Akt phosphorylation regulates the tumour-suppressor merlin through ubiquitination and degradation

The neurofibromatosis-2 (NF2) tumour-suppressor gene encodes an intracellular membrane-associated protein, called merlin, whose growth-suppressive function is dependent on its ability to form interactions through its intramolecular amino-terminal domain (NTD) and carboxy-terminal domain (CTD). Merlin phosphorylation plays a critical part in dictating merlin NTD/CTD interactions as well as in controlling binding to its effector proteins. Merlin is partially regulated by phosphorylation of Ser 518, such that hyperphosphorylated merlin is inactive and fails to form productive intramolecular and intermolecular interactions. Here, we show that the protein kinase Akt directly binds to and phosphorylates merlin on residues Thr 230 and Ser 315, which abolishes merlin NTD/CTD interactions and binding to merlin's effector protein PIKE-L and other binding partners. Furthermore, Akt-mediated phosphorylation leads to merlin degradation by ubiquitination. These studies demonstrate that Akt-mediated merlin phosphorylation regulates the function of merlin in the absence of an inactivating mutation.     

A conserved alternative splice in the von Recklinghausen neurofibromatosis (NF1) gene produces two neurofibromin isoforms, both of which have GTPase-activating protein activity.

Sequence analysis has shown significant homology between the catalytic regions of the mammalian ras GTPase-activating protein (GAP), yeast Ira1p and Ira2p (inhibitory regulators of the RAS-cyclic AMP pathway), and neurofibromin, the protein encoded by the NF1 gene. Yeast expression experiments have confirmed that a 381-amino-acid segment of neurofibromin, dubbed the GAP-related domain (GRD), can function as a GAP. Using the RNA polymerase chain reaction with primers flanking the NF1-GRD, we have identified evidence for alternative splicing in this region of the NF1 gene. In addition to the already published sequence (type I), an alternative RNA carrying a 63-nucleotide insertion (type II) is present in all tissues examined, although the relative amounts of types I and II vary. The insertion is conserved across species but is not present in GAP, IRA1, or IRA2. GenBank searches have failed to identify significant similarity between the inserted sequence and known DNA or protein sequences, although the basic amino acid composition of the insertion shares features with nuclear targeting sequences. Expression studies in yeasts show that despite the partial disruption of the neurofibromin-IRA-GAP homology by this insertion, both forms of the NF1-GRD can complement loss of IRA function. In vivo assays designed to compare the GAP activity of the two alternatively spliced forms of the NF1-GRD show that both can increase the conversion of GTP-bound ras to its GDP-bound form, although the insertion of the 21 amino acids weakens this effect. The strong conservation of this alternative splicing suggests that both type I and II isoforms mediate important biological functions of neurofibromin.     

Independence of changes in contraction properties and myofibrillar ATPase activity of denervated mammalian muscle on length of nerve stump.

1. Contractile properties of the fast extensor digitorum longus of one-month-old rats and of the fast peroneus longus muscles of adult rabbits were studied in vitro at 36 degrees C after nerve section close to the muscle. Changes in contraction properties (prolongation) are not observed until 48 hours after denervation in the rat and 14-30 days in the rabbit. 2. At no period after denervation are differences in twitch isometric contraction properties dependent on the length of the sectioned nerve stump. This lack of dependence of contractile behavior after denervation is in contrast to many metabolic changes which show a clear dependence on the length of the nerve stump. 3. It is concluded that the onset of denervation changes in contractile behavior are related to the loss of nerve-impulse activity, while the transient early metabolic changes are related to changes of fast axoplasmic flow, initiated after nerve section and therefore dependent on length of sectioned nerve stump.     

N-Hydroxylation of arylamides by the rat and guinea pig: Evidence for substrate specificity and participation of cytochrome P1-450

The hypothesis that N-hydroxylation of arylamides is essential for carcinogenicity was examined in vivo and in vitro with N-2-fluorenylacetamide, a potent carcinogen, and with N-3-fluorenylacetamide, an isomer with marginal carcinogenicity. About 10–20% of 2-[9-¹⁴C]fluorenylacetamide administered intraperitoneally to the rat was excreted in the bile as the N-hydroxy-2-[9-¹⁴C]-derivative, whereas <0.1% of 3-[G-³H]fluorenylacetamide was found as the N-hydroxy metabolite in bile and urine. N-Hydroxylation of the 2- isomer by hepatic microsomes of untreated or 3-methylcholanthrene-treated rats was 40 to 50-fold greater than that of the 3- isomer. The role of cytochromes P-450 and P₁-450 in N-hydroxylation of arylamides by rat liver microsomes was shown by inhibition of the reaction with carbon monoxide and cobaltous chloride. Interaction of the arylamides with cytochrome P₁-450 was also demonstrated by binding spectra obtained on addition on 2- and 3-fluorenylacetamide to hepatic chromosomes of methylcholanthrene-treated rats. There appeared to be no correlation between the magnitude of the spectra and the extent of N-hydroxylation. N-Hydroxylation of the 2- isomer by hepatic microsomes of the guinea pig, a species resistant to the carcinogenecity of this compound, was markedly less than N-hydroxylation by rat liver microsomes, even though, as judged by the appearance of the binding spectra, both 2- and 3- isomers were bound by cytochrome P₁-450 of guinea pig-liver microsomes. The results are in agreement with the view that the microsomal N-hydroxylation of arylamides parallels their carcinogenicity.     

Protein modifications by activated carcinogens. II. The acetylation of ribonuclease by N-acetoxy-3-fluorenylacetamide.

The reaction of N-[3H]acetoxy-3-fluorenylacetamide (N-[3H]acetoxy-3-FAA), a potent carcinogen for the rat, with RNAase yielded three modified proteins separable from RNAase by ion exchange chromatography on Bio-Gel CM-30 with a gradient of increasing sodium ion concentration. Only minor amounts of RNAase were recovered. The modified proteins were labeled with 3H to a varying degree, and their order of elution was inversely related to the extent of labeling. The modification of the proteins was the result of the transfer of the acetyl group from N-[3H]acetoxy-3-FAA to RNAase. The evidence for this conclusion was (a) the release of 84-86% of the radioactivity as [3H] acetic acid from the two major proteins upon acid hydrolysis and (b) the isolation of eplision-N-[3H] acetyl-L-lysine from enzymatic hydrolysates of these proteins. A comparison of the present data with those previously reported for the acetylaton of RNAase by the isomeric carcinogen, N-acetoxy-2-FAA, showed that N-acetoxy-3-FAA is the more potent acetyl-lating agent. The present study in conjunction with the previous results, suggests that structural alteration of cellular nucleopholes by acylation may be a biochemical mechanism underlying the biological activity of N-acetoxy-3-FAA and related activated carcinogens.     

A Multidrug ABC Transporter with a Taste for Salt

Background

LmrA is a multidrug ATP-binding cassette (ABC) transporter from Lactococcus lactis with no known physiological substrate, which can transport a wide range of chemotherapeutic agents and toxins from the cell. The protein can functionally replace the human homologue ABCB1 (also termed multidrug resistance P-glycoprotein MDR1) in lung fibroblast cells. Even though LmrA mediates ATP-dependent transport, it can use the proton-motive force to transport substrates, such as ethidium bromide, across the membrane by a reversible, H⁺-dependent, secondary-active transport reaction. The mechanism and physiological context of this reaction are not known.

Methodology/Principal Findings

We examined ion transport by LmrA in electrophysiological experiments and in transport studies using radioactive ions and fluorescent ion-selective probes. Here we show that LmrA itself can transport NaCl by a similar secondary-active mechanism as observed for ethidium bromide, by mediating apparent H⁺-Na⁺-Cl⁻ symport. Remarkably, LmrA activity significantly enhances survival of high-salt adapted lactococcal cells during ionic downshift.

Conclusions/Significance

The observations on H⁺-Na⁺-Cl⁻ co-transport substantiate earlier suggestions of H⁺-coupled transport by LmrA, and indicate a novel link between the activity of LmrA and salt stress. Our findings demonstrate the relevance of investigations into the bioenergetics of substrate translocation by ABC transporters for our understanding of fundamental mechanisms in this superfamily. This study represents the first use of electrophysiological techniques to analyze substrate transport by a purified multidrug transporter.