Raf-1 Protein Kinase Research Articles

P21-activated Kinase 1 (Pak1)-dependent Phosphorylation of Raf-1 Regulates Its Mitochondrial Localization, Phosphorylation of BAD, and Bcl-2 Association

Raf-1 protects cells from apoptosis, independently of its signals to MEK and ERK, by translocating to the mitochondria where it binds Bcl-2 and displaces BAD. However, the answer to the question of how Raf-1 is normally lured to the mitochondria and becomes activated remains elusive. p21-activated protein kinases (Paks) are serine/threonine protein kinases that phosphorylate Raf-1 at Ser-338 and Ser-339. Here we elucidate the molecular mechanism through which Pak1 signals to BAD through a Raf-1-activated pathway. Upon phosphorylation by Pak1, Raf-1 translocates to mitochondria and phosphorylates BAD at Ser-112. Moreover, the mitochondrial translocation of Raf-1 and the interaction between Raf-1 and Bcl-2 are regulated by Raf-1 phosphorylation at Ser-338/Ser-339. Notably, we show that formation of a Raf-1-Bcl-2 complex coincides with loss of an interaction between Bcl-2 and BAD. These signals are specific for Pak1, because Src-activated Raf-1 only stimulates the MAP kinase cascade. Thus, our data identify the molecular connections of a Pak1-Raf-1-BAD pathway that is involved in cell survival signaling.

Src tyrosine kinase inhibitor PP2 induces a sustained Ras-independent Activation of Raf-1 Protein Kinase by PMA and H2O2

Src tyrosine kinase inhibitor PP2 induces a sustained Ras-independent Activation of Raf-1 Protein Kinase by PMA and H2O2

Cell Signaling through the Protein Kinases cAMP-dependent Protein Kinase, Protein Kinase Cϵ, and RAF-1 Regulates Amphotropic Murine Leukemia Virus Envelope Protein-induced Syncytium Formation

Amphotropic murine leukemia virus (A-MuLV) utilizes the PiT2 sodium-dependent phosphate transporter as its cell surface receptor to infect mammalian cells. The process of A-MuLV infection requires cleavage of the R peptide from the envelope protein. This occurs within virions thereby rendering them competent to fuse with target cells. Envelope proteins lacking the inhibitory R peptide (e.g. envelope (R-) proteins) induce viral envelope-mediated cell-cell fusion (syncytium). Here we have performed studies to determine if cell signaling through protein kinases is involved in the regulation of PiT2-mediated A-MuLV envelope (R-)-induced syncytium formation. Truncated A-MuLV retroviral envelope protein lacking the inhibitory R peptide (R-) was used to induce viral envelope-mediated cell-cell fusion. Signaling through cyclic AMP to activate PKA was found to inhibit envelope-induced cell-cell fusion, whereas treatment of cells with PKA inhibitors H89, KT5720, and PKA Catalpha siRNA all enhanced this cell fusion process. It was noted that activation of PKC, as well as overexpression of PKCepsilon, up-regulated A-MuLV envelope protein-induced cell-cell fusion, whereas exposure to PKC inhibitors and expression of a kinase-inactive dominant-negative mutant of PKCepsilon (K437R) inhibited syncytium formation. v-ras transformed NIH3T3 cells were highly susceptible to A-MuLV envelope-induced cell-cell fusion, whereas expression of a dominant-negative mutant of Ras (N17Ras) inhibited this cell fusion process. Importantly, activation of Raf-1 protein kinase also is required for A-MuLV envelope-induced syncytium formation. Expression of constitutively active BXB Raf supported, whereas expression of a dominant-negative mutant of Raf-1 (Raf301) blocked, A-MuLV-induced cell-cell fusion. These results indicate that specific cell signaling components are involved in regulating PiT2-mediated A-MuLV-induced cell-cell fusion. Selective pharmacological modulation of these signaling components may be an effective means of altering cell susceptibility to viral-mediated cytopathic effects.

Surprises from Raf-1 Knockout

Ehrenreiter et al. explored the signaling roles of the Raf-1 protein kinase by creating mice that lacked Raf-1 expression in the epidermis. Although Raf-1 can function in activation of mitogen-activated protein kinases, phosphorylation of ERK was not altered in the keratinocytes lacking Raf-1. Instead, a critical role of Raf-1 in wound healing and cell migration was revealed. The development of the epidermis was normal in the knockout animals, but wound healing in vivo was strongly inhibited, as was keratinocyte migration in Boyden chamber assays. The defects were similar to those observed in cells lacking Rok-α (Rho-associated kinase, also called ROCK-II). Indeed Rok-α activity was increased in the knockout cells, and treatment of knockout fibroblasts with a Rok inhibitor or transfection with a catalytically inactive mutant of Rok-α restored normal morphology and migration of the cells. Rok-α was also mislocalized to the plasma membrane in the cells lacking Raf-1. Endogenous Raf-1 and Rok-α were shown to interact by immunoprecipitation of the proteins together from migrating wild-type cells. A catalytically inactive form of Raf-1 could also rescue the knockout cells, indicating that the physical presence of Raf-1, rather than its kinase activity, was required. K. Ehrenreiter, D. Piazzolla, V. Velamoor, I. Sobczak, J. V. Small, J. Takeda, T. Leung, M. Baccarini, Raf-1 regulates Rho signaling and cell migration. J. Cell Biol. 168 , 955-964 (2005). [Abstract] [Full Text]

Ethylene Signaling Pathway

The structural simplicity of the plant hormone ethylene contrasts with its dramatic effects in various developmental processes, as well as in the cellular processes that ethylene initiates in response to a diversity of environmental signals. A single well-conserved signaling cascade mediates this broad spectrum of responses. Ethylene is perceived by a family of two-component histidine kinase receptors that become inactivated upon ethylene binding. In the absence of the hormone, the receptors activate CTR1, a negative regulator of ethylene responses. Sequence similarity between CTR1 and the Raf protein kinases implies involvement of a mitogen-activated protein kinase cascade in this signaling pathway. The protein EIN2 acts downstream of CTR1 and the possible kinase cascade. Although the biochemical function of EIN2 is not understood, its critical role is manifested by the complete ethylene insensitivity of EIN2 loss-of-function mutants. Downstream of EIN2, a family of plant-specific EIN3-like transcription factors mediate ethylene responses. The regulation of EIN3 stability by ethylene is accomplished by F-box-containing proteins that participate in the formation of a SKP1/cullin/F-box complex that targets proteins for degradation by the proteasome. A large number of ethylene-regulated genes have been identified, including the APETALA2 domain-containing transcription factor genes ERF1 and EDF1 to 4, which suggests the participation of a transcriptional cascade in the ethylene response. The differential regulation of some components of this complex nuclear cascade by other signaling pathways provides a possible mechanism for interaction and signal integration. As new points of intersection with other pathways and additional participants in the pathway are identified, the Connections Map will be updated to include this new information.

Ras-Independent Activation of ERK Signaling via the Torso Receptor Tyrosine Kinase Is Mediated by Rap1

In Drosophila embryos, the Torso receptor tyrosine kinase (RTK) activates the small G protein Ras (D-Ras1) and the protein kinase Raf (D-Raf) to activate ERK to direct differentiation of terminal structures . However, genetic studies have demonstrated that Torso, and by extension other RTKs, can activate Raf and ERK independently of Ras . In mammalian cells, the small G protein Rap1 has been proposed to couple RTKs to ERKs. However, the ability of Rap1 to activate ERKs remains controversial, in part because direct genetic evidence supporting this hypothesis is lacking. Here, we present biochemical and genetic evidence that D-Rap1, the Drosophila homolog of Rap1, can activate D-Raf and ERK. We show that D-Rap1 binds D-Raf and activates ERKs in a GTP- and D-Raf-dependent manner. Targeted disruption of D-Rap1 expression decreased both Torso-dependent ERK activation and the ERK-dependent expression of the zygotic genes tailless and huckebein to levels similar to those achieved in D-Ras1 null embryos. Furthermore, combined deficiencies of D-Ras1 and D-Rap1 completely abolished expression of these genes, mimicking the phenotype observed in embryos lacking D-Raf. These studies provide the first direct genetic evidence of Rap1-mediated activation of the MAP kinase cascade in eukaryotic organisms.

Promoting and Protecting From Cell Death

Mutations in the protein kinase Raf are frequently associated with human tumors. Raf functions in growth-promoting signaling and also acts to protect cells from apoptosis, a key event that allows cancer cells to avoid normal fail-safe mechanisms that stop uncontrolled cell growth. Surprisingly, Raf's protective effect against apoptosis appears not to require enzymatic activity of Raf. O'Neill et al. describe an interaction between Raf-1 and another protein kinase, MST2, in mammalian cells. Raf-1 appears to directly inhibit activation of MST2 in vitro, and this effect, like that of Raf-1 on apoptosis, was independent of catalytic activity of Raf-1. Cells lacking Raf-1 showed increased activity of MST2, which correlated with an increased propensity to undergo apoptosis. Decreased expression of MST2 protected cells from apoptosis. Thus, MST2 represents an important new target of Raf-1 that may help in the control of cell proliferation and cell death in normal and cancerous cells. E. O'Neill, L. Rushworth, M. Baccarini, W. Kolch, Role of the kinase MST2 in suppression of apoptosis by the proto-oncogene product Raf-1. Science 306 , 2267-2270 (2004). [Abstract] [Full Text]

Role of the Kinase MST2 in Suppression of Apoptosis by the Proto-Oncogene Product Raf-1

The ablation of the protein kinase Raf-1 renders cells hypersensitive to apoptosis despite normal regulation of extracellular signal-regulated kinases, which suggests that apoptosis protection is mediated by a distinct pathway. We used proteomic analysis of Raf-1 signaling complexes to show that Raf-1 counteracts apoptosis by suppressing the activation of mammalian sterile 20-like kinase (MST2). Raf-1 prevents dimerization and phosphorylation of the activation loop of MST2 independently of its protein kinase activity. Depletion of MST2 from Raf-1-/- mouse or human cells abrogated sensitivity to apoptosis, whereas overexpression of MST2 induced apoptosis. Conversely, depletion of Raf-1 from Raf-1+/+ mouse or human cells led to MST2 activation and apoptosis. The concomitant depletion of both Raf-1 and MST2 prevented apoptosis.

Cooperative Regulation of the Cell Division Cycle by the Protein Kinases RAF and AKT

The RAS-activated RAF-->MEK-->extracellular signal-regulated kinase (ERK) and phosphatidylinositol 3'-kinase (PI3'-kinase)-->PDK1-->AKT signaling pathways are believed to cooperate to promote the proliferation of normal cells and the aberrant proliferation of cancer cells. To explore the mechanisms that underlie such cooperation, we have derived cells harboring conditionally active, steroid hormone-regulated forms of RAF and AKT. These cells permit the assessment of the biological and biochemical effects of activation of these protein kinases either alone or in combination with one another. Under conditions where activation of neither RAF nor AKT alone promoted S-phase progression, coactivation of both kinases elicited a robust proliferative response. Moreover, under conditions where high-level activation of RAF induced G(1) cell cycle arrest, activation of AKT bypassed the arrest and promoted S-phase progression. At the level of the cell cycle machinery, RAF and AKT cooperated to induce cyclin D1 and repress p27(Kip1) expression. Repression of p27(Kip1) was accompanied by a dramatic reduction in KIP1 mRNA and was observed in primary mouse embryo fibroblasts derived from mice either lacking SKP2 or expressing a T187A mutated form of p27(Kip1). Consistent with these observations, pharmacological inhibition of MEK or PI3'-kinase inhibited the effects of activated RAS on the expression of p27(Kip1) in NIH 3T3 fibroblasts and in a panel of bona fide human pancreatic cancer cell lines. Furthermore, we demonstrated that AKT activation led to sustained activation of cyclin/cdk2 complexes that occurred concomitantly with the removal of RAF-induced p21(Cip1) from cyclin E/cdk2 complexes. Cumulatively, these data strongly suggest that the RAF-->MEK-->ERK and PI3'K-->PDK-->AKT signaling pathways can cooperate to promote G(0)-->G(1)-->S-phase cell cycle progression in both normal and cancer cells.

Src Tyrosine Kinase Inhibitor PP2 Markedly Enhances Ras-independent Activation of Raf-1 Protein Kinase by Phorbol Myristate Acetate and H2O2

Recently we reported that simultaneous treatment of NIH 3T3 cells with the combination of phorbol myristate acetate (PMA) and hydrogen peroxide (H2O2) resulted in synergistic activation of Raf-1 kinase (Lee, M., Petrovics, G., and Anderson, W. B. (2003) Biochem. Biophys. Res. Commun. 311, 1026-1033). In this study we have demonstrated that PP2 (4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine), a potent and selective inhibitor of the Src-family tyrosine kinase, greatly potentiated the ability of PMA and/or H2O2 to activate Raf-1 kinase, whereas it blocked the tyrosine phosphorylation of Raf-1. Unlike PMA/H2O2 treatment, which showed transient activation, PP2-mediated Raf-1 activation was sustained and continued to increase through 4 h of treatment. Transient transfection studies with a dominant-negative mutant of Ras (N19Ras) indicated that this PP2-induced activation of Raf-1 was Ras-independent. Moreover, PP2 showed no effect on platelet-derived growth factor-induced Raf-1 activation. Interestingly, mutation of the reported Raf-1 Src family tyrosine kinase phosphorylation site by conversion of tyrosines 340 and 341 to phenylalanine (YY340/341FF Raf) had limited effect on the ability of PP2 to induce significant stimulation of Raf-1 kinase activity. Taken together, our results suggest that a tyrosine phosphorylation event is involved in the negative feedback regulation of Raf-1. Inhibition of a Src family tyrosine kinase by PP2 appears to alleviate this tyrosine kinase-mediated inhibition of Raf-1 and allow activating modification(s) of Raf-1 to proceed. This PP2 effect resulted in significant and sustained Ras-independent activation of Raf-1 by PMA and H2O2.

Gap Junctions Regulate Extracellular Signal-regulated Kinase Signaling to Affect Gene Transcription

Osteoblasts are highly coupled by gap junctions formed by connexin43. Overexpression of connexin45 in osteoblasts results in decreased chemical and electrical coupling and reduces gene transcription from connexin response elements (CxREs) in the osteocalcin and collagen Ialpha1 promoters. Here, we demonstrate that transcription from the gap junction-dependent osteocalcin CxRE is regulated by extracellular signal-regulated protein kinase (ERK) and phosphatidylinositol 3-kinase (PI3K) cascades. Overexpression of a constitutively active mitogen-activated protein kinase kinase (MEK), Raf, or Ras can increase transcription more than twofold of the CxRE, whereas inhibition of MEK or PI3K can decrease transcription threefold from the osteocalcin CxRE. Importantly, disruption of gap junctional communication by overexpression of connexin45 or treatment with pharmacological inhibitors of gap junctions results in reduced Raf, ERK, and Akt activation. The consequence of attenuated gap junction-dependent signal cascade activation is a decrease in Sp1 phosphorylation by ERK, resulting in decreased Sp1 recruitment to the CxRE and inhibited gene transcription. These data establish that ERK/PI3K signaling is required for the optimal elaboration of transcription from the osteocalcin CxRE, and that disruption of gap junctional communication attenuates the ability of cells to respond to an extracellular cue, presumably by limiting the propagation of second messengers among adjacent cells by connexin43-gap junctions.

Differential effects of echistatin and thrombin on collagen production and prolidase activity in human dermal fibroblasts and their possible implication in β 1-integrin-mediated signaling

Prolidase [E.C. 3.4.13.9] is a cytosolic imidodipeptidase that plays an important role in collagen biosynthesis. The enzyme contributes to the recovery of proline from protein degradation products (mainly collagen) for collagen resynthesis. Prolidase activity and collagen biosynthesis are supposed to be regulated by β 1-integrins, which initiate a signaling pathway in which several kinases and intracellular proteins are involved, including focal adhesion kinase pp125 FAK (FAK), Src, Shc, growth factor receptor bound protein 2 (Grb-2), son of sevenless protein (SOS), Ras, Raf and mitogen-activated protein kinases (MAPK), extracellular-signal regulated kinase 1 (ERK 1) and kinase 2 (ERK 2). We studied the effects of echistatin, a well-known disintegrin and thrombin, a serine protease capable of activation of platelet integrin α 2β 1 receptor on collagen production, prolidase activity, expression of prolidase, β 1-integrin receptor, FAK, SOS-protein and phosphorylated MAP-kinases (ERK 1 and ERK 2) in confluent human dermal fibroblasts. It has been found that treatment of the cells with 100 nM echistatin contributes to inhibition of collagen production, as well as prolidase activity and expression compared to control cells. These phenomena were accompanied by a decrease in the expression of FAK, SOS-protein and phosphorylated MAP-kinases, ERK 1 and ERK 2. An opposite phenomenon was observed in fibroblasts treated with 0.1 IU thrombin. In this case, a significant increase in collagen production and prolidase activity, accompanied by a distinct raise in the expression of prolidase, FAK and phosphorylated MAP-kinases and a slight increase in expression of SOS compared to controls were found. The results suggest that regulation of prolidase activity and collagen biosynthesis in human dermal fibroblasts may involve β 1-integrin-dependent signaling.

Activation of Raf1 and the ERK pathway in response to l-ascorbic acid in acute myeloid leukemia cells

l-ascorbic acid (LAA) shows cytotoxicity and induces apoptosis of malignant cells in vitro, but the mechanisms by which such effects occur have not been elucidated. In the present study, we provide evidence that the ERK MAP kinase pathway is activated in response to LAA (< 1 mM) in acute myeloid leukemia cell lines. LAA treatment of cells induces a dose-dependent phosphorylation of extracellular signal-regulated kinases (ERK) and results in activation of its catalytic domain. Our data also demonstrate that the small G protein Raf1 and MAPK-activated protein kinase 2 are activated by LAA as an upstream and a downstream regulator of ERK, respectively. Although the ERK pathway has been known to activate cell proliferation, pharmacologic inhibition of ERK reduces LAA-dependent apoptosis and growth inhibitory response of acute myeloid leukemia cell lines, suggesting that this signaling cascade positively regulates induction of apoptotic response by LAA.

Differentiation Induction of Human Keratinocytes by Phosphatidylethanolamine-binding Protein

Phosphatidylethanolamine-binding protein (PEBP) has been demonstrated to bind to Raf-1 and mitogen-activated protein kinase kinase, components of the extracellular signal-regulated protein kinase (ERK) pathway, thereby inhibiting the pathway and resulting in the suppression of cell proliferation. In the present study, we examined whether PEBP is involved in differentiation induction of human keratinocytes. PEBP expression was immunohistochemically examined in normal human skin and skin cancers with different differentiation properties. PEBP was not expressed in the basal layer of the epidermis but was expressed in the spinous and granular layers of normal skin. The protein was expressed in differentiated but not in undifferentiated carcinoma. PEBP expression was also examined in cultured normal human epidermal keratinocytes in which differentiation was induced by calcium treatment. Involucrin was used as a differentiation marker for spinous and granular cells. Northern blotting analysis indicated that both PEBP and involucrin mRNAs were enhanced 6 h after treatment with 2.0 mM CaCl(2). The protein amount of PEBP was also increased by this treatment. To investigate whether PEBP is involved in differentiation induction of keratinocytes, HaCaT keratinocytes were transfected with an expression vector. Fluorescent immunostain revealed that cells expressing PEBP exhibited enlarged and flattened cell shape, and induction of involucrin expression was demonstrated by immunoblot analysis. Although the protein amount of ERK was not altered, phosphorylated ERK levels were decreased and cell proliferation was partly inhibited by PEBP expression. These results indicate that PEBP not only inhibits cell proliferation but also induces differentiation of human keratinocytes.

Cation Diffusion Facilitator Proteins Modulate Raf-1 Activity

The Ras-extracellular signal-regulated kinase (ERK) cascade is a critical intracellular signaling pathway that regulates growth, survival, and differentiation. Previous work established that Ras-GTP binds to, and facilitates the activation of, the protein kinase Raf-1. Recently, it was demonstrated that the cation diffusion facilitator (CDF) proteins are involved in Ras-ERK signaling by use of a Caenorhabditis elegans genetic screen that identified suppressors of activated Ras. In the current work, we demonstrate that CDF proteins may function downstream of Ras, but upstream of Raf-1 in Xenopus oocytes. We also show that the C. elegans protein CDF-1 and its mammalian homologue ZnT-1 bind to the amino-terminal regulatory portion of Raf-1 and promote the biological and enzymatic activity of Raf-1. Furthermore, we show that Zn(2+) inhibits Raf-1 binding to ZnT-1. We propose a model in which CDF protein binding facilitates Raf-1 activation.

Enhanced therapeutic effects of doxorubicin and paclitaxel in combination with liposome-entrapped ends-modified raf antisense oligonucleotide against human prostate, lung and breast tumor models

Raf-1 protein kinase plays an important role in cell growth, proliferation and cell survival. We have previously described the use of liposome-entrapped antisense raf oligonucleotide (LErafAON) to inhibit Raf-1 expression resulting in tumor growth inhibition and radiosensitization. The present study was undertaken to evaluate the chemosensitization effects of LErafAON in combination with doxorubicin or paclitaxel on a panel of human tumor xenografts. LErafAON (25.0 mg/kg i.v. x 10) displayed significant antitumor activity (P<0.05) when administered as a single agent in prostate (PC-3), lung (A549) and breast (MDA-MB 231) carcinoma models. Doxorubicin (1.0-4.0 mg/kg i.v. per week x 3) and paclitaxel (1.0-4.0 mg/kg i.v. on alternate days x 3) were administered as single agents at non-toxic doses that led to only minimal to moderate antitumor activity. However, a combination of LErafAON with doxorubicin or paclitaxel led to significantly enhanced antitumor activity in all the tumor types tested (PC-3, P<0.03; A549, P<0.035; MDA-MB 231, P<0.045) as compared with LErafAON alone or chemotherapeutic agents alone treated groups. This effect of chemosensitization appeared to be sequence-specific because a mismatch control oligonucleotide continued to show significant tumor growth. Additionally, no inhibition in Raf-1 expression in MDA-MB 231 tumor tissue was observed with mismatch oligonucleotide treatment. On the other hand, LErafAON treatment led to >75% inhibition of Raf-1 expression in tumor tissue. These preclinical observations support the use of LErafAON in combination with chemotherapeutic agents to improve the treatment of human cancers.

Induction of p97MAPK expression regulates collagen mediated inhibition of proliferation and migration in human squamous cell carcinoma lines

The ability of cancer cells to proliferate abnormally and invade surrounding tissue is among the most important features of the malignant phenotype. The mechanisms by which the mitogen activated protein kinase (MAPK) cascade regulates these phenotypes have been investigated for many years. Activated GTP bound Ras binds the upstream protein kinases Raf-1 and B-Raf. The MAPK kinases 1 and 2, known as MKK or MEK, are phosphorylated and activated by Raf. MEKs phosphorylate the extracellular signal regulated kinases ERK1 and ERK2. A unique member of the MAPK family is p97MAPK, the human homolog of rat ERK3. p97MAPK is ubiquitously expressed but whether its cellular functions are different from ERK1 and ERK2 is unknown. p97MAPK is highly expressed in human cancer cell lines. In the present study, expression of p97MAPK was unique among the ERK family in that its expression was induced when human carcinoma cell lines are plated on type IV collagen. This increased expression correlated with slower cancer cell proliferation on collagen compared to plastic tissue culture dishes. Overexpression of p97MAPK was sufficient to inhibit cellular proliferation with concomitant changes in cell cycle regulatory protein expression. p97MAPK also inhibited cancer cell migration and invasion by decreasing Rac1 expression but not that of matrix metalloproteinase 9 which is regulated by other ERKs. These data represent the first reported function of p97MAPK in human cancer cells.

The transcriptional response to Raf activation is almost completely dependent on Mitogen-activated Protein Kinase Kinase activity and shows a major autocrine component.

The Raf protein kinases are major effectors of Ras GTPases and key components of the transcriptional response to serum factors, acting at least in part through the extracellular signal-regulated kinase/mitogen-activated protein kinase pathway. It has recently been suggested that Raf also may trigger other as yet uncharacterized signaling pathways. Here, we have used cDNA microarrays to dissect changes in gene expression induced by activation of inducible c-Raf-1 constructs in human mammary epithelial and ovarian epithelial cells. The majority of Raf-induced transcriptional responses are shown to be blocked by pharmacological inhibition of the Raf substrate mitogen-activated protein kinase kinase, indicating that potential mitogen-activated protein kinase kinase-independent Raf signaling pathways have no significant influence on gene expression. In addition, we used epidermal growth factor receptor inhibitory drugs to address the contribution of autocrine signaling by Raf-induced EGF family proteins to the Raf transcriptional response. At least one-half of the transcription induced by Raf activation requires epidermal growth factor (EGF) receptor function The EGF receptor-independent component of the Raf transcriptional response is entirely up-regulation of gene expression, whereas the EGF receptor-dependent component is an equal mixture of up- and down-regulation. The use of transcriptional profiling in this way allows detailed analysis of the architecture of signaling pathways to be undertaken.

Alternative, Nonapoptotic Programmed Cell Death: MEDIATION BY ARRESTIN 2, ERK2, AND Nur77

Programmed cell death (pcd) may take the form of apoptosis or of nonapoptotic pcd. Whereas cysteine aspartyl-specific proteases (caspases) mediate apoptosis, the mediators of nonapoptotic cell death programs are much less well characterized. Here we report that alternative, nonapoptotic pcd induced by the neurokinin-1 receptor (NK(1)R) activated by its ligand Substance P, is mediated by a MAPK phosphorylation cascade recruited by the scaffold protein arrestin 2. The activation of the protein kinases Raf-1, MEK2, and ERK2 is essential for this form of nonapoptotic pcd, leading to the phosphorylation of the orphan nuclear receptor Nur77. NK(1)R-mediated cell death was inhibited by a dominant negative form of arrestin 2, Raf-1, or Nur77, by MEK1/2-specific inhibitors, and by RNA interference directed against ERK2 or MEK2 but not ERK1 or MEK1 and against Nur77. The MAPK pathway is also activated in neurons in primary culture undergoing NK(1)R-mediated death, since the MEK inhibitor PD98059 inhibited Substance P-induced death in primary striatal neurons. These results suggest that Nur77, which is regulated by a MAPK pathway activated via arrestin 2, modulates NK(1)R-mediated nonapoptotic pcd.

The Canonical Ethylene Signaling Pathway

The structural simplicity of the plant hormone ethylene contrasts with its dramatic effects in various developmental processes, as well as in the responses to a diversity of environmental signals. This broad spectrum of responses is mediated by a single well-conserved signaling cascade. Ethylene is perceived by a family of two-component histidine kinase receptors that become inactivated upon ethylene binding. In the absence of the hormone, the receptors activate CTR1, a negative regulator of ethylene responses. Sequence similarity between CTR1 and the Raf protein kinases implies involvement of a mitogen-activated protein kinase (MAPK) cascade in this signaling pathway. The first candidate MAPKs in the ethylene response pathway have been identified. EIN2 acts downstream of CTR1 and the possible kinase cascade. Although the biochemical function of EIN2 is not understood, its critical role is manifested by the complete ethylene insensitivity of the EIN2 loss-of-function mutants. Downstream of EIN2 there is a family of plant-specific EIN3-like transcription factors that mediate all known ethylene responses. The regulation of EIN3 stability by ethylene is accomplished by F-box-containing proteins that participate in the formation of a SCF complex, which targets proteins for degradation by the proteasome. A large number of ethylene-regulated genes have been identified, among them the AP2-domain containing ERF1 and EDF family of transcription factors, which suggests the participation of a transcriptional cascade in the ethylene response. The differential regulation of some components of this complex nuclear cascade by other signaling pathways provides a possible mechanism for interaction and signal integration. As new points of intersection with other pathways and additional participants in the pathway are identified, the Connections Map will be updated to include this new information.