Phorbol 12-myristate 13-acetate and serum synergize to promote rapamycin-insensitive cell proliferation via protein kinase C-eta.

Previously, we have shown that PKC-eta (protein kinase C-eta) positively regulates glioblastoma proliferation and confers resistance to irradiation-induced apoptosis. In this study, we investigated the efficacy of rapamycin in inhibiting cell proliferation in two glioblastoma cell lines U-251MG (PKC-eta expressing) and U-1242MG (PKC-eta deficient) following PKC-eta activation. In U-251MG cells, rapamycin (10 nM) treatment was less effective as an antiproliferative agent when cells were concurrently stimulated with 10% serum and phorbol 12-myristate 13-acetate (PMA, 100 nM), a potent activator of PKC isozymes. Rapamycin-insensitive growth was owing to PKC-eta, as U-1242MG and U-251MG cells infected with a kinase-dead form of PKC-eta (U-251kr) were susceptible to rapamycin-induced inhibition of cell proliferation. Furthermore, U-251MG cells transfected with PKC-eta antisense oligonucleotides were sensitive to rapamycin. PKC-eta-expressing cells stimulated with PMA maintained p70S6K phosphorylation on Thr389 and phosphorylation of rpS6 (ser235/36), suggesting p70S6K kinase activity was still intact. Inhibition of p70S6K expression with small interfering RNA oligonucleotides inhibited cell proliferation greater than 50% in the presence of a combination of PMA and serum. Additionally, p70S6K co-precipitated with PKC-eta, suggesting a physical interaction between PKC-eta and p70S6K regulates the observed phosphorylation. Taken together, these data demonstrate that rapamycin-insensitive glioblastoma proliferation involves PKC-eta signaling.

Analyses of the effects of Rck2p mutants on Pbs2pDD-induced toxicity in Saccharomyces cerevisiae identify a MAP kinase docking motif, and unexpected functional inactivation due to acidic substitution of T379.

Rck2p is a Ser/Thr kinase that binds to, and is activated by, Hog1p. Expression of the MAP kinase kinase Pbs2pDD from a GAL1-driven plasmid hyperactivates the HOG MAP kinase pathway, and leads to cessation of growth. This toxic effect is reduced by deletion of RCK2. We studied the structural and functional basis for the role of Rck2p in mediating the growth arrest phenotype associated with overexpression of Pbs2pDD. Rck2p kinase activity is required for the effect, because Rck2p(Delta487-610), as well as full-length Rck2p, is toxic with Pbs2pDD, but kinase-defective versions of either protein with a K201R mutation are not. Thus, the C-terminal portion of Rck2p is not required provided the protein is activated by removal of the autoinhibitory domain. Relief of inhibition in Rck2p normally requires phosphorylation by Hog1p, and Rck2p contains a putative MAP kinase docking site (TILQR589R590KKVQ) in its C-terminal segment. The Rck2p double mutant R589A/R590A expressed from a centromeric plasmid did not detectably bind Hog1p-GFP and was functionally inactive in mediating the toxic effect of Pbs2pDD, equivalent to an RCK2 deletion. However, overexpression of Rck2p R589A/R590A from a multicopy plasmid restored function. In contrast, RCK2-K201R acted as a multicopy suppressor of PBS2DD, markedly reducing its toxicity. This suppressor activity required the K201R mutation, and the effect was largely lost when the docking site was mutated, suggesting suppression by inhibition of Hog1p functions. We also studied the effect of replacing the predicted T379 and established S520 phosphorylation sites in Rck2p by glutamic acid. Surprisingly, the T379E mutant markedly reduced Pbs2pDD toxicity, and toxicity was only partially rescued by S520E. Rck2 T379E was sufficiently inactive in an rck2Delta strain to allow some cells to survive PBS2DD toxicity even when overexpressed. The significance of these findings for our understanding of Rck2p function is discussed.

ERK2- and p90(Rsk2)-dependent pathways regulate the CCAAT/enhancer-binding protein-beta interaction with serum response factor.

The serum response element (SRE) of the c-fos promoter is a convergence point for mitogenic signaling pathways. Several transcription factors regulate SRE, including serum response factor (SRF), ternary complex factors, and CCAAT/enhancer-binding protein-beta (C/EBPbeta). C/EBPbeta can interact with both SRF and the ternary complex factor family member Elk-1, but only in response to activated Ras. Transactivation of the SRE by C/EBPbeta is also greatly stimulated by Ras. The Ras effectors that signal to C/EBPbeta are unknown. In this report, we demonstrate that a consensus MAPK site in C/EBPbeta is necessary for Ras stimulation of both C/EBPbeta-SRF interaction and transactivation of the SRE by C/EBPbeta. To dissect signaling pathways activated downstream of Ras, different Ras effector constructs were analyzed. We show that activated forms of Raf and phosphatidylinositol 3-kinase stimulate C/EBPbeta-SRF interaction. We also show a novel selectivity for the MAPK family member ERK2, where dominant-negative ERK2, but not dominant-negative ERK1, blocks Ras stimulation of C/EBPbeta-SRF interaction. In addition, recombinant C/EBPbeta protein is phosphorylated by ERK2, but not by ERK1, in vitro. Finally, we demonstrate a requirement for p90(Rsk2) in regulation of C/EBPbeta-SRF interaction. These data show that multiple Ras effectors are required to regulate C/EBPbeta and SRF association.

Identification of the S6 kinase activity stimulated in quiescent brine shrimp embryos upon entry to preemergence development as p70 ribosomal protein S6 kinase: isolation of Artemia franciscana p70S6k cDNA.

We previously demonstrated that a protein kinase responsible for phosphorylating 40S ribosomal subunits is activated in quiescent Artemia franciscana embryos within 15 min of restoration of normal tonicity and incubation at 30 degrees C. Here, we identify the activated S6 kinase as A. franciscana p70 ribosomal S6 kinase (p70S6k) subsequent to the isolation of an Artemia p70S6k cDNA. The protein conceptually translated from cDNA has 70% similarity and 64% identity to both Drosophila melanogaster and human p70S6k. Southern blot analysis is consistent with presence of a single p70S6k gene. Two transcripts of 5.4 and 2.7 kb were found. Abundance of both mRNAs increased dramatically around 4 h of preemergence development, and exhibited different steady-state level variation thereafter. Stimulated S6 kinase activity, partially purified by Superose 6 chromatography, correlated best with the slowest migrating, approximately 65 kDa, form detected by Western analysis using a specific polyclonal antibody made to a peptide from the predicted p70S6k NH2-terminus. Furthermore, the A. franciscana p70S6k was immunoprecipitated with the same antibody, showing in parallel an S6 kinase activity similar to peak profiles. We conclude that the stimulated S6 kinase activity is that of an ortholog of human p70S6k that may be involved in the regulation of protein synthesis during preemergence development in A. franciscana species.

A different function for a critical tryptophan in c-Raf and Hck.

The similarity of the catalytic domains of Raf and Src family members suggests that functions of homologous residues may be similar in both kinase families. A tryptophan residue, W260, in the WEI region of the Src family kinase Hck has an important role in regulating ATP binding. We tested the hypothesis that the tryptophan, W342, in the WEI region of c-Raf may have a similar role to the W260 of Hck. Mutation of W260 to A in Hck activates kinase activity, but we found that mutation of W342 to A in c-Raf inactivates the kinase activity. Mutating W342 to aspartate (D), lysine (K) or histidine (H) also inactivated c-Raf whether assayed as a purified immunoprecipitate or when recruited to the plasma membrane. A constitutively active c-Raf can be generated by mutating two regulatory tyrosines to aspartate. When placed into this active c-Raf mutant, mutation of W342 to D, K or H enabled phosphorylation and activation of the c-Raf substrate MEK at the plasma membrane but not in an immunoprecipitation assay. We conclude that (1) Tryptophan has a different role in the WEI regions of c-Raf and Hck, (2) W342 is not directly involved in MEK binding as both positive and negative residues at 342 are permissive for MEK activation at the membrane in a constitutively active c-Raf mutant, (3) Factors at the membrane are capable of potentiating activation of c-Raf containing mutated W342 in a hyperactivated c-Raf, but not in a wild type c-Raf and (4) There is a stringent structural requirement for W at residue 342 in c-Raf.

Creation of a stress-activated p90 ribosomal S6 kinase. The carboxyl-terminal tail of the MAPK-activated protein kinases dictates the signal transduction pathway in which they function.

Mitogen-activated protein kinase-activated protein kinases (MAPKAPKs) lie immediately downstream of the mitogen-activated protein kinases (MAPKs), extracellular signal-regulated kinase (ERK), and p38 MAPK. Although the family of MAPKAPKs shares sequence similarity, it demonstrates selectivity for the upstream activator. Here we demonstrate that each of the ERK- and p38 MAPK-regulated MAPKAPKs contains a MAPK docking site positioned distally to the residue(s) phosphorylated by MAPKs. The isolated MAPK docking sites show specificity for the upstream activator similar to that reported for the full-length proteins. Moreover, replacement of the ERK docking site of p90 ribosomal S6 kinase with the p38 MAPK docking site of MAPKAPK2 converts p90 ribosomal S6 kinase into a stress-activated kinase in vivo. It is apparent that mechanisms controlling events downstream of the proline-directed MAPKs involve specific MAPK docking sites within the carboxyl termini of the MAPKAPKs that determine the cascade in which the MAPKAPK functions.

Interaction among mitochondria, mitogen-activated protein kinases, and nuclear factor-kappaB in cellular models of Parkinson's disease.

Oxidative stress induced by acute complex I inhibition with 1-methyl-4-phenylpyridinium ion activated biphasically the stress-activated c-Jun N-terminal kinase (JNK) and the early transcription factor nuclear factor-kappaB (NF-kappaB) in SH-SY5Y neuroblastoma cells. Early JNK activation was dependent on mitochondrial adenine nucleotide translocator (ANT) activity, whereas late-phase JNK activation and the cleavage of signaling proteins Raf-1 and mitogen-activated protein kinase (MAPK) kinase (MEK) kinase (MEKK)-1 appeared to be ANT-independent. Early NF-kappaB activation depended on MEK, later activation required an intact electron transport chain (ETC), and Parkinson's disease (PD) cybrid (mitochondrial transgenic cytoplasmic hybrid) cells had increased basal NF-kappaB activation. Mitochondria appear capable of signaling ETC impairment through MAPK modules and inducing protective NF-kappaB responses, which are increased by PD mitochondrial genes amplified in cybrid cells. Irreversible commitment to apoptosis in this cell model may derive from loss of Raf-1 and cleavage/activation of MEKK-1, processes reported in other models to be caspase-mediated. Therapeutic strategies that reduce mitochondrial activation of proapoptotic MAPK modules, i.e., JNK, and enhance survival pathways, i.e., NF-kappaB, may offer neuroprotection in this debilitating disease.

Generation of constitutively active p90 ribosomal S6 kinase in vivo. Implications for the mitogen-activated protein kinase-activated protein kinase family.

p90 ribosomal S6 kinases (RSKs), containing two distinct kinase catalytic domains, are phosphorylated and activated by extracellular signal-regulated kinase (ERK). The amino-terminal kinase domain (NTD) of RSK phosphorylates exogenous substrates, whereas the carboxyl-terminal kinase domain (CTD) autophosphorylates Ser-386. A conserved putative autoinhibitory alpha helix is present in the carboxyl-terminal tail of the RSK isozymes ((697)HLVKGAMAATYSALNR(712) of RSK2). Here, we demonstrate that truncation (Delta alpha) or mutation (Y707A) of this helix in RSK2 resulted in constitutive activation of the CTD. In vivo, both mutants enhanced basal Ser-386 autophosphorylation by the CTD above that of wild type (WT). The enhanced Ser-386 autophosphorylation was attributed to disinhibition of the CTD because a CTD dead mutation (K451A) eliminated Ser-386 autophosphorylation even in conjunction with Delta alpha and Y707A. Constitutive activity of the CTD appears to enhance NTD activity even in the absence of ERK phosphorylation because basal phosphorylation of S6 peptide by Delta alpha and Y707A was approximately 4-fold above that of WT. A RSK phosphorylation motif antibody detected a 140-kDa protein (pp140) that was phosphorylated upon epidermal growth factor or insulin treatment. Ectopic expression of Delta alpha or Y707A resulted in increased basal phosphorylation of pp140 compared with that of WT, presenting the possibility that pp140 is a novel RSK substrate. Thus, it is clear that the CTD regulates NTD activity in vivo as well as in vitro.

Identification of an extracellular signal-regulated kinase (ERK) docking site in ribosomal S6 kinase, a sequence critical for activation by ERK in vivo.

Glutathione S-transferase (GST)-fusion proteins containing the carboxyl-terminal tails of three p90 ribosomal S6 kinase (RSK) isozymes (RSK1, RSK2, and RSK3) interacted with extracellular signal-regulated kinase (ERK) but not c-Jun-NH2-kinase (JNK) or p38 mitogen-activated protein kinase (MAPK). Within the carboxyl-terminal residues of the RSK isozymes is a region of high conservation corresponding to residues 722LAQRRVRKLPSTTL735 in RSK1. Truncation of the carboxyl-terminal 9 residues, 727VRKLPSTTL735, completely eliminated the interaction of the GST-RSK1 fusion protein with purified recombinant ERK2, whereas the truncation of residues 731PSTTL735 had no effect on the interaction with purified ERK2. ERK1 and ERK2 co-immunoprecipitated with hemagglutinin-tagged wild type RSK2 (HA-RSK2) in BHK cell cytosol. However, ERK did not co-immunoprecipitate with HA-RSK2((1-729)), a mutant missing the carboxyl-terminal 11 amino acids, similar to the minimal truncation that eliminated in vitro interaction of ERK with the GST-RSK1 fusion protein. Kinase activity of HA-RSK2 increased 6-fold in response to insulin. HA-RSK2((1-729)) had a similar basal kinase activity to that of HA-RSK2 but was not affected by insulin treatment. Immunoprecipitated HA-RSK2 and HA-RSK2((1-729)) could be activated to the same extent in vitro by active ERK2, demonstrating that HA-RSK2((1-729)) was properly folded. These data suggest that the conserved region of the RSK isozymes (722LAQRRVRKL730 of RSK1) provides for a specific ERK docking site approximately 150 amino acids carboxyl-terminal to the nearest identified ERK phosphorylation site (Thr573). Complex formation between RSK and ERK is essential for the activation of RSK by ERK in vivo. Comparison of the docking site of RSK with the carboxyl-terminal tails of other MAPK-activated kinases reveals putative docking sites within each of these MAPK-targeted kinases. The number and placement of lysine and arginine residues within the conserved region correlate with specificity for activation by ERK and p38 MAPKs in vivo.

pp90rsk1 regulates estrogen receptor-mediated transcription through phosphorylation of Ser-167.

The estrogen receptor alpha (ER), a member of the steroid receptor superfamily, contains an N-terminal hormone-independent transcriptional activation function (AF-1) and a C-terminal hormone-dependent transcriptional activation function (AF-2). Here, we used in-gel kinase assays to determine that pp90rsk1 activated by either epidermal growth factor (EGF) or phorbol myristate acetate specifically phosphorylates Ser-167 within AF-1. In vitro kinase assays demonstrated that pp90rsk1 phosphorylates the N terminus of the wild-type ER but not of a mutant ER in which Ser-167 was replaced by Ala. In vivo, EGF stimulated phosphorylation of Ser-167 as well as Ser-118. Ectopic expression of active pp90rsk1 increased the level of phosphorylation of Ser-167 compared to that of either a mutant pp90rsk1, which is catalytically inactive in the N-terminal kinase domain, or to that of vector control. The ER formed a stable complex with the mutant pp90rsk1 in vivo. Transfection of the mutant pp90rsk1 depressed ER-dependent transcription of both a wild-type ER and a mutant ER that had a defective AF-2 domain (ER TAF-1). Furthermore, replacing either Ser-118 or Ser-167 with Ala in ER TAF-1 showed similar decreases in transcription levels. A double mutant in which both Ser-118 and Ser-167 were replaced with Ala demonstrated a further decrease in transcription compared to either of the single mutations. Taken together, our results strongly suggest that pp90rsk1 phosphorylates Ser-167 of the human ER in vivo and that Ser-167 aids in regulating the transcriptional activity of AF-1 in the ER.

Ribosomal S6 kinase is activated as an early event in preemergence development of encysted embryos of Artemia salina.

Dormant Artemia salina cysts contain desiccated gastrulae that are metabolically inactive, and physiologically arrested. Following rehydration, embryos resume development via alterations in protein expression, in the complete absence of cell division. In mammals, activation of p70 ribosomal S6 kinase (p70S6k) has been implicated in translational control, in particular the selective up-regulation of translation of mRNAs with polypyrimidine tracts at their 5' start sites. We therefore investigated ribosomal S6 kinase activity in preemergence development. We demonstrate that an S6 kinase activity is rapidly stimulated (within < 15 min) following rehydration and coincides with the onset of ribosomal S6 subunit phosphorylation. This S6 kinase activity displays chromatographic and biochemical characteristics that are similar to those of mammalian p70S6k. Partially purified Artemia S6 kinase was inactivated by treatment with protein phosphatase 2A. Activation of S6 kinase activity was shown to be due to an enzymatic step(s), and not simply rehydration of stored, active enzyme. The temporal profile of activation of S6 kinase activity is compatible with a regulatory function for p70S6k in early preemergence development of encysted Artemia. These studies identify activated Artemia cysts as a system for biochemical studies of p70S6k regulation.

Activation in vitro of somatostatin receptor subtypes 2, 3, or 4 stimulates protein tyrosine phosphatase activity in membranes from transfected Ras-transformed NIH 3T3 cells: coexpression with catalytically inactive SHP-2 blocks responsiveness.

Somatostatin receptors (sstr) subtypes 1-5 were transiently expressed in NIH 3T3 cells stably transformed with Ha-Ras(G12V) to assess the ability of each receptor to stimulate protein tyrosine phosphatase (PTPase) activity in vitro. Treatment of membranes from sstr2-, sstr3-, or sstr4-expressing cells with somatostatin-14 plus guanyl-5'-yl imidodiphosphate (GMPPNP) increased PTPase activity, and this stimulation was pertussis toxin-sensitive. Somatostatin alone, GMPPNP alone, or somatostatin plus GDP were ineffective under these conditions. sstr1 and sstr5 failed to increase PTPase activity although both receptors were expressed, as assessed by appearance of high-affinity binding sites for [125I-Tyr11]somatostatin-14. Somatostatin plus GMPPNP stimulated PTPase activity in vitro when sstr2 was coexpressed with wild type PTP1B or a Cys to Ser (C/S), catalytically inactive PTP1B or with wild type SH2-domain containing PTPase SHP-2. However, coexpression with catalytically inactive C/S SHP-2 abrogated this response. Thus, three of the five cloned sstr's can couple to activate PTPase in this cellular background. Abrogation of the response by C/S SHP-2 strongly suggests, but does not prove, a role for SHP-2 in the mechanism.

The activating dual phosphorylation of MAPK by MEK is nonprocessive.

Activation of mitogen-activated protein kinases (MAPKs), also known as extracellular-signal-regulated kinases (ERKs), by MAPK/extracellular protein kinase kinases (MEKs) requires phosphorylation at two sites. The first step in MAPK activation by MEK must be the formation of a MEK x MAPK enzyme-substrate complex, followed by phosphorylation producing monophosphorylated MAPK (pMAPK). Subsequently, one of two events may occur. (1) MEK catalyzes the second and fully activating phosphorylation of MAPK, producing ppMAPK (a processive mechanism). (2) The complex of MEK x pMAPK dissociates before the second phosphorylation occurs, full activation requiring a reassociation of pMAPK with MEK (a nonprocessive or distributive mechanism). Simulations indicate that these two mechanisms predict different kinetics of MAPK activation. Specifically, the nonprocessive mechanism predicts that there will be a paradoxical decrease in the rate of MAPK activation as the MAPK concentration is increased. The present study uses p42 MAPK, also known as ERK2, and MEK1 as representatives of their respective classes of enzymes. We find that increasing the ERK2 concentration decreases the rate of activation by a mechanism which does not involve inhibition of MEK1 function. The accumulation of the active, doubly phosphorylated ERK2 (ppERK2) was directly assessed using a phosphorylation-state-specific antibody. The rate of accumulation of ppERK2 is decreased by increasing the ERK2 concentration. Therefore, the mechanism of ERK2 activation by MEK1 in vitro is nonprocessive.

Divergence in signal transduction pathways of platelet-derived growth factor (PDGF) and epidermal growth factor (EGF) receptors. Involvement of sphingosine 1-phosphate in PDGF but not EGF signaling.

Platelet-derived growth factor (PDGF) and serum, but not epidermal growth factor (EGF), stimulated sphingosine kinase activity in Swiss 3T3 fibroblasts and increased intracellular concentrations of sphingosine 1-phosphate (SPP), a sphingolipid second messenger (Olivera, A., and Spiegel, S. (1993) Nature 365, 557-560). We report herein that DL-threo-dihydrosphingosine (DHS), a competitive inhibitor of sphingosine kinase that prevents PDGF-induced SPP formation, specifically inhibited the activation of two cyclin-dependent kinases (p34(cdc2) kinase and Cdk2 kinase) induced by PDGF, but not by EGF. SPP reversed the inhibitory effects of DHS on PDGF-stimulated cyclin-dependent kinases and DNA synthesis, demonstrating that the DHS effects were mediated via inhibition of sphingosine kinase. DHS also markedly reduced PDGF-stimulated but not EGF-stimulated mitogen-activated protein kinase activity and DNA binding activity of activator protein-1. Examination of the early signaling events of PDGF action revealed that DHS did not affect PDGF-induced autophosphorylation of the growth factor receptor or phosphorylation of the SH2/SH3 adaptor protein Shc and its association with Grb2. This sphingosine kinase inhibitor did not abrogate activation of phosphatidylinositol 3-kinase by PDGF. In agreement, treatment with SPP had no effect on these responses but did, however, potently stimulate phosphorylation of Crk, another SH2/SH3 adaptor protein. Moreover, DHS inhibited PDGF-stimulated, but not EGF-stimulated, Crk phosphorylation. Thus, regulation of sphingosine kinase activity defines divergence in signal transduction pathways of PDGF and EGF receptors leading to mitogen-activated protein kinase activation.

Identification of Raf-1 Ser621 kinase activity from NIH 3T3 cells as AMP-activated protein kinase.

Raf-1 is extensively phosphorylated on Ser621 in both quiescent and mitogen-stimulated cells. To identify the responsible kinase(s), cytosolic fractions of NIH 3T3 cells were analyzed for Ser621 peptide kinase activity. One major peak of activity was detected and identified as AMP-activated protein kinase (AMPK) by immunodepletion experiments. AMPK phosphorylated the catalytic domain of Raf-1, expressed in Escherichia coli as a soluble GST fusion protein, to generate a single tryptic [32P]phosphopeptide containing exclusively phospho-Ser621. AMPK also phosphorylated full-length, kinase-defective Raf-1 (K375M) to generate two [32P]phosphopeptides, one co-migrating with synthetic tryptic peptide containing phospho-Ser621 and the other with phospho-Ser259.

S49 cells endogenously express subtype 2 somatostatin receptors which couple to increase protein tyrosine phosphatase activity in membranes and down-regulate Raf-1 activity in situ.

S49 cells expressed type 2 somatostatin receptors (sstr2) by immunoblotting. Analysis by reverse transcription and polymerase chain reaction (RT-PCR) methodologies showed that S49 cells express predominantly sstr2A and sstr2B mRNAs; other subtypes were either not detected, in the case of sstr1, sstr3, sstr4, or variably detected, in the case of sstr5. No mutations were present in S49 cells at codon 12, 13, or 61 of the N-, K-, or H-ras genes. Nevertheless, randomly growing S49 cells contained Raf-1 activity by specific immune complex kinase assays. Treatment of S49 cells with somatostatin transiently inactivated the basal activity of Raf-1, but not that of B-Raf. Addition of somatostatin plus guanyl-5'-yl imidodiphosphate (GMPPNP) to S49 membranes stimulated PTPase activity. The concentration dependence for stimulation of PTPase activity correlated with high affinity binding of [125I-Tyr11]somatostatin-14. Both the effect of somatostatin to stimulate PTPase activity and to inactivate Raf-1 were abrogated by PTx. PTPase activity stimulated by somatostatin plus GMPPNP was recovered in a peak of high apparent M(r) (670,000) after solubilisation with Triton X-100 and Superose 6 chromatography. Furthermore, addition of activated, brain G alpha i/o subunits to fractions from control membranes stimulated PTPase activity in the high M(r) peak. Thus, S49 membranes contain a G-protein regulated PTPase (PTPase-G), and PTPase-G in these cells may reside in a high molecular weight complex.

Activation of Raf by ionizing radiation.

The critical pathways through which ionizing radiation induces malignant transformation and cell death are not well defined. Raf-1, a cytoplasmic serine-threonine protein kinase, mediates the transmission of mitogenic signals initiated at the cell membrane to the nucleus, resulting in the activation of transcription factors that regulate cell growth and proliferation. Moreover, Raf-1 overexpression and activation increases the survival response of mammalian cells to the toxic effects of ionizing radiation by an as-yet unknown mechanism (refs 3, 4 and V. Soldatenkov et al.; manuscript submitted). Somewhat analogous to mitogen-induced signalling, radiation stimulates protein-tyrosine kinase(s) and transcription factors. No direct biochemical link has been established, however, between radiation-stimulated protein tyrosine phosphorylation and downstream signals. Here we report a series of radiation-responsive events in which protein-tyrosine phosphorylation is followed by membrane recruitment, then tyrosine phosphorylation and activation of Raf-1 in vivo. Our results show that radiation-stimulated protein-tyrosine kinase(s) modify Raf-1, and implicate Raf-1 in the ionizing-radiation signal-transduction pathway.

Developmental and tissue-specific expression of mouse pelle-like protein kinase.

The NF-kappaB/c-Rel proteins are a family of evolutionarily conserved transcription factors activated during development that in the adult, mediate many processes including the immune response. A high degree of sequence similarity is shared between the NF-kappaB/c-Rel family of transcription factors and the Drosophila Dorsal protein as well as between its cytoplasmic inhibitor, IkappaBalpha, and the Drosophila Cactus protein. Genetic analyses of Dorsal have defined components of a signaling pathway for Dorsal activation, including a serine/threonine kinase, Pelle, placed upstream of Dorsal and Cactus. We demonstrate that this pathway is likely to be conserved in mammals by the isolation of a cDNA that encodes a novel mouse protein highly related to Pelle, mPLK (mouse Pelle-like protein kinase). Expression of mPLK mRNA is developmentally regulated in the mouse and in adult tissue mPLK expression is greatest in the liver, a tissue that expresses a high level of NF-kappaB. Recombinant mPLK produced in bacteria is a protein kinase capable of autophosphorylating and phosphorylating IkappaBalpha.

Activation of a protein tyrosine phosphatase and inactivation of Raf-1 by somatostatin.

Human somatostatin receptor 3 ('hsstr3') was transiently expressed in NIH 3T3 cells stably transformed with Ha-Ras (G12V). Somatostatin activated a protein tyrosine phosphatase and inactivated the constitutively active, membrane-associated form of the Raf-1 serine kinase present in these cells in vivo and in vitro.

Ras-induced activation of Raf-1 is dependent on tyrosine phosphorylation.

Although Rafs play a central role in signal transduction, the mechanism(s) by which they become activated is poorly understood. Raf-1 activation is dependent on the protein's ability to bind Ras, but Ras binding is insufficient to activate Raf-1 tyrosine phosphorylation to this Ras-induced activation, in the absence of an over-expressed tyrosine kinase. We demonstrate that Raf-1 purified form Sf9 cells coinfected with baculovirus Ras but not Src could be inactivated by protein tyrosine phosphatase PTP-1B. 14-3-3 and Hsp90 proteins blocked both the tyrosine dephosphorylation and inactivation of Raf-1, suggesting that Raf-1 activity is phosphotyrosine dependent. In Ras-transformed NIH 3T3 cells, a minority of Raf-1 protein was membrane associated, but essentially all Raf-1 activity and Raf-1 phosphotyrosine fractionated with plasma membranes. Thus, the tyrosine-phosphorylated and active pool of Raf-1 constitute a membrane-localized subfraction which could also be inactivated with PTP-1B. By contrast, B-Raf has aspartic acid residues at positions homologous to those of the phosphorylated tyrosines (at 340 and 341) of Raf-1 and displays a high basal level of activity. B-Raf was not detectably tyrosine phosphorylated, membrane localized, or further activated upon Ras transformation, even though B-Raf has been shown to bind to Ras in vitro. We conclude that tyrosine phosphorylation is an essential component of the mechanism by which Ras activates Raf-1 kinase activity and that steady-state activated Ras is insufficient to activate B-Raf in vivo.