The Src-like adaptor protein 2 regulates colony-stimulating factor-1 receptor signaling and down-regulation

Src-like adaptor protein 2 (SLAP-2) is a hematopoietic adaptor protein previously implicated as a negative regulator of T-cell antigen receptor (TCR)-mediated signaling. SLAP-2 contains an SH3 and an SH2 domain, followed by a unique carboxyl-terminal tail, which is important for c-Cbl binding. Here we describe a novel role for SLAP-2 in regulation of the colony-stimulating factor 1 receptor (CSF-1R), a receptor tyrosine kinase important for growth and differentiation of myeloid cells. SLAP-2 co-immunoprecipitates with c-Cbl and CSF-1R in primary bone marrow-derived macrophages. Using murine myeloid cells expressing CSF-1R (FD-Fms cells), we show that SLAP-2 is tyrosine-phosphorylated upon stimulation with CSF-1 and associates constitutively with both c-Cbl and CSF-1R. In addition, we show that expression of a dominant negative form of SLAP-2 impairs c-Cbl association with the CSF-1R and receptor ubiquitination. Impaired c-Cbl recruitment also correlated with changes in the kinetics of CSF-1R down-regulation and trafficking. CSF-1-mediated differentiation of FD-Fms cells and activation of downstream signaling events was also enhanced in cells stably expressing dominant negative SLAP-2. Together, these results demonstrate that SLAP-2 plays a role in c-Cbl-dependent down-regulation of CSF-1R signaling.     

Inhibition of B56-containing protein phosphatase 2As by the early response gene IEX-1 leads to control of Akt activity

The importance of PP2A in the regulation of Akt/PKB activity has long been recognized but the nature of the holoenzyme involved and the mechanisms controlling dephosphorylation are not yet known. We identified IEX-1, an early gene product with proliferative and survival activities, as a specific inhibitor of B56 regulatory subunit-containing PP2A. IEX-1 inhibits B56-PP2A activity by allowing the phosphorylation of B56 by ERK. This leads to sustained ERK activation. IEX-1 has no effect on PP2A containing other B family subunits. Thus, studying IEX-1 contribution to signaling should help the discovery of new pathways controlled by B56-PP2A. By using overexpression and RNA interference, we show here that IEX-1 increases Akt/PKB activity in response to various growth factors by preventing Akt dephosphorylation on both Thr(308) and Ser(473) residues. PP2A-B56beta and gamma subunits have the opposite effect and reverse IEX-1-mediated Akt activation. The effect of IEX-1 on Akt is ERK-dependent. Indeed: (i) a IEX-1 mutant deficient in ERK binding had no effect on Akt; (ii) ERK dominant-negative mutants reduced IEX-1-mediated increase in pAkt; (iii) a B56beta mutant that cannot be phosphorylated in the ERK.IEX-1 complex showed an enhanced ability to compete with IEX-1. These results identify B56-containing PP2A holoenzymes as Akt phosphatases. They suggest that IEX-1 behaves as a general inhibitor of B56 activity, enabling the control of both ERK and Akt signaling downstream of ERK.     

Identification, expression, and functional analyses of a thylakoid ATP/ADP carrier from Arabidopsis

In plants the chloroplast thylakoid membrane is the site of light-dependent photosynthetic reactions coupled to ATP synthesis. The ability of the plant cell to build and alter this membrane system is essential for efficient photosynthesis. A nucleotide translocator homologous to the bovine mitochondrial ADP/ATP carrier (AAC) was previously found in spinach thylakoids. Here we have identified and characterized a thylakoid ATP/ADP carrier (TAAC) from Arabidopsis.(i) Sequence homology with the bovine AAC and the prediction of chloroplast transit peptides indicated a putative carrier encoded by the At5g01500 gene, as a TAAC. (ii) Transiently expressed TAAC-green fluorescent protein fusion construct was targeted to the chloroplast. Western blotting using a peptide-specific antibody together with immunogold electron microscopy revealed a major location of TAAC in the thylakoid membrane. Previous proteomic analyses identified this protein in chloroplast envelope preparations. (iii) Recombinant TAAC protein specifically imports ATP in exchange for ADP across the cytoplasmic membrane of Escherichia coli. Studies on isolated thylakoids from Arabidopsis confirmed these observations. (iv) The lack of TAAC in an Arabidopsis T-DNA insertion mutant caused a 30-40% reduction in the thylakoid ATP transport and metabolism. (v) TAAC is readily expressed in dark-grown Arabidopsis seedlings, and its level remains stable throughout the greening process. Its expression is highest in developing green tissues and in leaves undergoing senescence or abiotic stress. We propose that the TAAC protein supplies ATP for energy-dependent reactions during thylakoid biogenesis and turnover in plants.     

The LIM domains of WLIM1 define a new class of actin bundling modules

Actin filament bundling, i.e. the formation of actin cables, is an important process that relies on proteins able to directly bind and cross-link subunits of adjacent actin filaments. Animal cysteine-rich proteins and their plant counterparts are two LIM domain-containing proteins that were recently suggested to define a new family of actin cytoskeleton regulators involved in actin filament bundling. We here identified the LIM domains as responsible for F-actin binding and bundling activities of the tobacco WLIM1. The deletion of one of the two LIM domains reduced significantly, but did not entirely abolish, the ability of WLIM1 to bind actin filaments. Individual LIM domains were found to interact directly with actin filaments, although with a reduced affinity compared with the native protein. Variants lacking the C-terminal or the inter-LIM domain were only weakly affected in their F-actin stabilizing and bundling activities and trigger the formation of thick cables containing tightly packed actin filaments as does the native protein. In contrast, the deletion of one of the two LIM domains negatively impacted both activities and resulted in the formation of thinner and wavier cables. In conclusion, we demonstrate that the LIM domains of WLIM1 are new autonomous actin binding and bundling modules that cooperate to confer WLIM1 high actin binding and bundling activities.     

Differential expression of sarcoplasmic proteins in four heterogeneous ovine skeletal muscles

Fiber-type distribution is known to vary widely within and between muscles according to differences in muscle functions. 2-DE and MALDI-MS were used to investigate the molecular basis of muscle fiber type-related variability. We compared four lamb skeletal muscles with heterogeneous fiber-type composition that are relatively rich in fast-twitch fiber types, i.e., the semimembranosus, vastus medialis, longissimus dorsi, and tensor fasciae latae (TL). Our results clearly showed that none of the glycolytic metabolism enzymes detected, including TL which was most strongly glycolytic, made intermuscular differentiation possible. Muscle differentiation was based on the differential expression of proteins involved in oxidative metabolism, including not only citric acid cycle enzymes but also other classes of proteins with functions related to oxidative metabolism, oxidative stress, and probably to higher protein turnover. Detected proteins were involved in transport (carbonate dehydratase, myoglobin, fatty acid-binding protein), repair of misfolding damage (heat shock protein (HSP) 60 kDa, HSP-27 kDa, alpha-crystallin beta subunit, DJ1, stress-induced phosphoprotein), detoxification or degradation of impaired proteins (GST-Pi, aldehyde dehydrogenase, peroxiredoxin, ubiquitin), and protein synthesis (tRNA-synthetase). The fractionating method led to the detection of proteins involved in different functions related to oxidative metabolism that have not previously been shown concomitancy.     

Evaluation of the social bond: a new method tested in Mus spicilegus

Innovative and fruitful studies of social bonds have been developed in recent years, although the methods used to establish the existence of a social bond between two individuals have not evolved significantly. Two types of paradigms have been currently used: the separation-reunion paradigm, which evaluates the distress caused by the disruption of the social bond, and choice paradigms, which test the specificity of the bond to a given individual. We have developed a new paradigm based on the idea that the cost an individual was ready to pay in order to gain access to a conspecific depended on the strength of the social bond between the two individuals. To test our paradigm we used mound-building mice, Mus spicilegus that present, in both males and females, a level of tolerance that differs greatly according to the degree of familiarity between the individuals. Our new method for testing social bond revealed unsuspected differences between males and females. Our results suggested that, at least in Mus spicilegus, strong social bonds were not necessary to the development of a high level of tolerance between individuals.     

15N-metabolic labeling for comparative plasma membrane proteomics in Arabidopsis cells

An important goal for proteomic studies is the global comparison of proteomes from different genotypes, tissues, or physiological conditions. This has so far been mostly achieved by densitometric comparison of spot intensities after protein separation by 2-DE. However, the physicochemical properties of membrane proteins preclude the use of 2-DE. Here, we describe the use of in vivo labeling by the stable isotope 15N as an alternative approach for comparative membrane proteomic studies in plant cells. We confirm that 15N-metabolic labeling of proteins is possible and efficient in Arabidopsis suspension cells. Quantification of 14N versus 15N MS signals reflects the relative abundance of 14N and 15N proteins in the sample analyzed. We describe the use of 15N-metabolic labeling to perform a partial comparative analysis of Arabidopsis cells following cadmium exposure. By focusing our attention on plasma membrane proteins, we were able to confidently identify proteins showing up to 5-fold regulation compared to unexposed cells. This study provides a proof of principle that 15N-metabolic labeling is a useful technique for comparative membrane proteome studies.