Leukotriene D4 mediates survival and proliferation via separate but parallel pathways in the human intestinal epithelial cell line Int 407

We demonstrated previously that leukotriene D4 (LTD4) regulates proliferation of intestinal epithelial cells through a CysLT receptor by protein kinase C (PKC)epsilon-dependent stimulation of the mitogen-activated protein kinase ERK1/2. Our current study provides the first evidence that LTD4 can activate 90-kDa ribosomal S6 kinase (p90RSK) and cAMP-responsive element-binding protein (CREB) via pertussis-toxin-sensitive Gi protein pathways. Transfection and inhibitor experiments revealed that activation of p90RSK, but not CREB, is a PKCepsilon/Raf-1/ERK1/2-dependent process. LTD4-mediated CREB activation was not affected by expression of kinase-dead p90RSK but was abolished by transfection with the regulatory domain of PKCalpha (a specific dominant-inhibitor of PKCalpha). Kinase-negative mutants of p90RSK and CREB (K-p90RSK and K-CREB) blocked the LTD4-induced increase in cell number and DNA synthesis (thymidine incorporation). Compatible with these results, flow cytometry showed that LTD4 caused transition from the G0/G1 to the S+G2/M cell cycle phase, indicating increased proliferation. Similar treatment of cells transfected with K-p90RSK resulted in cell cycle arrest in the G0/G1 phase, consistent with a role of p90RSK in LTD4-induced proliferation. On the other hand, expression of K-CREB caused a substantial buildup in the sub-G0/G1 phase, suggesting a role for CREB in mediating LTD4-mediated survival in intestinal epithelial cells. Our results show that LTD4 regulates proliferation and survival via distinct intracellular signaling pathways in intestinal epithelial cells.     

Spatially separate docking sites on ERK2 regulate distinct signaling events in vivo

Inhibitors of the oncogenic Ras-MAPK pathway have been intensely pursued as therapeutics. Targeting this pathway, however, presents challenges due to the essential role of MAPK in homeostatic functions. The phosphorylation and activation of MAPK substrates is regulated by protein-protein interactions with MAPK docking sites. Active ERK1/2 (extracellular signal-regulated kinase 1/2)-MAPKs localize to effectors containing DEF (docking site for ERK, (F)/(Y) -X-(F)/(Y) -P)- or D-domain (docking domain) motifs. We have examined the in vivo activity of ERK2 mutants with impaired ability to signal via either docking site. Mutations in the DEF-domain binding pocket prevent activation of DEF-domain-containing effectors but not RSK (90 kDa ribosomal S6 kinase), which contains a D domain. Conversely, mutation of the ERK2 CD domain, which interacts with D domains, prevents RSK activation but not DEF-domain signaling. Uncoupling docking interactions does not compromise ERK2 phosphotransferase activity. ERK2 DEF mutants undergo regulated nuclear translocation but are defective for Elk-1/TCF transactivation and target gene induction. Thus, downstream branches of ERK2 signaling can be selectively inhibited without blocking total pathway activity. Significantly, several protooncogenes contain DEF domains and are regulated by ERK1/2. Therefore, disrupting ERK-DEF domain interactions could be an alternative to inhibiting oncogenic Ras-MAPK signaling.     

RAS/ERK signaling promotes site-specific ribosomal protein S6 phosphorylation via RSK and stimulates cap-dependent translation

Converging signals from the mammalian target of rapamycin (mTOR) and phosphoinositide 3-kinase (PI3K) pathways are well established to modulate translation initiation. Less is known regarding the molecular basis of protein synthesis regulated by other inputs, such as agonists of the Ras/extracellular signal-regulated kinase (ERK) signaling cascade. Ribosomal protein (rp) S6 is a component of the 40S ribosomal subunit that becomes phosphorylated at several serine residues upon mitogen stimulation, but the exact molecular mechanisms regulating its phosphorylation and the function of phosphorylated rpS6 is poorly understood. Here, we provide evidence that activation of the p90 ribosomal S6 kinases (RSKs) by serum, growth factors, tumor promoting phorbol esters, and oncogenic Ras is required for rpS6 phosphorylation downstream of the Ras/ERK signaling cascade. We demonstrate that while ribosomal S6 kinase 1 (S6K1) phosphorylates rpS6 at all sites, RSK exclusively phosphorylates rpS6 at Ser(235/236) in vitro and in vivo using an mTOR-independent mechanism. Mutation of rpS6 at Ser(235/236) reveals that phosphorylation of these sites promotes its recruitment to the 7-methylguanosine cap complex, suggesting that Ras/ERK signaling regulates assembly of the translation preinitiation complex. These data demonstrate that RSK provides an mTOR-independent pathway linking the Ras/ERK signaling cascade to the translational machinery.     

The tumor suppressor DAP kinase is a target of RSK-mediated survival signaling

The viability of vertebrate cells depends on a complex signaling interplay between survival factors and cell-death effectors. Subtle changes in the equilibrium between these regulators can result in abnormal cell proliferation or cell death, leading to various pathological manifestations. Death-associated protein kinase (DAPK) is a multidomain calcium/calmodulin (CaM)-dependent Ser/Thr protein kinase with an important role in apoptosis regulation and tumor suppression. The molecular signaling mechanisms regulating this kinase, however, remain unclear. Here, we show that DAPK is phosphorylated upon activation of the Ras-extracellular signal-regulated kinase (ERK) pathway. This correlates with the suppression of the apoptotic activity of DAPK. We demonstrate that DAPK is a novel target of p90 ribosomal S6 kinases (RSK) 1 and 2, downstream effectors of ERK1/2. Using mass spectrometry, we identified Ser-289 as a novel phosphorylation site in DAPK, which is regulated by RSK. Mutation of Ser-289 to alanine results in a DAPK mutant with enhanced apoptotic activity, whereas the phosphomimetic mutation (Ser289Glu) attenuates its apoptotic activity. Our results suggest that RSK-mediated phosphorylation of DAPK is a unique mechanism for suppressing the proapoptotic function of this death kinase in healthy cells as well as Ras/Raf-transformed cells.     

Modulation of cell-cycle regulatory signaling network by 2-methoxyestradiol in prostate cancer cells is mediated through multiple signal transduction pathways

2-Methoxyestradiol (2-ME(2)), a promising anticancer drug, induces growth arrest and apoptosis in various androgen-dependent (LNCaP) and -independent (DU145 and PC-3) prostate cancer cell lines. Moreover, flow cytometric analysis indicated a novel dual impact of 2-ME(2) on the cell division cycle of prostate cancer cells. Chronic exposure of high doses of 2-ME(2) enhance the accumulation of cells in S and G2/M phases, while cell numbers in the G1 phase were reduced significantly by this treatment. Because cyclin B1 overexpression, induction of cdc2 phosphorylation, and its regulatory proteins wee1 and phospho-cdc25C (interphase and mitotic forms) by 2-ME(2) treatment correlated with the induction of apoptosis, growth arrest at the G2/M phase, and accumulation of the S phase, we reasoned that cyclin B1 and cdc2 phosphorylation and its upstream regulatory molecular networks may be associated with the ultimate impacts of 2-ME(2). Because phosphorylation of cdc2 and upregulation of wee1 by 2-ME(2) can be abolished by both extracellular receptor kinase (ERK) inhibitor (U0126) and c-Jun N-terminal kinase (JNK) inhibitor (SP600125), our studies indicate that the 2-ME(2)-induced upregulation of wee1 and subsequent cdc2 phosphorylation are mediated through mitogen-activated protein kinase (MAPK)-ERK-JNK signaling pathways.     

Time Courses of Changes in Phospho- and Total- MAP Kinases in the Cochlea after Intense Noise Exposure

Mitogen-activated protein kinases (MAP kinases) are intracellular signaling kinases activated by phosphorylation in response to a variety of extracellular stimuli. Mammalian MAP kinase pathways are composed of three major pathways: MEK1 (mitogen-activated protein kinase kinase 1)/ERK 1/2 (extracellular signal-regulated kinases 1/2)/p90 RSK (p90 ribosomal S6 kinase), JNK (c-Jun amino (N)-terminal kinase)/c-Jun, and p38 MAPK pathways. These pathways coordinately mediate physiological processes such as cell survival, protein synthesis, cell proliferation, growth, migration, and apoptosis. The involvement of MAP kinase in noise-induced hearing loss (NIHL) has been implicated in the cochlea; however, it is unknown how expression levels of MAP kinase change after the onset of NIHL and whether they are regulated by transient phosphorylation or protein synthesis. CBA/J mice were exposed to 120-dB octave band noise for 2 h. Auditory brainstem response confirmed a component of temporary threshold shift within 0–24 h and significant permanent threshold shift at 14 days after noise exposure. Levels and localizations of phospho- and total- MEK1/ERK1/2/p90 RSK, JNK/c-Jun, and p38 MAPK were comprehensively analyzed by the Bio-Plex® Suspension Array System and immunohistochemistry at 0, 3, 6, 12, 24 and 48 h after noise exposure. The phospho-MEK1/ERK1/2/p90 RSK signaling pathway was activated in the spiral ligament and the sensory and supporting cells of the organ of Corti, with peaks at 3–6 h and independently of regulations of total-MEK1/ERK1/2/p90 RSK. The expression of phospho-JNK and p38 MAPK showed late upregulation in spiral neurons at 48 h, in addition to early upregulations with peaks at 3 h after noise trauma. Phospho-p38 MAPK activation was dependent on upregulation of total-p38 MAPK. At present, comprehensive data on MAP kinase expression provide significant insight into understanding the molecular mechanism of NIHL, and for developing therapeutic models for acute sensorineural hearing loss.     

Retinoic acid increases amount of phosphorylated raf; Ectopic expression of cFMS reveals that retinoic acid-induced differentiation is more strongly dependent on ERK2 signaling than induced go arrest is

Summary Retinoic acid is known to cause the myeloid differentiation and G1/0 cell cycle arrest of HL-60 cells in a process that requires mitogen-activated protein/extracellular signal regulated kinase (MEK)-dependent extracellular signal regulated kinase (ERK)2 activation. It has also been shown that ectopic expression of cFMS, a platelet-derived growth factor (PDGF)-family transmembrane tyrosine kinase receptor, enhances retinoic acid-induced differentiation and G1/0 arrest. The mechanism of how the retinoic acid and cFMS signaling pathways intersect is not known. The present data show that the ectopic expression of cFMS results in the differential loss of sensitivity of retinoic acid-induced differentiation or G1/0 arrest to inhibition of ERK2 activation. PD98059 was used to inhibit MEK and consequently ERK2. In wild-type HL-60 cells, PD98059 blocked retinoic acid-induced differentiation; but in cFMS stable transfectants, PD98059 only attenuated the induced differentiation, with the resulting response resembling that of retinoic acid-treated wild-type HL-60. In wild-type HL-60, PD98059 greatly attenuated the retinoic acid-induced G1/0 arrest allied with retinoblastoma (RB) hypophosphorylation; but in cFMS stable transfectants, PD98059 had no inhibitory effect on RB hypophosphorylation and G1/0 arrest. This differential sensitivity to PD98059 and uncoupling of retinoic acid-induced differentiation and G1/0 arrest in cFMS transfectants is associated with changes in mitogen-activated protein kinase signaling molecules. The cFMS transfectants had more activated ERK2 than did the wild-type cells, which surprisingly was not attributable to enhanced mitogen-activated protein-kinase-kinase-kinase (RAF) phosphorylation. Retinoic acid increased the amount of activated ERK2 and phosphorylated RAF in both cell lines. But PD98059 eliminated detectable ERK2 activation, as well as inhibited RAF phosphorylation, in untreated and retinoic acid-treated wild-type HL-60 and cFMS transfectants, consistent with MEK or ERK feedback-regulation of RAF, in all four cases. Since PD98059 blocks the cFMS-conferred enhancement of the retinoic acid-induced differentiation, but not growth arrest, the data indicate that cFMS-enhanced differentiation acts primarily through MEK and ERK2, but cFMS-enhanced G1/0 arrest allied with RB hypophosphorylation depends on another cFMS signal route, which by itself can effect G1/0 arrest without activated ERK2. Ectopic expression of cFMS and differential sensitivity to ERK2 inhibition thus reveal that retinoic acid-induced HL-60 cell differentiation and G1/0 arrest are differentially dependent on ERK2 and can be uncoupled. A significant unanticipated finding was that retinoic acid caused a MEK-dependent increase in the amount of phosphorylated RAF. This increase may help sustain prolonged ERK2 activation.     

Truncated MEK1 is required for transient activation of MAPK signalling in G2 phase cells

The primary endpoint of signalling through the canonical Raf–MEK–ERK MAP kinase cascade is ERK activation. Here we report a novel signalling outcome for this pathway. Activation of the MAP kinase pathway by growth factors or phorbol esters during G2 phase results in only transient activations of ERK and p90RSK, then suppression to below control levels. A small peak of ERK and p90RSK activation in early G2 phase cells was identified, and inhibition of this delayed entry into mitosis. The previously identified, proteolytically cleaved form of MEK1 termed tMEK (truncated MEK1), is also induced with G2 phase MAPK pathway activation. We demonstrate that addition of recombinant mutants of MEK1 with an N-terminal truncation similar to that of tMEK also inhibited ERK and p90RSK activations and delayed progression into mitosis. Only catalytically inactive forms of tMEK were capable of these effects, but surprisingly, phosphorylation on the activating Ser218/222 sites was also required. A lack of MEK1 or ability to accumulate tMEK resulted in the absence of the feedback inhibition of ERK and p90RSK activations. tMEK is a novel output from the canonical MAP kinase signalling pathway, acting in a MAPK signalling-regulated dominant negative manner to inhibit ERK and p90RSK activations, acting as a dampening mechanism to reduce the magnitude or duration of MAPK pathway signalling in G2/M phase.     

Signaling pathways regulating murine cardiac CREB phosphorylation

Using the mouse Langendorff heart perfusion model, the signaling pathways that regulate cardiac CREB-S133 phosphorylation have been defined. In mouse hearts stimulated with isoproterenol (ISO) (10(-8) M), endothelin-1 (ET-1) (10(-8) M), and phorbol 12-myristate 13-acetate (TPA) (10(-7) M), CREB-S133 phosphorylation was attained only by TPA-treatment. Activation of protein kinase A (PKA) was achieved by ISO. ISO- and ET-1-stimulation activated Ca2+/calmodulin-dependent kinase II (CaMKII). Protein kinase C (PKC) and p90(RSK) were activated with all three stimuli. Inhibition of ERK1/2 with PD98059 (10(-5) M) completely inhibited the activation of p90(RSK), but did not block CREB-S133 phosphorylation in TPA-perfused heart, indicating that PKA, CaMKII, and p90(RSK) do not phosphorylate CREB-S133 in the murine heart. PKC activation is signal specific. Analyses of PKC isoforms suggest that CREB phosphorylation is mediated by PKC epsilon translocating into nucleus only with TPA stimulation. These results, unlike those reported in other tissues, demonstrate that cardiac CREB is not a multi-signal target.     

ERK1/2 Signaling Plays an Important Role in Topoisomerase II Poison-Induced G2/M Checkpoint Activation

Topo II poisons, which target topoisomerase II (topo II) to generate enzyme mediated DNA damage, have been commonly used for anti-cancer treatment. While clinical evidence demonstrate a capability of topo II poisons in inducing apoptosis in cancer cells, accumulating evidence also show that topo II poison treatment frequently results in cell cycle arrest in cancer cells, which was associated with subsequent resistance to these treatments. Results in this report indicate that treatment of MCF-7 and T47D breast cancer cells with topo II poisons resulted in an increased phosphorylation of extracellular signal-regulated kinase 1 and 2 (ERK1/2) and an subsequent induction of G2/M cell cycle arrest. Furthermore, inhibition of ERK1/2 activation using specific inhibitors markedly attenuated the topo II poison-induced G2/M arrest and diminished the topo II poison-induced activation of ATR and Chk1 kinases. Moreover, decreased expression of ATR by specific shRNA diminished topo II poison-induced G2/M arrest but had no effect on topo II poison-induced ERK1/2 activation. In contrast, inhibition of ERK1/2 signaling had little, if any, effect on topo II poison-induced ATM activation. In addition, ATM inhibition by either incubation of cells with ATM specific inhibitor or transfection of cells with ATM specific siRNA did not block topo II poison-induced G2/M arrest. Ultimately, inhibition of ERK1/2 signaling greatly enhanced topo II poison-induced apoptosis. These results implicate a critical role for ERK1/2 signaling in the activation of G2/M checkpoint response following topo II poison treatment, which protects cells from topo II poison-induced apoptosis.     

Ribosomal S6 kinase 1 (RSK1) activation requires signals dependent on and independent of the MAP kinase ERK.

BACKGROUND: The rsk1 gene encodes the 90 kDa ribosomal S6 kinase 1 (RSK1) protein, which contains two kinase domains. RSK1, which is involved in regulating cell survival and proliferation, lies at the end of the signaling cascade mediated by the extracellular signal-regulated kinase (ERK) subfamily of mitogen-activated protein (MAP) kinases. ERK activation and subsequent phosphorylation of the RSK1 carboxy-terminal catalytic loop stimulates phosphotransferase activity in the RSK1 amino-terminal kinase domain. When activated, RSK1 phosphorylates both nuclear and cytoplasmic substrates through this amino-terminal catalytic domain. It is thought that stimulation of the ERK/MAP kinase pathway is sufficient for RSK1 activation, but how ERK phosphorylation activates the RSK1 amino-terminal kinase domain is not known. RESULTS: The individual isolated RSK1 kinase domains were found to be under regulatory control. In vitro kinase assays established that ERK phosphorylates RSK1 within the carboxy-terminal kinase domain, and the phosphoinositide-dependent kinase 1 (PDK1) phosphorylates RSK1 within the amino-terminal kinase domain. In transiently transfected HEK 293E cells, PDK1 alone stimulated phosphotransferase activity of an isolated RSK1 amino-terminal kinase domain. Nevertheless, activation of full-length RSK1 in the absence of serum required activation by both PDK1 and ERK. CONCLUSIONS: RSK1 is phosphorylated by PDK1 in the amino-terminal kinase-activation loop, and by ERK in the carboxy-terminal kinase-activation loop. Activation of phosphotransferase activity of full-length RSK1 in vivo requires both PDK1 and ERK. RSK1 activation is therefore regulated by both the mitogen-stimulated ERK/MAP kinase pathway and a PDK1-dependent pathway.     

GLP-1 Mediates Antiapoptotic Effect by Phosphorylating Bad through a ��-Arrestin 1-mediated ERK1/2 Activation in Pancreatic ��-Cells

Strategies based on activating GLP-1 receptor (GLP-1R) are intensively developed for the treatment of type 2 diabetes. The exhaustive knowledge of the signaling pathways linked to activated GLP-1R within the β-cells is of major importance. In β-cells, GLP-1 activates the ERK1/2 cascade by diverse pathways dependent on either Gα_s/cAMP/cAMP-dependent protein kinase (PKA) or β-arrestin 1, a scaffold protein. Using pharmacological inhibitors, β-arrestin 1 small interfering RNA, and islets isolated from β-arrestin 1 knock-out mice, we demonstrate that GLP-1 stimulates ERK1/2 by two temporally distinct pathways. The PKA-dependent pathway mediates rapid and transient ERK1/2 phosphorylation that leads to nuclear translocation of the activated kinases. In contrast, the β-arrestin 1-dependent pathway produces a late ERK1/2 activity that is restricted to the β-cell cytoplasm. We further observe that GLP-1 phosphorylates the cytoplasmic proapoptotic protein Bad at Ser-112 but not at Ser-155. We find that the β-arrestin 1-dependent ERK1/2 activation engaged by GLP-1 mediates the Ser-112 phosphorylation of Bad, through p90RSK activation, allowing the association of Bad with the scaffold protein 14-3-3, leading to its inactivation. β-Arrestin 1 is further found to mediate the antiapoptotic effect of GLP-1 in β-cells through the ERK1/2-p90RSK-phosphorylation of Bad. This new regulatory mechanism engaged by activated GLP-1R involving a β-arrestin 1-dependent spatiotemporal regulation of the ERK1/2-p90RSK activity is now suspected to participate in the protection of β-cells against apoptosis. Such signaling mechanism may serve as a prototype to generate new therapeutic GLP-1R ligands.     

Phosphoproteomic analysis of thrombin- and p38 MAPK-regulated signaling networks in endothelial cells

Endothelial dysfunction is a hallmark of inflammation and is mediated by inflammatory factors that signal through G protein–coupled receptors including protease-activated receptor-1 (PAR1). PAR1, a receptor for thrombin, signals via the small GTPase RhoA and myosin light chain intermediates to facilitate endothelial barrier permeability. PAR1 also induces endothelial barrier disruption through a p38 mitogen-activated protein kinase–dependent pathway, which does not integrate into the RhoA/MLC pathway; however, the PAR1-p38 signaling pathways that promote endothelial dysfunction remain poorly defined. To identify effectors of this pathway, we performed a global phosphoproteome analysis of thrombin signaling regulated by p38 in human cultured endothelial cells using multiplexed quantitative mass spectrometry. We identified 5491 unique phosphopeptides and 2317 phosphoproteins, four distinct dynamic phosphoproteome profiles of thrombin-p38 signaling, and an enrichment of biological functions associated with endothelial dysfunction, including modulators of endothelial barrier disruption and a subset of kinases predicted to regulate p38-dependent thrombin signaling. Using available antibodies to detect identified phosphosites of key p38-regulated proteins, we discovered that inhibition of p38 activity and siRNA-targeted depletion of the p38α isoform increased basal phosphorylation of extracellular signal–regulated protein kinase 1/2, resulting in amplified thrombin-stimulated extracellular signal–regulated protein kinase 1/2 phosphorylation that was dependent on PAR1. We also discovered a role for p38 in the phosphorylation of α-catenin, a component of adherens junctions, suggesting that this phosphorylation may function as an important regulatory process. Taken together, these studies define a rich array of thrombin- and p38-regulated candidate proteins that may serve important roles in endothelial dysfunction.