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Research proposes a model for the action of endothelin-1 in the cell cycle process, which suggests that the diversity of the endothelin receptors is linked to the diversity of cellular responses elicited by endothelin-1 in human lung fibroblasts. Results describe endothelin-1 stimulation of cell cycle progression through mitogenic effects.

The authors have investigated p38 mitogen-activated protein (MAP) kinase activated by different protein kinases. The role of these other protein kinases in p38 MAP kinase activation mechanism has been studied via the analysis of the disruption effect of the murine genes on the p38 MAP kinase signaling pathway.

Research has been conducted on nuclear thyroid hormone receptor TR(beta)1.The authors report the identification of the thyroid hormone receptor's serine phosphorylation site in receptor's DNA-binding domain, and the identifications of mitogen-activated protein kinase docking site on thyroid hormone receptor induced by L-thyroxine.

Results demonstrate that Janus kinase 2-induced phosphorylation of calmodulin is crucial for the activation of sodium/proton exchange type 1 by angiotensin II and 5-hydroxytryptamine (serotonin) in vascular smooth muscle cells. Data support the role of mitogens in the activation of the sodium/proton exchange type 1 signal transduction pathway.

Research has been conducted on specialized gene expression programs induced by signaling pathways acting on transcription factors. The authors report the results of their study of the mechanism by which transcription factors can regulate gene expression programs in response to signaling.

Herein, we investigated the activity of mitogen-activated protein kinase (MAPK), a key component of downstream signaling events, which is activated subsequent to platelet-derived growth factor (PDGF)-BB stimulation. Specifically, [p42.sup.MAPK] activity peaked 60 min after addition of PDGF-BB, declined thereafter, and was determined not to be a direct or necessary component of glycosaminoglycan (GAG) synthesis. PDGF-BB also activated MAPK kinase 2 (MAPKK2) but had no effect on MAPKK1 and Raf-1 activity. Chemical inhibition of Janus kinase, phosphatidylinositol 3-kinase, Src kinase, or tyrosine phosphorylation inhibition of the PDGF [Beta]-receptor (PDGFR-[Beta]) did not abrogate PDGF-BB-induced [p42.sup.MAPK] activation or its threonine or tyrosine phosphorylation. A dominant negative cytoplasmic receptor for hyaluronan-mediated motility variant 4 (RHAMMv4), a regulator of MAPKK-MAPK interaction and activation, did not inhibit PDGF-BB-induced [p42.sup.MAPK] activation nor did a construct expressing PDGFR-[Beta] with cytoplasmic tyrosines mutated to phenylalanine. However, overexpression of a dominant negative PDGFR-[Beta] lacking the cytoplasmic signaling domain abrogated [p42.sup.MAPK] activity. These results suggest that PDGF-BB-mediated activation of [p42.sup.MAPK] requires the PDGFR-[Beta] but is independent of its tyrosine phosphorylation. mitogen-activated protein kinase; platelet-derived growth factor receptor; fetal development; lung fibroblasts; tyrosine phosphorylation

We have previously shown that interleukin-1 receptor-generated ceramide induces growth arrest in smooth muscle pericytes by inhibiting an upstream kinase in the extracellular signal-regulated kinase (ERK) cascade. Here, we now report the mechanism by which ceramide inhibits ERK activity. Ceramide renders the human embryonic kidney 293 cells (HEK 293) resistant to the mitogenic actions of growth factors and activators of protein kinase C (PKC). A role for PKC to mediate ceramide inhibition of growth factor-induced ERK activity and mitogenesis is suggested, as exogenous ceramide directly inhibits both immunoprecipitated and recombinant PKC-[Epsilon] activities. To confirm that PKC-[Epsilon] is necessary for ceramide-inhibited ERK activity, HEK 293 cells were transfected with a dominant-negative mutant of PKC-[Epsilon] ([Delta] PKC-[Epsilon]). These transfected cells respond to insulin-like growth factor I (IGF-I) with a significantly decreased ERK activity that is not further reduced by ceramide treatment. Coimmunoprecipitation studies reveal that the treatment with IGF-I induces the association of ERK with PKC-[Epsilon] but not with PKC-[Zeta]. Ceramide treatment significantly inhibits the IGF-I-induced PKC-[Epsilon] interaction with bioactive phosphorylated ERK. Ceramide also inhibits IGF-I-induced PKC-[Epsilon] association with Raf-1, an upstream kinase of ERK. Together, these studies demonstrate that ceramide exerts anti-mitogenic actions by limiting the ability of PKC-[Epsilon] to form a signaling complex with Raf-1 and ERK. protein kinase C; extracelular signal-regulated kinase; mitogen-activated protein kinase; ceramide; Raf-1

Long-term adaptation of renal cells to hypertonicity: role of MAP kinases and Na-K-ATPase. Am J Physiol Renal Physiol 280: F768-F776, 2001.--Renal cells in culture have low viability when exposed to hypertonicity. We developed cell lines of inner medullary collecting duct cells adapted to live at 600 and 900 mosmol/kg[H.sub.2]O. We studied the three modules of the mitogen-activated protein (MAP) kinase family in the adapted cells. These cells had no increase in either extracellular signal-regulated kinase, c-Jun [NH.sub.2]-terminal kinase, or p38 MAP kinase protein or basal activity. When acutely challenged with further increments in tonicity, they had blunted activation of these kinases, which was not due to enhanced phosphatase activity. In contrast, the cells adapted to the hypertonicity displayed a marked increment in Na-K-ATPase expression (5-fold) and ouabain-sensitive Na-K-ATPase activity (10-fold). The changes were reversible on return to isotonic conditions. Replacement of 300 mosmol/kg[H.sub.2]O of NaCl by urea in cells adapted to 600 mosmol/kg[H.sub.2]O resulted in marked decrement in Na-K-ATPase and failure to maintain the cell line. Replacement of NaCl for urea in cells adapted to 900 mosmol/kg[H.sub.2]O did not alter either Na-K-ATPase expression, or the viability of the cells. The in vivo modulation of Na-K-ATPase was studied in the renal papilla of water-deprived mice (urinary osmolality 2,900 mosmol/ kg[H.sub.2]O), compared with that of mice drinking dextrose in water (550 mosmol/kg[H.sub.2]O). Increased water intake was associated with a ~30% decrement in Na-K-ATPase expression (P [is less than] 0.02, n = 6), suggesting that this enzyme is osmoregulated in vivo. We conclude that whereas MAP kinases play a role in the response to acute changes in tonicity, they are not central to the chronic adaptive response. Rather, in this setting there is upregulation of other osmoprotective proteins, among which Na-K-ATPase appears to be an important component of the adaptive process. mitogen-activated protein; kidney cells; hyperosmolality; sodium pump

The cAMP-responsive element binding protein (CREB), a key regulator of gene expression, is activated by phosphorylation on Ser-133. Several different protein kinases possess the capability of driving this phosphorylation, making it a point of potential convergence for multiple intracellular signaling cascades. Previous work in neurons has indicated that physiologic synaptic stimulation recruits a fast calmodulin kinase IV (CaMKIV)-dependent pathway that dominates early signaling to CREB. Here we show in hippocampal neurons that the fast, CaMK-dependent pathway can be followed by a slower pathway that depends on Ras/mitogen-activated protein kinase (MAPK), along with CaMK. This pathway was blocked by dominant-negative Ras and was specifically recruited by depolarizations that produced strong intracellular [Ca.sup.2+] transients. When both pathways were recruited, phosphorylated CREB (pCREB) formation was overwhelmingly dominated by the CaMK pathway between 0 and 10 min, and by the MAPK pathway at 60 min, whereas the two pathways acted in concert at 30 min. The [Ca.sup.2+] signals that produced only rapid CaMK signaling to pCREB or both rapid CaMK and slow MAPK signaling deviated significantly for only [approximately equals] 1 min, yet their differential impact on pCREB extended over a much longer period, between 20 and 60 min and beyond, which is of likely significance for gene expression. The CaMK-dependent MAPK pathway may inform the nucleus about stimulus amplitude. In contrast, the CaMKIV pathway may be well suited to conveying information on the precise timing of localized synaptic stimuli, befitting its greater speed and sensitivity, whereas the previously described calcineurin pathway may carry information about stimulus duration.

Regulation of MAP kinase by calcium-sensing receptor in bovine parathyroid and CaR-transfected HEK293 cells. Am J Physiol Renal Physiol 280: F291-F302, 2001.--Regulation of the extracellular signal-regulated kinase 1 and 2 (ERK1/2) pathway by the extracellular calcium ([MATHEMATICAL EXPRESSION NOT REPRODUCIBLE IN ASCII])-sensing receptor (CaR) was investigated in bovine parathyroid and CaR-transfected human embryonic kidney (HEKCaR) cells. Elevating [MATHEMATICAL EXPRESSION NOT REPRODUCIBLE IN ASCII] or adding the selective CaR activator NPS R-467 elicited rapid, dose-dependent phosphorylation of ERK1/2. These phosphorylations were attenuated by pretreatment with pertussis toxin (PTX) or by treatment with the phosphotyrosine kinase (PTK) inhibitors genistein and herbimycin, the phosphatidylinositol-specific phospholipase C (PI-PLC)inhibitor U-73122, or the protein kinase C (PKC) inhibitor GF109203X and were enhanced by the PKC activator phorbol 12-myristate 13-acetate. Combined treatment with PTX and inhibitors of both PKC and PTK nearly abolished high [MATHEMATICAL EXPRESSION NOT REPRODUCIBLE IN ASCII]-evoked ERK1/2 activation in HEKCaR cells, demonstrating CaR-mediated coupling via both [G.sub.q] and [G.sub.i]. High [MATHEMATICAL EXPRESSION NOT REPRODUCIBLE IN ASCII] increased serine phosphorylation of the 85-kDa cytosolic phospholipase [A.sub.2] ([cPLA.sub.2]) in both parathyroid and HEKCaR cells. The selective mitogen-activated protein kinase (MAPK) inhibitor PD98059 abolished high-[MATHEMATICAL EXPRESSION NOT REPRODUCIBLE IN ASCII]-induced ERK1/2 activation and reduced [cPLA.sub.2] phosphorylation in both cell types, documenting MAPK's role in [cPLA.sub.2] activation. Thus our data suggest that the CaR activates MAPK through PKC, presumably through [G.sub.q/11]-mediated activation of PI-PLC, as well as through [G.sub.i]- and PTK-dependent pathway(s) in bovine parathyroid and HEKCaR cells and indicate the importance of MAPK in [cPLA.sub.2] activation. calcium-sensing receptor; mitogen-activated protein kinase; signal transduction; phospholipase [A.sub.2]; kidney

PC-cell-derived growth factor (PCDGF) is an 88-kDa glycoprotein corresponding to the granulin precursor. We have reported that PCDGF was expressed in human breast cancer cells. In estrogen-receptor positive cells, 17-[Beta]-estradiol ([E.sub.2]) transcriptionally stimulated PCDGF expression in a dose- and time-dependent fashion. We demonstrate here that PCDGF mediates the mitogenic effect of [E.sub.2] in MCF-7 cells. PCDGF substituted for [E.sub.2] to stimulate DNA synthesis. The [E.sub.2] mitogenic effect was inhibited in a dose-dependent fashion by anti-PCDGF neutralizing antibody. Inhibition of PCDGF expression by antisense transfection also inhibited the [E.sub.2] mitogenic effect. In contrast, overexpression of PCDGF in MCF-7 cells resulted in cells that were able to proliferate in the absence of estrogen and were tamoxifen resistant. The PCDGF signaling pathway was examined. Like [E.sub.2], PCDGF stimulated mitogen-activated protein kinase activity. PCDGF could substitute for [E.sub.2] in stimulating cyclin D1 expression. The cyclin D1 stimulation by [E.sub.2] was 50% inhibited by anti-PCDGF antibody. In contrast, PCDGF did not stimulate c-myc expression, another molecular target of [E.sub.2]. We conclude that autocrine PCDGF mediates the [E.sub.2] mitogenic effect via stimulation of cyclin D1. These studies provide information on estrogen action and identify an autocrine molecular target in human breast cancer cells.

Quiescent full-grown Xenopus oocytes remain arrested at the [G.sub.2]/M border of meiosis I until exposed to progesterone, their natural mitogen. Progesterone triggers rapid, nontranscriptional responses that lead to the translational activation of stored mRNAs, resumption of the meiotic cell cycles, and maturation of the oocyte into a fertilizable egg. It has long been presumed that progesterone activates the oocyte through a novel nontranscriptional signaling receptor. Here, we provide evidence that a conventional transcriptional progesterone receptor cloned from Xenopus oocytes, XPR-1, is required for oocyte activation, Overexpression of XPR-1 through mRNA injection increases sensitivity to progesterone and accelerates progesterone-activated cell cycle reentry. Injection of XPR-1 antisense oligonucleotides blocks the ability of oocytes to respond to progesterone; these oocytes are rescued by subsequent injection of XPR-1 or the human progesterone receptor PR-B. Antisense-treated oocytes can be activated in response to inhibition of protein kinase A, one of the earliest known changes occurring downstream of progesterone stimulation. These results argue that the conventional progesterone receptor also functions as the signaling receptor that is responsible for the rapid nontranscriptional activation of frog oocytes.

Translation initiation is one of the key events regulated in response to mitogenic stimulation and nutrient availability, tightly coupled to mammalian cell cycle progression and growth. FKBP12-rapamycin-associated protein (FRAP; also named mTOR or RAFT1), a member of the ataxia telangiectasia mutated (ATM)-related kinase family, governs a rapamycin-sensitive membrane-to-cytoplasm signaling cascade that modulates translation initiation via p70 S6 kinase ([p70.sup.s6k]) and eIF-4E binding protein 1 (4E-BP1). Our studies reported here reveal a surprising regulatory mechanism of this signaling, which involves cytoplasmic-nuclear shuttling of FRAP. By using leptomycin B (LMB), a specific inhibitor of nuclear export receptor Crm1, we show that FRAP is a cytoplasmic-nuclear shuttling protein. Inhibition of FRAP nuclear export by LMB coincides with diminished [p70.sup.s6k] activation and 4E-BP1 phosphorylation. Further investigation by altering FRAP's nuclear shuttling activity with exogenous nuclear import and export signals has yielded results that are consistent with a direct link between nuclear shuttling of FRAP and mitogenic stimulation of [p70.sup.s6k] activation and 4E-BP1 phosphorylation. Furthermore, by using a reporter system, we provide evidence suggesting that nuclear shuttling of FRAP regulates mitogen-stimulated rapamycin-sensitive translation initiation. These findings uncover a function for the nucleus in the direct regulation of the protein synthesis machinery via extracellular signals.

Thibonnier, Marc, Doreen M. Conarty, and Christine L. Plesnicher. Mediators of the mitogenic action of human [V.sub.1] vascular vasopressin receptors. Am J Physiol Heart Circ Physiol 279: H2529-H2539, 2000.--Arginine vasopressin (AVP) activation of [V.sub.1] vascular receptors ([V.sub.1]Rs) stimulates cell growth and proliferation in different tissues via cellular signaling pathways that remain to be identified. To explore the intracellular mediators of the mitogenic action of [V.sub.1]R, Chinese hamster ovary (CHO) cells were stably transfected with the human [V.sub.1]R cDNA clone we isolated previously. We assessed AVP effects on kinase activation (immunoblotting with phosphospecific antibodies), DNA synthesis (tritiated thymidine uptake), cell cycle progression (flow cytometry analysis after nuclear labeling with propidium iodide), and cell proliferation (conversion of the colorimetric reagent MTS) in the presence or absence of various pathway inhibitors. AVP stimulation of [V.sub.1]Rs leads to the phosphorylation of several kinases, an increase in DNA synthesis, a progression through the S and [G.sub.2]-M phases of the cell cycle, and an increase in cell proliferation. The mediators of the mitogenic action of [V.sub.1]R activation included calcium mobilization, coupling to a [G.sub.q] protein, and the simultaneous and parallel activation of several kinases, mainly calcium/calmodulindependent kinase II, phosphatidylinositol 3 kinase, protein kinase C, and p42/p44 mitogen-activated protein kinase. kinase activation; cell cycle; cell proliferation; arginine vasopressin

Tian, Wei, Zheng Zhang, and David M. Cohen. MAPK signaling and the kidney. Am J Physiol Renal Physiol 279: F593-F604, 2000.--Following an overview of the biochemistry of mitogen-activated protein kinase (MAPK) pathways, the relevance of these signaling events to specific models of renal cell function and pathophysiology, both in vitro and in vivo, will be emphasized. In in vitro model systems, events activating the principal MAPK families [extracellular signal-regulated and c-Jun [NH.sub.2]-terminal kinase and p38] have been best characterized in mesangial and tubular epithelial cell culture systems and include peptide mitogens, cytokines, lipid mediators, and physical stressors. Several in vivo models of proliferative or toxic renal injury are also associated with aberrant MAPK regulation. It is anticipated that elucidation of downstream effector signaling mechanisms and a clearer understanding of the immediate and remote upstream activating pathways, when applied to these highly clinically relevant model systems, will ultimately provide much greater insight into the basis for specificity now seemingly absent from these signaling events. urea; tubule; mesangial; extracellular signal-regulated kinase; stress-activated protein kinase; c-Jun [NH.sub.2]-terminal kinase; p38; mitogen-activated protein kinase

Vascular endothelial growth factor (VEGF) is a secreted mitogen which specifically stimulates proliferation of vascular endothelial cells in vitro and in vivo. Its expression pattern is consistent with it being an important regulator of vasculogenesis and angiogenesis, and targeted disruption of VEGF-A has demonstrated that it is essential for vascular development. To determine if VEGF-A was sufficient to alter vascularization in the eye we generated transgenic mice which express human VEGF-[A.sub.165] specifically in the lens. Expression of transgenic VEGF-A led to excessive proliferation and accumulation of disorganized angioblasts and endothelial cells around the lens. The results support the hypothesis that VEGF-A can initiate the process of vascularization by stimulating chemoattraction and proliferation of angioblasts and endothelial cells and that VEGF-A expression can stimulate angiogenic remodeling. However, VEGF-A alone was not sufficient to direct blood vessel organization or maturation. [C] 2000 Academic Press Key Words: VEGF-A; transgenic mice; angioblasts; angiogenesis; vascular development; neovascularization; eye development.

Low [[[Mg.sup.2+]].sub.o] induces contraction of cerebral arteries: roles of tyrosine and mitogen-activated protein kinases. Am J Physiol Heart Circ Physiol 279: H185-H194, 2000.--The present study was designed to investigate the mechanism of action of low extracellular magnesium ion concentration ([[[Mg.sup.2+]].sub.o]) on isolated canine basilar arteries and single cerebral vascular smooth muscle cells from these arteries. Low-[[[Mg.sup.2+]].sub.o] medium (0-0.6 mM) produces endothelium-independent contractions in isolated canine basilar arteries in a concentration-dependent manner; the lower the concentration of [[[Mg.sup.2+]].sub.o], the stronger the contractions. The low-[[[Mg.sup.2+]].sub.o] medium-induced contractions are significantly attenuated by pretreatment of the arteries with low concentrations of either SB-203580, U-0126, PD98059, genistein, or an Src homology 2 (SH2) domain inhibitor peptide. [IC.sub.50] levels obtained for these five antagonists are consistent with reported inhibitor constant ([K.sub.i]) values for these tyrosine kinase and mitogen-activated protein kinase (MAPK) antagonists. Low-[[[Mg.sup.2+]].sub.o] medium (0-0.6 mM) produces transient intracellular calcium ion concentration ([[[Ca.sup.2+]].sub.i]) peaks followed by a slow, sustained, and elevated plateau of [[[Ca.sup.2+]].sub.i] in primary single smooth muscle cells from canine basilar arteries. Low-[[[Mg.sup.2+]].sub.o] medium induces rapid and stable increases in [[[Ca.sup.2+]].sub.i]; these increases are inhibited markedly in the presence of either SB-203580, U-0126, PD98059, genistein or a SH2 domain inhibitor peptide. Several specific antagonists of known endogenously formed vasoconstrictors do not inhibit or attenuate either the low-[[[Mg.sup.2+]].sub.o]-induced contractions or the elevation of [[[Ca.sup.2+]].sub.i]. The present study suggests that activation of several cellular signaling pathways, such as protein tyrosine kinases (including the Src family) and MAPK, appears to play important roles in low-[[[Mg.sup.2+]].sub.o]-induced contractions and the elevation of [[[Ca.sup.2+]].sub.i] in smooth muscle cells from canine basilar arteries. canine basilar arterial rings; extracellular magnesium ion deficiency; intracellular calcium ion concentration; protein tyrosine kinase; Src homology 2 adaptor proteins

Mechanical strain-induced proliferation and signaling in pulmonary epithelial H441 cells. Am J Physiol Lung Cell Mol Physiol 279: L43-L51, 2000.--Pulmonary epithelial cells are exposed to mechanical strain during physiological breathing and mechanical ventilation. Strain regulates pulmonary growth and development and is implicated in volutrauma-induced fibrosis. The mechanisms of strain-induced effects are not well understood. It was hypothesized that mechanical strain induces proliferation of pulmonary epithelial cells and that this is mediated by signals initiated within seconds of strain. To test this hypothesis, human pulmonary adenocarcinoma H441 cells were strained in vitro. Cyclic as well as tonic strain resulted in increased cellular proliferation. Western blot analysis of strained cells demonstrated three newly phosphorylated tyrosine residues within 30 s of strain. Phosphorylation of mitogen-activated protein kinases p42/44 increased, electrophoretic mobility shift assay demonstrated activation of transcription factor activating protein-1, and immunohistochemistry demonstrated increased phosphorylation of c-jun in response to strain. The tyrosine kinase inhibitor genistein blocked the strain-induced proliferation. We conclude that strain induces proliferation in pulmonary epithelial cells and that tyrosine kinase activity is necessary to signal the proliferative response to mechanical strain. lung; tyrosine kinase; mitogen-activating protein

In addition to preventing crosstalk among related signaling pathways, scaffold proteins might facilitate signal transduction by preforming multimolecular complexes that can be rapidly activated by incoming signal. In many cases, such as mitogen-activated protein kinase (MAPK) cascades, scaffold proteins are necessary for full activation of a signaling pathway. To date, however, no detailed biochemical model of scaffold action has been suggested. Here we describe a quantitative computer model of MAPK cascade with a generic scaffold protein. Analysis of this model reveals that formation of scaffold-kinase complexes can be used effectively to regulate the specificity, efficiency, and amplitude of signal propagation. In particular, for any generic scaffold there exists a concentration value optimal for signal amplitude. The location of the optimum is determined by the concentrations of the kinases rather than their binding constants and in this way is scaffold independent. This effect and the alteration of threshold properties of the signal propagation at high scaffold concentrations might alter local signaling properties at different subcellular compartments. Different scaffold levels and types might then confer specialized properties to tune evolutionarily conserved signaling modules to specific cellular contexts.

[Ca.sup.2+] and p38 MAP kinase regulate mAChR-mediated c-Fos expression in avian exocrine cells. Am J Physiol Cell Physiol 278: C879-C884, 2000.--Muscarinic acetylcholine receptors (mAChRs) in exocrine tissue from the avian nasal salt gland are coupled to phospholipase C and generate inositol phosphate and [Ca.sup.2+] signals upon activation. An early effect of receptor activation in the secretory cells is a transient accumulation of c-Fos protein. In cooperation with constitutively expressed Jun, Fos presumably serves as a transcription factor altering gene expression during cell growth and differentiation processes in the gland associated with adaptation to osmotic stress in animals. Nothing is known, however, about the mAChR-dependent signaling pathways leading to Fos expression in these cells. By incubation of isolated nasal gland tissue in short-term culture with activators or inhibitors of signaling pathways and quantitative Western blot analysis of Fos abundance, we have now identified the sustained elevation of the intracellular [Ca.sup.2+] concentration and the activation of the p38 mitogen-activated protein (MAP) kinase as intermediate signaling elements for the regulation of c-Fos by muscarinic receptor activation. It is suggested that p38 MAP kinase, rather than exclusively mediating stress responses, is involved in the regulation of cellular growth and differentiation controlled by G protein-coupled receptors. muscarinic acetylcholine receptors; signal transduction; protooncogenes; Jun; intracellular calcium concentration; protein kinases; mitogen-activated protein kinase

Genetic and molecular characterization of the dominant suppressors of [D-raf.sup.C110] on the second chromosome identified two gain-of-function alleles of rolled (rl), which encodes a mitogen-activated protein (MAP) kinase in Drosophila. One of the alleles, [rl.sup.Su23], was found to bear the same molecular lesion as [rl.sup.Sem] which has been reported to be dominant female sterile. However, [rl.sup.Su23] and the current stock of [rl.sup.Sem] showed only a weak dominant female sterility. Detailed analyses of the rl mutations demonstrated moderate dominant activities of these alleles in the Torso (Tor) signaling pathway, which explains the weak dominant female sterility observed in this study. The dominant rl mutations failed to suppress the terminal class maternal-effect mutations, suggesting that activation of Rl is essential, but not sufficient, for Tor signaling. Involvement of rl in cell proliferation was also demonstrated by clonal analysis. Branching and integration of signals in the MAP kinase cascade is discussed.