Calmodulin binds to K-Ras, but not to H- or N-Ras, and modulates its downstream signaling.

Activation of Ras induces a variety of cellular responses depending on the specific effector activated and the intensity and amplitude of this activation. We have previously shown that calmodulin is an essential molecule in the down-regulation of the Ras/Raf/MEK/extracellularly regulated kinase (ERK) pathway in cultured fibroblasts and that this is due at least in part to an inhibitory effect of calmodulin on Ras activation. Here we show that inhibition of calmodulin synergizes with diverse stimuli (epidermal growth factor, platelet-derived growth factor, bombesin, or fetal bovine serum) to induce ERK activation. Moreover, even in the absence of any added stimuli, activation of Ras by calmodulin inhibition was observed. To identify the calmodulin-binding protein involved in this process, calmodulin affinity chromatography was performed. We show that Ras and Raf from cellular lysates were able to bind to calmodulin. Furthermore, Ras binding to calmodulin was favored in lysates with large amounts of GTP-bound Ras, and it was Raf independent. Interestingly, only one of the Ras isoforms, K-RasB, was able to bind to calmodulin. Furthermore, calmodulin inhibition preferentially activated K-Ras. Interaction between calmodulin and K-RasB is direct and is inhibited by the calmodulin kinase II calmodulin-binding domain. Thus, GTP-bound K-RasB is a calmodulin-binding protein, and we suggest that this binding may be a key element in the modulation of Ras signaling.

MEK kinase activity is not necessary for Raf-1 function.

Raf-1 protein kinase has been identified as an integral component of the Ras/Raf/MEK/ERK signalling pathway in mammals. Activation of Raf-1 is achieved by RAS:GTP binding and other events at the plasma membrane including tyrosine phosphorylation at residues 340/341. We have used gene targeting to generate a 'knockout' of the raf-1 gene in mice as well as a rafFF mutant version of endogenous Raf-1 with Y340FY341F mutations. Raf-1(-/-) mice die in embryogenesis and show vascular defects in the yolk sac and placenta as well as increased apoptosis of embryonic tissues. Cell proliferation is not affected. Raf-1 from cells derived from raf-1(FF/FF) mice has no detectable activity towards MEK in vitro, and yet raf-1(FF/FF) mice survive to adulthood, are fertile and have an apparently normal phenotype. In cells derived from both the raf-1(-/-) and raf-1(FF/FF) mice, ERK activation is normal. These results strongly argue that MEK kinase activity of Raf-1 is not essential for normal mouse development and that Raf-1 plays a key role in preventing apoptosis.

S338 phosphorylation of Raf-1 is independent of phosphatidylinositol 3-kinase and Pak3.

The Raf-1 serine/threonine protein kinase requires phosphorylation of the serine at position 338 (S338) for activation. Ras is required to recruit Raf-1 to the plasma membrane, which is where S338 phosphorylation occurs. The recent suggestion that Pak3 could stimulate Raf-1 activity by directly phosphorylating S338 through a Ras/phosphatidylinositol 3-kinase (Pl3-K)/-Cdc42-dependent pathway has attracted much attention. Using a phospho-specific antibody to S338, we have reexamined this model. Using LY294002 and wortmannin, inhibitors of Pl3-K, we find that growth factor-mediated S338 phosphorylation still occurs, even when Pl3-K activity is completely blocked. Although high concentrations of LY294002 and wortmannin did suppress S338 phosphorylation, they also suppressed Ras activation. Additionally, we show that Pak3 is not activated under conditions where S338 is phosphorylated, but when Pak3 is strongly activated, by coexpression with V12Cdc42 or by mutations that make it independent of Cdc42, it did stimulate S338 phosphorylation. However, this occurred in the cytosol and did not stimulate Raf-1 kinase activity. The inability of Pak3 to activate Raf-1 was not due to an inability to stimulate phosphorylation of the tyrosine at position 341 but may be due to its inability to recruit Raf-1 to the plasma membrane. Taken together, our data show that growth factor-stimulated Raf-1 activity is independent of Pl3-K activity and argue against Pak3 being a physiological mediator of S338 phosphorylation in growth factor-stimulated cells.

Regulation of Ras.GTP loading and Ras-Raf association in neonatal rat ventricular myocytes by G protein-coupled receptor agonists and phorbol ester. Activation of the extracellular signal-regulated kinase cascade by phorbol ester is mediated by Ras.

The small G protein Ras has been implicated in hypertrophy of cardiac myocytes. We therefore examined the activation (GTP loading) of Ras by the following hypertrophic agonists: phorbol 12-myristate 13-acetate (PMA), endothelin-1 (ET-1), and phenylephrine (PE). All three increased Ras.GTP loading by 10-15-fold (maximal in 1-2 min), as did bradykinin. Other G protein-coupled receptor agonists (e.g. angiotensin II, carbachol, isoproterenol) were less effective. Activation of Ras by PMA, ET-1, or PE was reduced by inhibition of protein kinase C (PKC), and that induced by ET-1 or PE was partly sensitive to pertussis toxin. 8-(4-Chlorophenylthio)-cAMP (CPT-cAMP) did not inhibit Ras.GTP loading by PMA, ET-1, or PE. The association of Ras with c-Raf protein was increased by PMA, ET-1, or PE, and this was inhibited by CPT-cAMP. However, only PMA and ET-1 increased Ras-associated mitogen-activated protein kinase kinase 1-activating activity, and this was decreased by PKC inhibition, pertussis toxin, and CPT-cAMP. PMA caused the rapid appearance of phosphorylated (activated) extracellular signal-regulated kinase in the nucleus, which was inhibited by a microinjected neutralizing anti-Ras antibody. We conclude that PKC- and Gi-dependent mechanisms mediate the activation of Ras in myocytes and that Ras activation is required for stimulation of extracellular signal-regulated kinase by PMA.

Lovastatin decreases prolactin and growth hormone gene expression in GH4C1 cells through a cAMP dependent mechanism.

The heterotrimeric G protein Gs couples several surface ligand receptors to cAMP production, as well as to both growth hormone (GH) and prolactin (PRL) gene expression in pituitary and GH cells. It has been shown that constitutively active alpha s stimulates transient expression of both PRL- and GH- chloramphenicol acetyl transferase (CAT) constructions, which indicates that both the PRL and GH promoter regions are under the influence of signal pathways mediated by alpha s. We have previously shown that the cholesterol lowering drug lovastatin decreases both the amount of G alpha s subunit in the membrane and the adenylyl cyclase activity in GH4C1 cells. Thus, we tried to verify whether that decrease in alpha s levels could affect PRL and GH secretion, as well as the expression of PRL- and GH-CAT constructions. Since the regulation of these two genes is dependent on the pituitary specific transcription factor Pit-1, the effect of lovastatin on the expression of Pit-1-CAT constructions was also studied. Our results show that lovastatin decreased the basal expression of these three cAMP-responsive genes in GH4C1 cells, being partially reversed by the addition of mevalonate to the culture medium. This effect of lovastatin on the promoter activities of the transfected constructions was also observed in PRL and GH secretion to the medium, suggesting that this drug produces similar changes in the endogenous promoters of both hormones. Moreover, the presence of lovastatin did not prevent the response to the cAMP activator forskolin, indicating that the main effect of this drug could be exerted through upstream adenylyl cyclase. In conclusion, our data indicate that lovastatin decreases the basal expression of Pit-1 and consequently of both GH and PRL genes through a mechanism probably mediated by the decrease of G alpha s levels in the cell membrane. Taken together, these results suggest that the activity of membrane heterotrimeric G proteins regulates the basal transcription of specific cellular genes in GH4C1 cells. Moreover the effects of lovastatin may be taken into account in the study of constitutively endocrine disorders associated with an increased secretion of either PRL or GH.

Identification of a protein-tyrosine phosphatase (SHP1) different from that associated with acid phosphatase in rat prostate.

Using [32P]poly(Glu,Tyr) as substrate, we have identified, for the first time, in the rat prostatic gland a protein-tyrosine phosphatase activity different from that associated with prostatic acid phosphatase. Concanavalin A-Sepharose 4B was used to separate the two protein-tyrosyl phosphatases activities. The activity retained by the lectin had characteristics of the prostatic acid phosphatase. It was sensitive to inhibition by PNPP and the optimum pH shifted towards physiological values when [32P]poly(Glu,Tyr) was used as substrate. However, the major protein-tyrosine phosphatase activity was not retained by the lectin, and corresponded, at least in part, to SHP1 as probed by the presence of the protein, its mRNA and the loss of PTPase activity after immunodepletion of SHP1. This enzyme is localized within the epithelial cells. Thus, the coexistence of two protein-tyrosine phosphatase activities in rat prostate, one associated with the acid phosphatase and the other related to SHP1, makes it necessary to analyze the importance of both activities in vivo and their possible function regarding prostatic cell growth and its regulation.