Hypoglycaemia, liver necrosis and perinatal death in mice lacking all isoforms of phosphoinositide 3-kinase p85 alpha.

Phosphoinositide 3-kinases produce 3'-phosphorylated phosphoinositides that act as second messengers to recruit other signalling proteins to the membrane. Pi3ks are activated by many extracellular stimuli and have been implicated in a variety of cellular responses. The Pi3k gene family is complex and the physiological roles of different classes and isoforms are not clear. The gene Pik3r1 encodes three proteins (p85 alpha, p55 alpha and p50 alpha) that serve as regulatory subunits of class IA Pi3ks (ref. 2). Mice lacking only the p85 alpha isoform are viable but display hypoglycaemia and increased insulin sensitivity correlating with upregulation of the p55 alpha and p50 alpha variants. Here we report that loss of all protein products of Pik3r1 results in perinatal lethality. We observed, among other abnormalities, extensive hepatocyte necrosis and chylous ascites. We also noted enlarged skeletal muscle fibres, brown fat necrosis and calcification of cardiac tissue. In liver and muscle, loss of the major regulatory isoform caused a great decrease in expression and activity of class IA Pi3k catalytic subunits; nevertheless, homozygous mice still displayed hypoglycaemia, lower insulin levels and increased glucose tolerance. Our findings reveal that p55 alpha and/or p50 alpha are required for survival, but not for development of hypoglycaemia, in mice lacking p85 alpha.

Alginate beads as a storage, delivery and containment system for genetically modified PCB degrader and PCB biosensor derivatives of Pseudomonas fluorescens F113.

AIMS: Pseudomonas fluorescens F113Rifpcb is a genetically engineered rhizosphere bacterium with the potential to degrade polychlorinated biphenyls (PCBs). F113Rifpcbgfp and F113L::1180gfp are biosensor strains capable of detecting PCB bioavailability and biodegradation. The aim of this paper is to evaluate the use of alginate beads as a storage, delivery and containment system for use of these strains in PCB contaminated soils. METHODS AND RESULTS: The survival and release of Ps. fluorescens F113Rifpcb from alginate beads were evaluated. Two Ps. fluorescens F113-based biosensor strains were encapsulated, and their ability to detect 3-chlorobenzoate (3-CBA) and 3-chlorobiphenyl (3-CBP) degradation in soil was assessed. After 250 days of storage, 100% recovery of viable F113Rifpcb cells was possible. Amendments to the alginate formulation allowed for the timed release of the inoculant. Encapsulation of the F113Rifpcb cells provided a more targeted approach for the inoculation of plants and resulted in lower inoculum populations in the bulk soil, which may reduce the risk of unintentional spread of these genetically modified micro-organisms in the environment. Encapsulation of the biosensor strains in alginate beads did not interfere with their ability to detect either 3-CBA or 3-CBP degradation. In fact, detection of 3-CBP degradation was enhanced in encapsulated biosensors. CONCLUSIONS: Alginate beads are an effective storage and delivery system for PCB degrading inocula and biosensors. SIGNIFICANCE AND IMPACT OF THE STUDY: Pseudomonas fluorescens F113Rifpcb and the F113 derivative PCB biosensor strains have excellent potential for detecting and bioremediation of PCB contaminated soils. The alginate bead delivery system could facilitate the application of these strains as biosensors.

Carboxyl-terminal modulator protein (CTMP), a negative regulator of PKB/Akt and v-Akt at the plasma membrane.

The PKB (protein kinase B, also called Akt) family of protein kinases plays a key role in insulin signaling, cellular survival, and transformation. PKB is activated by phosphorylation on residues threonine 308, by the protein kinase PDK1, and Serine 473, by a putative serine 473 kinase. Several protein binding partners for PKB have been identified. Here, we describe a protein partner for PKBalpha termed CTMP, or carboxyl-terminal modulator protein, that binds specifically to the carboxyl-terminal regulatory domain of PKBalpha at the plasma membrane. Binding of CTMP reduces the activity of PKBalpha by inhibiting phosphorylation on serine 473 and threonine 308. Moreover, CTMP expression reverts the phenotype of v-Akt-transformed cells examined under a number of criteria including cell morphology, growth rate, and in vivo tumorigenesis. These findings identify CTMP as a negative regulatory component of the pathway controlling PKB activity.

Ten years of protein kinase B signalling: a hard Akt to follow.

It is ten years since the publication of three papers describing the cloning of a new proto-oncogene serine/threonine kinase termed protein kinase B (PKB)/Akt. Key roles for this protein kinase in cellular processes such as glucose metabolism, cell proliferation, apoptosis, transcription and cell migration are now well established. The explosion of publications involving PKB/Akt in the past three years emphasizes the high level of current interest in this signalling molecule. This review focuses on tracing the characterization of this kinase, through the elucidation of its mechanism of regulation, to its role in regulating physiological and pathophysiological processes, to our current understanding of the biology of PKB/Akt, and prospects for the future.

Cell polarity: Scaffold proteins par excellence.

Par proteins are involved in determining cellular asymmetry. Recent studies have identified one of these proteins, Par6, as a key regulator of cell polarity and transformation via its interactions with small GTPases and atypical forms of protein kinase C.

Evidence for the presence of G-proteins, adenylyl cyclase and phospholipase C activities in lymphatic smooth muscle cell membranes.

In plasma membranes derived from bovine mesenteric lymphatic smooth muscle cells, guanine nucleotide and forskolin stimulated adenylyl cyclase (AC) activity in a concentration-dependent manner, indicative of the presence of the stimulatory G-protein Gs linked to AC. There was no significant enzyme inhibition by low concentrations of guanine nucleotide and no effect on basal or guanine nucleotide-stimulated activity following pertussis toxin treatment of cells, suggesting the absence of Gi linked to inhibition of AC. Furthermore, there was no effect of adrenaline, isoprenaline or clonidine on basal or forskolin-stimulated activities, nor was there any specific binding of the beta-adrenoceptor ligand [125I]cyanopindolol to membranes, suggesting that catecholamine receptors do not modulate AC activity in these membranes. Pertussis toxin-mediated ADP ribosylation of membrane proteins and Western immunoblotting analysis revealed the presence of G-protein subunits G alpha i2, G alpha q, and G beta 1. In experiments designed to identify a possible effector enzyme for these G-proteins, membranes were screened with a range of antibodies raised against phospholipase C (PLC) beta, gamma and delta isozymes. Though no evidence was obtained by Western blotting for any of these proteins, PLC activity was concentration-dependently stimulated by Ca2+, but not by AIF4-, GTP[S], or purified G beta gamma subunits. Finally, no specific binding to membranes of the alpha 1-adrenoceptor ligand [3H]prazosin or the alpha 2-adrenoceptor ligand [3H]yohimbine was obtained. In conclusion, this study provides evidence for a Gs-dependent stimulation of AC, and for the presence of Gi2 and Gq/11, which do not appear to regulate a PLC activity also identified in lymphatic smooth muscle cell membranes. Furthermore, neither AC nor PLC appear to be associated with catecholamine receptors.

Construction of a modified mini-Tn5 lacZY non-antibiotic marker cassette: ecological evaluation of a lacZY marked Pseudomonas strain in the sugarbeet rhizosphere.

In order to monitor the fate of genetically manipulated fluorescent pseudomonads following release into the environment, a lacZY transposable cassette, lacking antibiotic resistance genes, was constructed using a pUT suicide plasmid delivery system. The resulting plasmid, pUTLacZY, can be easily used to generate lacZY marked pseudomonads without having to use antibiotic resistance determinants. The lacZY transposon generates random, stable transcriptional/translational fusions on integration into the target genome. Pseudomonas fluorescens strain F113 was marked with lacZY and was unaltered with respect to ecological fitness in the rhizosphere. Although lateral gene transfer of the chromosomally integrated lacZY marker could be detected in vitro, it was not detected in rhizosphere microcosms.

Interindividual-intergroup discontinuity reduction through the anticipation of future interaction.

Consistent with the role of a long-term perspective in reducing the tendency of intergroup relations to be more competitive than interindividual relations in the context of noncorrespondent outcomes, an experiment demonstrated that anticipated future interaction reduced intergroup but not interindividual competitiveness. Further results indicated that the effect was present only for groups composed of members high in abstractness (Openness-Intellect on the Big 5 Inventory and Intuition on the Myers-Briggs Type Inventory) who trusted their opponents.

On the mechanism of the inhibition of transducin function by farnesylcysteine analogs.

The gamma subunits of heterotrimeric G proteins are isoprenylated/methylated on their carboxy termini. The photoreceptor G protein, transducin, is farnesylated/methylated at this position. Since the isoprenyl group is required for G protein function, it is of great interest to determine the mechanism by which the farnesyl group of Tgamma interacts with the other transducin subunits and/or the activated photoreceptor, rhodopsin. Farnesylcysteine derivatives (N-acetyl-S-farnesyl-L-cysteine and farnesylated peptides) have been previously shown to have effects on transducin activity at high concentrations. Here, an extensive survey is done of farnesylcysteine analogs and other lipid molecules, which are tested for their ability to inhibit GTP/GDP exchange in transducin catalyzed by photolyzed rhodopsin. These studies are carried out to determine the nature of the inhibition process. While it does not appear that these molecules exhibit the specificity which would characterize a ligand-receptor type mechanism, the results suggest that these compounds are not acting in a nonspecific detergent-like manner either. The most likely mode of action of famesylcysteine analogs is that they interfere with the lipid-lipid based association of Talpha and Tbetagamma through the lipid modifications present on each subunit.

Phase and microstructural development in alumina sol-gel coatings on CoCr alloy.

Phase transformation of gamma-Al(2)O(3) to alpha-Al(2)O(3) in alumina sol gel coatings on biomedical CoCr alloy was studied as function of heat treatment temperature and time. Transformation in unseeded coatings was significant only above approximately 1200 degrees C. Addition of alpha-Al(2)O(3) seed particles having an average size of approximately 40 nm lowered the phase transformation temperature to around 800 degrees C. These particles were considered to act as heterogeneous nucleation sites for epitaxial growth of the alpha-Al(2)O(3) phase. The kinetics and activation energy (420 kJ/mol) for the phase transformation in the seeded coatings were similar to those reported for seeded monolithic alumina gels indicating that the transformation mechanism is the same in the two material configurations. Avrami growth parameters indicated that the mechanism was diffusion controlled and invariant over the temperature range studied but that growth was possibly constrained by the finite size of the seed particles and/or coating thickness. The phase transformation occurred by the growth of alpha-Al(2)O(3) grains at the expense of the precursor fine-grained gamma-Al(2)O(3) matrix and near-complete transformation coincided with physical impingement of the growing grains. The grain size at impingement was approximately 100 nm which agreed well with that predicted from the theoretical linear spacing of seed particles in the initial sol.

Stimulation of phospholipase C-beta 2 by recombinant guanine-nucleotide-binding protein beta gamma dimers produced in a baculovirus/insect cell expression system. Requirement of gamma-subunit isoprenylation for stimulation of phospholipase C.

Recombinant wild-type beta 1 gamma 1 dimers of signal-transducing guanine nucleotide-binding proteins (G proteins) and beta 1 gamma 1 dimers carrying a mutation known to block gamma-subunit isoprenylation (beta 1 gamma 1 C71S) were expressed in baculovirus-infected insect cells. Both wild-type and mutant beta 1 gamma 1 dimers were found in soluble fractions of infected cells upon subcellular fractionation. Anion exchange chromatographic and metabolic-radiolabeling studies revealed that the soluble beta 1 gamma 1 preparation contained approximately equal amounts of non-isoprenylated and isoprenylated beta 1 gamma 1 dimers. Soluble wild-type and mutant beta 1 gamma 1 dimers and native beta 1 gamma 1 dimers purified from bovine retina were reconstituted with recombinant phospholipase C-beta 2. Only isoprenylated beta 1 gamma 1 dimers were capable of stimulating phospholipase C-beta 2. The results show that gamma-subunit isoprenylation and/or additional post-translational processing of the protein are required for beta gamma subunit stimulation of phospholipase C.

Interaction of oestrogen receptor with the regulatory subunit of phosphatidylinositol-3-OH kinase.

Oestrogen produces diverse biological effects through binding to the oestrogen receptor (ER). The ER is a steroid hormone nuclear receptor, which, when bound to oestrogen, modulates the transcriptional activity of target genes. Controversy exists, however, concerning whether ER has a role outside the nucleus, particularly in mediating the cardiovascular protective effects of oestrogen. Here we show that the ER isoform, ER alpha, binds in a ligand-dependent manner to the p85alpha regulatory subunit of phosphatidylinositol-3-OH kinase (PI(3)K). Stimulation with oestrogen increases ER alpha-associated PI(3)K activity, leading to the activation of protein kinase B/Akt and endothelial nitric oxide synthase (eNOS). Recruitment and activation of PI(3)K by ligand-bound ER alpha are independent of gene transcription, do not involve phosphotyrosine adapter molecules or src-homology domains of p85alpha, and extend to other steroid hormone receptors. Mice treated with oestrogen show increased eNOS activity and decreased vascular leukocyte accumulation after ischaemia and reperfusion injury. This vascular protective effect of oestrogen was abolished in the presence of PI(3)K or eNOS inhibitors. Our findings define a physiologically important non-nuclear oestrogen-signalling pathway involving the direct interaction of ER alpha with PI(3)K.

Identification of tyrosine phosphorylation sites on 3-phosphoinositide-dependent protein kinase-1 and their role in regulating kinase activity.

3-Phosphoinositide-dependent protein kinase-1 (PDK1) plays a central role in signal transduction pathways that activate phosphoinositide 3-kinase. Despite its key role as an upstream activator of enzymes such as protein kinase B and p70 ribosomal protein S6 kinase, the regulatory mechanisms controlling PDK1 activity are poorly understood. PDK1 has been reported to be constitutively active in resting cells and not further activated by growth factor stimulation (Casamayor, A., Morrice, N. A., and Alessi, D. R. (1999) Biochem. J. 342, 287-292). Here, we report that PDK1 becomes tyrosine-phosphorylated and translocates to the plasma membrane in response to pervanadate and insulin. Following pervanadate treatment, PDK1 kinase activity increased 1.5- to 3-fold whereas the activity of PDK1 associated with the plasma membrane increased approximately 6-fold. The activity of PDK1 localized to the plasma membrane was also increased by insulin treatment. Three tyrosine phosphorylation sites of PDK1 (Tyr-9 and Tyr-373/376) were identified using in vivo labeling and mass spectrometry. Using site-directed mutants, we show that, although phosphorylation on Tyr-373/376 is important for PDK1 activity, phosphorylation on Tyr-9 has no effect on the activity of the kinase. Both of these residues can be phosphorylated by v-Src tyrosine kinase in vitro, and co-expression of v-Src leads to tyrosine phosphorylation and activation of PDK1. Thus, these data suggest that PDK1 activity is regulated by reversible phosphorylation, possibly by a member of the Src kinase family.

Insulin-stimulated protein kinase B phosphorylation on Ser-473 is independent of its activity and occurs through a staurosporine-insensitive kinase.

Full activation of protein kinase B (PKB, also called Akt) requires phosphorylation on two regulatory sites, Thr-308 in the activation loop and Ser-473 in the hydrophobic C-terminal regulatory domain (numbering for PKB alpha/Akt-1). Although 3'-phosphoinositide-dependent protein kinase 1 (PDK1) has now been identified as the Thr-308 kinase, the mechanism of the Ser-473 phosphorylation remains controversial. As a step to further characterize the Ser-473 kinase, we examined the effects of a range of protein kinase inhibitors on the activation and phosphorylation of PKB. We found that staurosporine, a broad-specificity kinase inhibitor and inducer of cell apoptosis, attenuated PKB activation exclusively through the inhibition of Thr-308 phosphorylation, with Ser-473 phosphorylation unaffected. The increase in Thr-308 phosphorylation because of overexpression of PDK1 was also inhibited by staurosporine. We further show that staurosporine (CGP 39360) potently inhibited PDK1 activity in vitro with an IC(50) of approximately 0.22 microm. These data indicate that agonist-induced phosphorylation of Ser-473 of PKB is independent of PDK1 or PKB activity and occurs through a distinct Ser-473 kinase that is not inhibited by staurosporine. Moreover, our results suggest that inhibition of PKB signaling is involved in the proapoptotic action of staurosporine.

Isoprenylation of the G protein gamma subunit is both necessary and sufficient for beta gamma dimer-mediated stimulation of phospholipase C.

We have previously shown that isoprenylation and/or additional post-translational processing of the G protein gamma 1 subunit carboxyl terminus is required for beta 1 gamma 1 subunit stimulation of phospholipase C-beta 2 (PLC beta 2) [Dietrich, A., Meister, M., Brazil, D., Camps, M., & Gierschik, P. (1994) Eur. J. Biochem. 219, 171-178]. To examine whether isoprenylation of the gamma 1 subunit alone is sufficient for beta 1 gamma 1-mediated PLC beta 2 stimulation or whether any of the two subsequent modifications, proteolytic removal of the carboxyl-terminal tripeptide and/or carboxylmethylation, is required for this effect, nonisoprenylated recombinant beta 1 gamma 1 dimers were produced in baculovirus-infected insect cells, purified to near homogeneity, and then isoprenylated in vitro using purified recombinant protein farnesyltransferase. Analysis of the beta 1 gamma 1 dimer after in vitro farnesylation by reversed phase high-performance liquid chromatography followed by delayed extraction matrix-assisted laser desorption/ionization mass spectrometry confirmed that the gamma 1 subunit was carboxyl-terminally farnesylated but not proteolyzed and carboxylmethylated. Functional reconstitution of in vitro-farnesylated beta 1 gamma 1 dimers with a recombinant PLC beta 2 isozyme revealed that farnesylation rendered recombinant nonisoprenylated beta 1 gamma 1 dimers capable of stimulating PLC beta 2 and that the degree of this stimulation was only approximately 45% lower for in vitro-farnesylated beta 1 gamma 1 dimers than for fully modified native beta 1 gamma 1 purified from bovine retinal rod outer segments. Taken together, these results suggest that isoprenylation of the gamma subunit is both necessary and sufficient for beta gamma dimer-mediated stimulation of phospholipase C.