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Rho-like GTPases play a pivotal role in the orchestration of changes in the actin cytoskeleton in response to receptor stimulation, and have been implicated in transcriptional activation, cell growth regulation, and oncogenic transformation. Recently, a role for RhoA in the regulation of cardiac contractility and hypertrophic cardiomyocyte growth has been suggested but the mechanisms underlying RhoA function in the heart remain undefined. We now report that transcription factor GATA-4, a key regulator of cardiac genes, is a nuclear mediator of RhoA signaling and is involved in the control of sarcomere assembly in cardiomyocytes. Both RhoA and GATA-4 are essential for sarcomeric reorganization in response to hypertrophic growth stimuli and overexpression of either protein is sufficient to induce sarcomeric reorganization. Consistent with convergence of RhoA and GATA signaling, RhoA potentiates the transcriptional activity of GATA-4 via a p38 MAPK-dependent pathway that phosphorylates GATA-4 activation domains and GATA binding sites mediate RhoA activation of target cardiac promoters. Moreover, a dominant-negative GATA-4 protein abolishes RhoA-induced sarcomere reorganization. The identification of transcription factor GATA-4 as a RhoA mediator in sarcomere reorganization and cardiac gene regulation provides a link between RhoA effects on transcription and cell remodeling.

Treatment of vascular smooth muscle cells (SMC) with angiotensin II (AII) leads to an increase in the tyrosine phosphorylation of multiple cellular substrates. Here, we have demonstrated that AII stimulates tyrosine phosphorylation of the focal adhesion-associated protein paxillin in rat aortic SMC. AII-induced phosphorylation of paxillin was detectable within 1 min and was sustained up to 60 min. Preincubation with the AT1-selective antagonist losartan abolished this response. The stimulatory effect of AII on paxillin tyrosine phosphorylation was observed only in aortic SMC and not in other target cells such as adrenal zona glomerulosa cells, chromaffin cells, or hepatocytes. The effect of AII was dependent on the activation of phospholipase C. Chelation of intracellular calcium completely inhibited the ability of AII to stimulate paxillin tyrosine phosphorylation, while selective inhibition of protein kinase C partially attenuated the response. In contrast, treatment of the cells with pertussis toxin had no effect on AII-induced paxillin tyrosine phosphorylation. These findings identify paxillin as a new substrate for AII-stimulated tyrosine phosphorylation and suggest a role for cytoskeleton-associated proteins in the growth response of aortic SMC.

Angiotensin II (AII) is a growth factor that stimulates protein synthesis and induces cellular hypertrophy in aortic smooth muscle cells (SMC). This trophic effect is mediated by the AT1 subtype of AII receptors. However, very little is known about the cellular signaling pathways involved in this response. In the present study, we examined the role of protein tyrosine phosphorylation in the growth-promoting effects of AII on rat aortic SMC. The addition of AII to quiescent aortic SMC induced tyrosine phosphorylation of multiple substrates, as revealed by antiphosphotyrosine immunoblotting. This response was blocked by preincubation with the AT1-selective antagonist losartan. To explore the functional role of this signaling pathway, we performed experiments with two mechanistically distinct tyrosine kinase inhibitors. Treatment of quiescent aortic SMC with genistein and herbimycin A abolished the stimulatory effect of AII on overall protein tyrosine phosphorylation. Similarly, the two inhibitors prevented AII-induced tyrosine phosphorylation of the cytoskeletal protein paxillin. Under the same conditions, incubation with genistein or herbimycin A did not interfere with AII binding to the AT1 receptor and did not significantly affect AII-stimulated inositol-1,4,5-trisphosphate production and Ca2+ mobilization. In parallel to their selective action on tyrosine phosphorylation, both genistein and herbimycin A completely inhibited AII-stimulated protein synthesis in a dose-dependent manner. In contrast, the two inhibitors were much less potent in preventing the trophic effect of phorbol-12-myristate 13-acetate in these cells. We further demonstrate that genistein and herbimycin A did not prevent mitogen-activated protein kinase activation and c-fos gene induction, which is consistent with the notion that these downstream effectors do not link AII-induced tyrosine phosphorylation to protein synthesis. These results provide evidence that tyrosine phosphorylation has a critical role in cellular hypertrophy and is involved in AII action in vascular SMC.

We have characterized two atrial natriuretic factor (ANF) receptor subtypes, designated ANF-R1 and ANF-R2, in two established cell lines that express exclusively one receptor subtype. The ANF-R1 receptor is selectively expressed by the kidney epithelial cell line LLC-PK1. It is a 130-kDa protein that has a much higher affinity for the biologically active forms of ANF than for its metabolites. The binding of ANF to this subtype is potentiated by amiloride and by divalent cations. The activation of the ANF-R1 receptor leads to an accumulation of cyclic GMP that is only partially inhibited by methylene blue. The ANF-R2 receptor, which is expressed selectively by the fibroblast cell line NIH-3T3, is a 130-kDa protein composed of two disulfide-linked subunits of 64-kDa. Activation of this subtype by saturating concentrations of ANF does not appear to elicit cyclic GMP production. However, supraphysiological concentrations of ANF induce a nonsaturable accumulation of cyclic GMP with an apparent ED50 in the high micromolar range. In contrast to the ANF-R1 subtype, the stimulation of cyclic GMP production is completely abolished by methylene blue. This subtype recognizes the active forms of ANF as well as its metabolites, and the binding is insensitive to amiloride and is decreased by divalent cations. These two cell lines can serve as models for studying the differential regulatory properties of ANF-R1 and ANF-R2 subtypes. In addition, we have also characterized the two ANF receptor subtypes in rat kidney glomeruli, where they show the same structure and pharmacological characteristics as in the two model cell lines.

The atrial natriuretic factor (ANF) receptor has been solubilized from bovine adrenal zona glomerulosa membranes with the nonionic detergent octyl-beta-D-glucoside. Mathematical analysis of competition binding curves with solubilized receptor revealed the presence of two classes of binding sites with pK of 10.4 (Kd = 40 pM) and 8.2 (kd = 6000 pM), similar to the native receptor of intact membranes. The hydrodynamic properties of the ANF receptor were determined by prelabeling the membrane receptor with 125I-ANF prior to solubilization. The solubilized 125I-ANF-receptor complex eluted as a major peak with a Stokes radius of 50.8 A from a Superose 6 steric exclusion column. A partial specific volume of 0.770 ml/g and a sedimentation coefficient (S20,w) of 6.34 S were determined by sucrose density gradient centrifugation in H2O and D2O. These data were used to calculate a molecular weight of 158,000 and a frictional ratio of 1.25 for the labeled receptor-detergent complex. The amount of detergent bound to the receptor was estimated to be 0.45 g/g of protein, assuming a partial specific volume of 0.730 ml/g for the protein. Correction for the mass contributed by the bound detergent yielded a molecular weight of 109,000 for the receptor protein. Affinity cross-linking of 125I-ANF to its binding sites in zona glomerulosa membranes and analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography revealed that one single band with apparent Mr 130,000 was specifically labeled. These results indicate that the ANF receptor from bovine adrenal cortex is a membrane protein with a total molecular weight of 110,000-130,000 and suggest that the native protein contains only one single polypeptide chain.

In the present study, we have examined the effect of increased cyclic AMP (cAMP) levels on the stimulatory action of angiotensin II (Ang II) on protein synthesis. Treatment with cAMP-elevating agents potently inhibited Ang II-induced protein synthesis in rat aortic smooth muscle cells and in rat fibroblasts expressing the human AT1 receptor. The inhibition was dose-dependent and was observed at all concentrations of the peptide. To explore the mechanism of cAMP action, we have analyzed the effects of forskolin and 3-isobutyl-1-methylxanthine on various receptor-mediated responses. Elevation of cAMP did not alter the binding properties of the AT1 receptor and did not interfere with the activation of phospholipase C or the induction of early growth response genes by Ang II. Likewise, Ang II-dependent activation of the mitogen-activated protein kinases ERK1/ERK2 and p70 S6 kinase was unaffected by cAMP. In contrast, we found that increased concentration of cAMP strongly inhibited the stimulatory effect of Ang II on protein tyrosine phosphorylation. Specifically, cAMP abolished Ang II-induced tyrosine phosphorylation of the focal adhesion-associated protein paxillin and of the tyrosine kinase Tyk2. These results identify a novel mechanism by which the cAMP signaling system may exert growth-inhibitory effects in specific cell types.

The effects of amiloride on the molecular characteristics of the atrial natriuretic factor (ANF) receptor from bovine adrenal zona glomerulosa were studied by computer modeling of competitive binding data, by affinity labeling experiments, and by steric exclusion high performance liquid chromatography of solubilized receptor. The order of potency of a series of truncated ANF analogs in competing for 125I-ANF binding to bovine adrenal zona glomerulosa membranes was the same as that obtained for inhibition of aldosterone secretion. Deletion of amino acids at the COOH-terminal end drastically reduced the affinities of the peptides. Computer analysis of competition curves revealed that all ANF analogs tested show similar binding characteristics: shallow competition curves, discrimination of varying proportions of high and low affinity binding states, and sensitivity to amiloride which increases the proportion of the high affinity binding component. These results from binding studies are suggestive of potential heterogeneity of ANF binding sites. In contrast, results from affinity cross-linking experiments are consistent with the notion of a single receptor protein. Incubation of membranes with increasing concentrations of 125I-ANF-(99-126) up to 3 nM resulted in the labeling of a single band of Mr 130,000. The ability of ANF analogs to compete for the labeling of the Mr 130,000 band by 125I-ANF-(99-126) agreed well with their potency as inhibitors of 125I-ANF binding to intact membranes. Addition of amiloride caused a dose-dependent increase in the labeling of the Mr 130,000 band. A single Mr 130,000 band was also labeled in bovine aorta and LLC-PK1 cell membranes. In order to further investigate the molecular basis for the apparent heterogeneity of ANF binding we have prelabeled the membrane receptor with 125I-ANF-(99-126) prior to solubilization with octyl-beta-D-glucoside and chromatography on a Superose 6 steric exclusion column. The elution profile of the prelabeled receptor consistently showed two peaks of radioactivity with mean Stokes radii of 70 and 50 A. When amiloride was added to the incubation medium, the elution profile consisted almost exclusively of the 70-A peak. Quantitative analysis of the chromatographic profiles revealed that amiloride increases by 2-3 times the area of the 70-A peak. We conclude that the 70-A form represents a ternary complex of the receptor with an amiloride-sensitive effector protein.

The NF-kappaB/Rel transcription factors participate in the activation of immune system regulatory genes and viral early genes including the human immunodeficiency virus type 1 long terminal repeat. NF-kappaB/Rel proteins are coupled to inhibitory molecules, collectively termed IkappaB, which are responsible for cytoplasmic retention of NF-kappaB. Cell activation leads to the phosphorylation and degradation of IkappaBalpha, permitting NG-kappaB/Rel translocation to the nucleus and target gene activation. To further characterize the signaling events that contribute to IkappaBalpha phosphorylation, a kinase activity was isolated from Jurkat T cells that specifically interacted with IkappaBalpha in an affinity chromatography step and phosphorylated IkappaBalpha with high specificity in vitro. By using an in-gel kinase assay with recombinant IkappaBalpha as substrate, two forms of the kinase (43 and 38 kDa) were identified. Biochemical criteria and immunological cross-reactivity identified the kinase activity as the alpha catalytic subunit of casein kinase II (CKII). Deletion mutants of IkappaBalpha delta1 to delta4) localized phosphorylation to the C-terminal PEST domain of IkappaBalpha. Point mutation of residues T-291, S-283, and T-299 dramatically reduced phosphorylation of IkappaBalpha by the kinase in vitro. NIH-3T3 cells that stably expressed wild-type IkappaBalpha (wtIkappaB), double-point-mutated IkappaBalpha (T291A, S283A), or triple-point-mutated IkappaBalpha (T291A, S283A, T299A) under the control of the tetracycline-responsive promoter were generated. Constitutive phosphorylation of the triple point mutant was eliminated in vivo, although tumor necrosis factor-inducible IkappaBalpha degradation was unaffected. In cell lines and in transiently transfected cells, mutation of the CKII sites in IkappaBalpha resulted in a protein with increased intrinsic stability. Together with results demonstrating a role for N-terminal sites in inducer-mediated phosphorylation and degradation of IkappaBalpha, these studies indicate that CKII sites in the C-terminal PEST domain are important for constitutive phosphorylation and intrinsic stability of IkappaBalpha.

Extracellular signal-regulated protein kinase (ERK, or mitogen-activated protein kinase [MAPK]) regulatory cascades in fungi turn on transcription factors that control developmental processes, stress responses, and cell wall integrity. CEK1 encodes a Candida albicans MAPK homolog (Cek1p), isolated by its ability to interfere with the Saccharomyces cerevisiae MAPK mating pathway. C. albicans cells with a deletion of the CEK1 gene are defective in shifting from a unicellular budding colonial growth mode to an agar-invasive hyphal growth mode when nutrients become limiting on solid medium with mannitol as a carbon source or on glucose when nitrogen is severely limited. The same phenotype is seen in C. albicans mutants in which the homologs (CST20, HST7, and CPH1) of the S. cerevisiae STE20, STE7, and STE12 genes are disrupted. In S. cerevisiae, the products of these genes function as part of a MAPK cascade required for mating and invasiveness of haploid cells and for pseudohyphal development of diploid cells. Epistasis studies revealed that the C. albicans CST20, HST7, CEK1, and CPH1 gene products lie in an equivalent, canonical, MAPK cascade. While Cek1p acts as part of the MAPK cascade involved in starvation-specific hyphal development, it may also play independent roles in C. albicans. In contrast to disruptions of the HST7 and CPH1 genes, disruption of the CEK1 gene adversely affects the growth of serum-induced mycelial colonies and attenuates virulence in a mouse model for systemic candidiasis.

To investigate the molecular basis of the hypertrophic action of angiotensin II (AII) in vascular smooth muscle cells (SMC), we have examined the ability of the hormone to regulate the function of the translational repressor 4E-binding protein 1 (4E-BP1). Addition of AII to quiescent aortic SMC potently increased the phosphorylation of 4E-BP1 as revealed by a decreased electrophoretic mobility and an increased phosphate content of the protein. The stimulation of 4E-BP1 phosphorylation was maximal at 15 min and persisted up to 120 min. Results from affinity chromatography on m7GTP-agarose demonstrated that AII-induced phosphorylation of 4E-BP1 promotes its dissociation from eIF4E in target cells. Further characterization of 4E-BP1 phosphorylation by phosphoamino acid analysis and phosphopeptide mapping revealed that 4E-BP1 is phosphorylated on eight distinct peptides containing serine and threonine residues in AII-treated cells. The combination of results obtained from kinetics experiments, phosphopeptide analysis of in vitro and in vivo phosphorylated 4E-BP1, and pharmacological studies with the MAP kinase kinase inhibitor PD 98059 provided strong evidence that the MAP kinases ERK1/ERK2 are not involved in the regulation of 4E-BP1 phosphorylation in aortic SMC. Together, our results demonstrate that AII treatment of vascular SMC leads to hyperphosphorylation of the translational regulator 4E-BP1 and to its dissociation from eIF4E by a MAP kinase-independent mechanism.

A common response of cells to mitogenic and hypertrophic factors is the activation of high rates of protein synthesis. To investigate the molecular basis of this action, we have used the recently developed MAP kinase/extracellular signal-regulated kinase (ERK) kinase (MEK) inhibitor PD 98059 to examine the involvement of the ERK pathway in the regulation of global protein synthesis by growth factors in rat aortic smooth muscle cells (SMC). Incubation with PD 98059 blocked angiotensin II (AII)-dependent phosphorylation and enzymatic activity of both MEK1 and MEK2 isoforms, leading to inhibition of the phosphorylation and activation of p44(mapk) and p42(mapk). The compound was found to selectively inhibit activation of the ERK pathway by AII, but not the stimulation of p70 S6 kinase, phospholipase C, or tyrosine phosphorylation. Most importantly, treatment of aortic SMC with PD 98059 potently inhibited AII-stimulated protein synthesis with a half-maximal inhibitory concentration of 4.3 microM. The effect of PD 98059 was not restricted to AII, since the compound also blocked to various extent the induction of protein synthesis by growth factors acting through tyrosine kinase receptors, G protein-coupled receptors, or protein kinase C. These results provide strong evidence that activation of ERK isoforms is an obligatory step for growth factor-induced protein synthesis in aortic SMC.

Angiotensin II (AII) is a growth factor which induces cellular hypertrophy in cultured vascular smooth muscle cells (SMC). To understand the molecular basis of this action, we have examined the role of the 70-kDa S6 kinases (p70S6K) in the hypertrophic response to AII in aortic SMC. AII potently stimulated the phosphotransferase activity of p70S6K, which reached a maximal value at 15 min and persisted for at least 4 h. This response was completely abolished when the cells were incubated in the presence of the AT1-selective receptor antagonist losartan. The enzymatic activation of p70S6K was associated with increased phosphorylation of the enzyme on serine and threonine residues. The immunosuppressant drug rapamycin was found to selectively inhibit the activation of p70S6K by AII, but not the activation of mitogen-activated protein kinase or the induction of c-fos mRNA expression. Treatment of aortic SMC with rapamycin also potently inhibited AII-stimulated protein synthesis with a half-maximal concentration similar to that required for inhibition of p70S6K. These results provide strong evidence that p70S6K plays a critical role in the signaling pathways by which AII induces hypertrophy of vascular SMC.

Without Abstract:

125I-Labeled atrial natriuretic factor (ANF) was covalently cross-linked to its binding sites in bovine adrenal zona glomerulosa membranes using the hydrophilic cross-linker bis(sulfosuccinimidyl) suberate. Analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis in the presence of 2-mercaptoethanol revealed that two protein bands with apparent Mr 68,000 and 114,000 were specifically labeled. The labeling of the two bands was prevented in a dose-dependent fashion by unlabeled ANF with a significant inhibition observed at 10(-10) M. High concentrations of angiotensin II and adrenocorticotropic hormone did not compete with 125I-ANF for binding and cross-linking. The dose-response curve for inhibition of covalent cross-linking of 125I-ANF by unlabeled ANF coincided with the dose-response curve for inhibition of binding to the receptor. No radioactive bands were observed in liver membranes. Experiments in which adrenal membranes were prepared and incubated in the presence of protease inhibitors showed no difference in the labeling pattern. Electrophoresis in the absence of reductant showed that the affinity-labeled species are not derived from larger disulfide-linked components. The apparent molecular weight of the two labeled species was not affected by a 100-fold variation in cross-linker concentration, and the labeling of both species increased in parallel. Possible relationships between the two labeled species are discussed.