 or OAG, while the second and third groups showed increased phosphorylation following stimulation with IL 2 but not OAG, and with OAG but not IL 2, respectively. Here, we characterize the kinetics of phosphorylation of one cellular substrate, p68, which appears to be phosphorylated in response to direct activators of PK-C or lymphoid or myeloid growth factors in their respective lineage cell lines. The observation that IL 2 also stimulates a unique series of phosphoproteins in addition to those induced by direct PK-C activators suggests that IL 2 may initiate additional protein kinase activities, unrelated to PK-C, which may also be critical for the ligand-receptor signal transduction process regulating growth and gene expression.     

MKP3 negatively modulates PDGF-induced Akt and Erk5 phosphorylation as well as chemotaxis

MAP kinase phosphatase-3 (MKP3), also known as DUSP6 or Pyst1, is a dual specificity phosphatase considered to selectively dephosphorylate extracellular-signal-regulated kinase 1/2 (Erk1/2). Here, we report that in NIH3T3 cells, MKP3 is induced in response to platelet-derived growth factor (PDGF)-BB treatment in an Erk1/2- and phosphatidylinositol 3-kinase (PI3K)-dependent manner, but independently of Erk5 expression. Silencing of MKP3 expression did not affect PDGF-BB-induced Erk1/2 or p38 phosphorylation; however, their basal level of phosphorylation was elevated. Furthermore, we found that PDGF-BB-mediated activation of Erk5 and Akt was enhanced when the MKP3 expression was reduced. Interfering with Mek1/2 or PI3K using the inhibitors CI-1040 and LY-294002, respectively, inhibited PDGF-BB-induced MKP3 expression. Functionally, we found that MKP3 silencing did not affect cell proliferation, but enhanced the chemotactic response toward PDGF-BB. Although both Akt and Erk5 have been linked to increased cell survival, downregulation of MKP3 did not alter the ability of PDGF-BB to protect NIH3T3 cells from starvation-induced apoptosis. However, we observed an increased apoptosis in untreated cells with reduced MKP3 expression. In summary, our data indicate that there is negative cross-talk between Erk1/2 and Erk5 that involves regulation of MKP3 expression, and that PI3K in addition to promoting Akt phosphorylation also negatively modulates Akt, through MKP3 expression.     

Rheb GTPase Controls Apoptosis by Regulating Interaction of FKBP38 with Bcl-2 and Bcl-XL

FKBP38 is a member of the family of FK506-binding proteins that acts as an inhibitor of the mammalian target of rapamycin (mTOR). The inhibitory action of FKBP38 is antagonized by Rheb, an oncogenic small GTPase, which interacts with FKBP38 and prevents its association with mTOR. In addition to the role in mTOR regulation, FKBP38 is also involved in binding and recruiting Bcl-2 and Bcl-X_L, two anti-apoptotic proteins, to mitochondria. In this study, we investigated the possibility that Rheb controls apoptosis by regulating the interaction of FKBP38 with Bcl-2 and Bcl-X_L. We demonstrate in vitro that the interaction of FKBP38 with Bcl-2 is regulated by Rheb in a GTP-dependent manner. In cultured cells, the interaction is controlled by Rheb in response to changes in amino acid and growth factor conditions. Importantly, we found that the Rheb-dependent release of Bcl-X_L from FKBP38 facilitates the association of this anti-apoptotic protein with the pro-apoptotic protein Bak. Consequently, when Rheb activity increases, cells become more resistant to apoptotic inducers. Our findings reveal a novel mechanism through which growth factors and amino acids control apoptosis.     

Src-mediated coupling of focal adhesion kinase to integrin alpha(v)beta5 in vascular endothelial growth factor signaling

The p38 mitogen-activated protein kinase (p38) is activated in response to environmental stress and inflammatory cytokines. Although several growth factors, including fibroblast growth factor (FGF)-2, mediate activation of p38, the consequences for growth factor-dependent cellular functions have not been well defined. We investigated the role of p38 activation in FGF-2-induced angiogenesis. In collagen gel cultures, bovine capillary endothelial cells formed tubular growth-arrested structures in response to FGF-2. In these collagen gel cultures, p38 activation was induced more potently by FGF-2 treatment compared with that in proliferating cultures. Treatment with the p38 inhibitor SB202190 enhanced FGF-2-induced tubular morphogenesis by decreasing apoptosis, increasing DNA synthesis and cell proliferation, and enhancing the kinetics of cell differentiation including increased expression of the Notch ligand Jagged1. Overexpression of dominant negative mutants of the p38-activating kinases MKK3 and MKK6 also supported FGF-2-induced tubular morphogenesis. Sustained activation of p38 by FGF-2 was identified in vascular endothelial cells in vivo in the chick chorioallantoic membrane (CAM). SB202190 treatment enhanced FGF-2-induced neovascularization in the CAM, but the vessels displayed abnormal features indicative of hyperplasia of endothelial cells. These results implicate p38 in organization of new vessels and suggest that p38 is an essential regulator of FGF-2-driven angiogenesis.     

Signal pathways involved in the regulation of prostaglandin E synthase-1 in human gingival fibroblasts

Microsomal prostaglandin E synthase-1 (mPGES-1) is the terminal enzyme regulating the synthesis of prostaglandin E2 (PGE2) in inflammatory conditions. In this study we investigated the regulation of mPGES-1 in gingival fibroblasts stimulated with the inflammatory mediators interleukin-1 beta (IL-1beta) and tumour necrosis factor alpha (TNFalpha). The results showed that IL-1beta and TNFalpha induce the expression of mPGES-1 without inducing the expression of early growth response factor-1 (Egr-1). Treatment of the cells with the PLA2 inhibitor 4-bromophenacyl bromide (BPB) decreased the cytokine-induced mPGES-1 expression accompanied by decreased PGE2 production whereas the addition of arachidonic acid (AA) upregulated mPGES-1 expression and PGE2 production. The protein kinase C (PKC) activator PMA did not upregulate the expression of mPGES-1 in contrast to COX-2 expression and PGE2 production. In addition, inhibitors of PKC, tyrosine and p38 MAP kinase markedly decreased the cytokine-induced PGE2 production but not mPGES-1 expression. Moreover, the prostaglandin metabolites PGE2 and PGF2alpha induced mPGES-1 expression as well as upregulated the cytokine-induced mPGES-1 expression indicating positive feedback regulation of mPGES-1 by prostaglandin metabolites. The peroxisome proliferator-activated receptor-gamma (PPARgamma) ligand, 15-deoxy-Delta12,14-prostaglandin J2 (15d-PGJ2), decreased mPGES-1 expression but not COX-2 expression or PGE2 production. The results indicate that the inflammatory-induced mPGES-1 expression is regulated by PLA2 and 15d-PGJ2 but not by PKC, tyrosine kinase or p38 MAP kinase providing new insights into the regulation of mPGES-1.     

Tyrosine Phosphorylation of Extracellular Signal-Regulated Protein Kinase 4 in Response to Growth Factors

Abstract: Extracellular signal-regulated protein kinases (ERKs) are members of the mitogen-activated protein kinase family that are rapidly phosphorylated and activated in response to various extracellular stimuli, including growth factors. Of these, the ERK1 and ERK2 forms are by far the most abundant and the most studied. Much less is known about other ERK forms, including one previously designated ERK4 on the basis of its cross-reactivity with ERK1 and ERK2. We report here that ERK4 in rat PC12 pheochromocytoma cells can be immunoprecipitated by anti-ERK antiserum R2 and have used this reagent to characterize this species further. We find that ERK4 rapidly becomes tyrosine-phosphorylated in response to nerve growth factor (NGF) and epidermal growth factor (EGF) and, to a lesser degree, in response to insulin and a permeant cyclic AMP analogue. As in the case of ERK1 and ERK2, tyrosine phosphorylation of ERK4 occurs by a ras-dependent pathway in response to NGF and EGF and shows prolonged kinetics for NGF but not EGF treatment. Recognition by multiple antisera directed against various domains of ERK1 supports classification of ERK4 within the ERK family; however, two-dimensional gel analysis clearly distinguishes ERK4 from isoforms of ERK1. These findings thus reveal an additional member of the ERK family that is responsive to growth factors and that could play a distinct role in intracellular signaling.     

Ectopic Expression of eIF-4E in Human Colon Cancer Cells Promotes the Stimulation of Adhesion Molecules by Transforming Growth Factorβ

Transforming growth factor β1 (TGFβ) inhibits cellular proliferation, promotes differentiation, and stimulates the expression and secretion of the extracellular matrix adhesion molecules fibronectin and laminin and the colon-associated intercellular adhesion molecule carcinoembryonic antigen. This is collectively called the TGFβ-mediated adhesion response and occurs in the human colon cancer cell line Moser while the cell line KM12SM is relatively unresponsive to TGFβ. We have previously shown that TGFβ rapidly stimulates protein kinase C (PKC) phosphotransferase activity in the Moser cells and that the induction of the adhesion response (but not antiproliferation) by TGFβ is dependent on PKC. Because resistance to growth factors may be due to translational suppression and the translation initiation factor eIF-4E may alleviate translational suppression, we determined the effect of eIF-4E expression on the responses of Moser and KM12SM cells to TGFβ. Ectopic expression of eIF-4E in the TGFβ-responsive Moser cells enhanced the activation of PKC by TGFβ and the induction of the adhesion response, especially the secretion of adhesion molecules, but not the antiproliferative response. Ectopic expression of eIF-4E in the TGFβ-resistant KM12SM cells increased TGFβ stimulation of PKC and the TGFβ-mediated adhesion response (but not antiproliferation). The secretion of adhesion molecules was significantly increased by TGFβ. These results showed in these cells that eIF-4E promotes TGFβ-regulated adhesion but not antiproliferation in a PKC-dependent manner.     

Differential involvement of ERK1-2 and p38MAPK activation on Swiss 3T3 cell proliferation induced by prostaglandin F2alpha

Prostaglandin F2alpha (PGF2alpha) induces cyclin D1 expression and DNA synthesis in Swiss 3T3 cells. In order to assess which signaling mechanisms are implicated in these processes, we have used both a pharmacological approach and interfering mutants. We demonstrate that PGF2alpha induces extracellular-signal-regulated kinase (ERK1-2) and p38MAPK activation, and inhibition of any of these signaling pathways completely blocks PGF2alpha-stimulated DNA synthesis. We also show that ERK1-2, but not p38MAPK activation is required to induce cyclin D1 expression, strongly suggesting that the concerted action of cyclin D1 gene expression and other events are required to induce complete phosphorylation of retinoblastoma protein and S-phase entry in response to PGF2alpha.     

Loss of HAUSP-mediated deubiquitination contributes to DNA damage-induced destabilization of Hdmx and Hdm2

The p53 tumor suppressor protein has a major role in protecting the integrity of the genome. In unstressed cells, p53 is maintained at low levels by the ubiquitin-proteasome pathway. A balance between ubiquitin ligase activity (Hdm2, COP1, and Pirh2) and the ubiquitin protease activity of the Herpes virus-associated ubiquitin-specific protease (HAUSP) determines the half-life of p53. HAUSP also modulates p53 stability indirectly by deubiquitination and stabilization of Hdm2. The Hdmx protein affects p53 stability as well through its interaction with and regulation of Hdm2. Vice versa, Hdmx is a target for Hdm2-mediated ubiquitination and degradation. Here, we show that HAUSP also interacts with Hdmx, resulting in its direct deubiquitination and stabilization. HAUSP activity is required to maintain normal Hdmx protein levels. Therefore, the balance between HAUSP and Hdm2 activity determines Hdmx protein stability. Importantly, impaired deubiquitination of Hdmx/Hdm2 by HAUSP contributes to the DNA damage-induced degradation of Hdmx and transient instability of Hdm2.     

Two closely related human members of chitinase-like family, CHI3L1 and CHI3L2, activate ERK1/2 in 293 and U373 cells but have the different influence on cell proliferation

The activation of extracellular signal-regulated kinases (ERK1/2) has been associated with specific outcomes. Sustained activation of ERK1/2 by nerve growth factor (NGF) is associated with translocation of ERKs to the nucleus of PC12 cells and precedes their differentiation into sympathetic-like neurons whereas transient activation by epidermal growth factor (EGF) leads to cell proliferation. It was demonstrated that different growth factors initiating the same cellular signaling pathways may lead to the different cell destiny, either to proliferation or to the inhibition of mitogenesis and apoptosis. Thus, further investigation on kinetic differences in activation of certain signal cascades in different cell types by biologically different agents are necessary for understanding the mechanisms as to how cells make a choice between proliferation and differentiation.It was reported that chitinase 3-like 1 (CHI3L1) protein promotes the growth of human synovial cells as well as skin and fetal lung fibroblasts similarly to insulin-like growth factor 1 (IGF1). Both are involved in mediating the mitogenic response through the signal-regulated kinases ERK1/2. In addition, CHI3L1 which is highly expressed in different tumors including glioblastomas possesses oncogenic properties. As we found earlier, chitinase 3-like 2 (CHI3L2) most closely related to human CHI3L1 also showed increased expression in glial tumors at both the RNA and protein levels and stimulated the activation of the MAPK pathway through phosphorylation of ERK1/2 in 293 and U87 MG cells. The work described here demonstrates the influence of CHI3L2 and CHI3L1 on the duration of MAPK cellular signaling and phosphorylated ERK1/2 translocation to the nucleus. In contrast to the activation of ERK1/2 phosphorylation by CHI3L1 that leads to a proliferative signal (similar to the EGF effect in PC12 cells), activation of ERK1/2 phosphorylation by CHI3L2 (similar to NGF) inhibits cell mitogenesis and proliferation.