Molecular evolution of the MAGUK family in metazoan genomes

Background  

Development, differentiation and physiology of metazoans all depend on cell to cell communication and subsequent intracellular signal transduction. Often, these processes are orchestrated via sites of specialized cell-cell contact and involve receptors, adhesion molecules and scaffolding proteins. Several of these scaffolding proteins important for synaptic and cellular junctions belong to the large family of membrane-associated guanylate kinases (MAGUK). In order to elucidate the origin and the evolutionary history of the MAGUKs we investigated full-length cDNA, EST and genomic sequences of species in major phyla.     

Preventing farnesylation of the dynein adaptor Spindly contributes to the mitotic defects caused by farnesyltransferase inhibitors

The clinical interest in farnesyltransferase inhibitors (FTIs) makes it important to understand how these compounds affect cellular processes involving farnesylated proteins. Mitotic abnormalities observed after treatment with FTIs have so far been attributed to defects in the farnesylation of the outer kinetochore proteins CENP-E and CENP-F, which are involved in chromosome congression and spindle assembly checkpoint signaling. Here we identify the cytoplasmic dynein adaptor Spindly as an additional component of the outer kinetochore that is modified by farnesyltransferase (FTase). We show that farnesylation of Spindly is essential for its localization, and thus for the proper localization of dynein and its cofactor dynactin, to prometaphase kinetochores and that Spindly kinetochore recruitment is more severely affected by FTase inhibition than kinetochore recruitment of CENP-E and CENP-F. Molecular replacement experiments show that both Spindly and CENP-E farnesylation are required for efficient chromosome congression. The identification of Spindly as a new mitotic substrate of FTase provides insight into the causes of the mitotic phenotypes observed with FTase inhibitors.     

Two alanine aminotranferases link mitochondrial glycolate oxidation to the major photorespiratory pathway in Arabidopsis and rice

The major photorespiratory pathway in higher plants is distributed over chloroplasts, mitochondria, and peroxisomes. In this pathway, glycolate oxidation takes place in peroxisomes. It was previously suggested that a mitochondrial glycolate dehydrogenase (GlcDH) that was conserved from green algae lacking leaf-type peroxisomes contributes to photorespiration in Arabidopsis thaliana. Here, the identification of two Arabidopsis mitochondrial alanine:glyoxylate aminotransferases (ALAATs) that link glycolate oxidation to glycine formation are described. By this reaction, the mitochondrial side pathway produces glycine from glyoxylate that can be used in the glycine decarboxylase (GCD) reaction of the major pathway. RNA interference (RNAi) suppression of mitochondrial ALAAT did not result in major changes in metabolite pools under standard conditions or enhanced photorespiratroy flux, respectively. However, RNAi lines showed reduced photorespiratory CO(2) release and a lower CO(2) compensation point. Mitochondria isolated from RNAi lines are incapable of converting glycolate to CO(2), whereas simultaneous overexpression of GlcDH and ALAATs in transiently transformed tobacco leaves enhances glycolate conversion. Furthermore, analyses of rice mitochondria suggest that the side pathway for glycolate oxidation and glycine formation is conserved in monocotyledoneous plants. It is concluded that the photorespiratory pathway from green algae has been functionally conserved in higher plants.     

Resin-based investigation of acyl carrier protein interaction networks in Escherichia coli

Protein-protein interactions play an integral role in metabolic regulation. Elucidation of these networks is complicated by the changing identity of the proteins themselves. Here we demonstrate a resin-based technique that leverages the unique tools for acyl carrier protein (ACP) modification with non-hydrolyzable linkages. ACPs from Escherichia coli and Shewanella oneidensis MR-1 are bound to Affigel-15 with varying acyl groups attached and introduced to proteomic samples. Isolation of these binding partners is followed by MudPIT analysis to identify each interactome with the variable of ACP-tethered substrates. These techniques allow for investigation of protein interaction networks with the changing identity of a given protein target.     

Activity-based protein profiling implicates urokinase activation as a key step in human fibrosarcoma intravasation

Entry of malignant cells into the vasculature (i.e. intravasation) requires proteolytic remodeling of the extracellular matrix so that tumor cells may pass through the local stroma and penetrate the vessel wall. The circulatory system then provides a means of transporting tumor cells to distant sites where they extravasate and establish metastatic lesions. This study utilizes activity-based protein profiling to compare the active serine hydrolase repertoire in high intravasating (HT-hi/diss) and low intravasating (HT-lo/diss) variants of the human fibrosarcoma HT-1080 cell line to determine which enzyme(s) play a role in intravasation. Activity-based protein profiling revealed multiple serine hydrolases with altered activity between HT-hi/diss and HT-lo/diss cells, with the largest difference being the activity of urokinase-type plasminogen activator (uPA). Levels of inactive uPA zymogen were similar between the two cell variants, but only HT-hi/diss conditioned medium contained active uPA, suggesting that uPA activation may contribute to the enhanced intravasation of HT-hi/diss cells. To analyze the role of uPA activity specifically in the process of intravasation, we grafted cells from the two HT-1080 variants onto the chorioallantoic membrane of chick embryos and measured levels of tumor cell intravasation in the distal chorioallantoic membrane using quantitative human-specific Alu PCR. Inhibition of uPA activity with natural (plasminogen activator inhibitor-1) or synthetic (amiloride) inhibitors diminished HT-hi/diss Matrigel invasion in vitro and intravasation and metastasis in vivo. Additionally, treatment of HT-lo/diss tumors with exogenous active uPA increased the number of intravasated cells in vivo. These results indicate that active uPA promotes tumor cell intravasation and that uPA activation appears to be a key step in tumor progression.     

Differential Effects of Protein Kinase B/Akt Isoforms on Glucose Homeostasis and Islet Mass

Protein kinase B (PKB)/Akt is considered to be a key target downstream of insulin receptor substrate 2 (IRS2) in the regulation of β-cell mass. However, while deficiency of IRS2 in mice results in diabetes with insulin resistance and severe failure of β-cell mass and function, only loss of the PKBβ isoform leads to a mild metabolic phenotype with insulin resistance. Other isoforms were reported not to be required for metabolic regulation. To clarify the roles of the three PKB isoforms in the regulation of islet mass and glucose homeostasis, we assessed the metabolic and pancreatic phenotypes of Pkbα, Pkbβ, and Pkbγ-deficient mice. Our study uncovered a novel role for PKBα in the regulation of glucose homeostasis, whereas it confirmed that Pkbβ^−/− mice are insulin resistant with compensatory increase of islet mass. Pkbα^−/− mice displayed an opposite phenotype with improved insulin sensitivity, lower blood glucose, and higher serum glucagon concentrations. Pkbγ^−/− mice did not show metabolic abnormalities. Additionally, our signaling analyses revealed that PKBα, but not PKBβ or PKBγ, is specifically activated by overexpression of IRS2 in β-cells and is required for IRS2 action in the islets.Adaptation of pancreatic islet mass and function relative to metabolic demand maintains glucose homeostasis and may prevent the development of type 2 diabetes. β-Cell proliferation, apoptosis, growth, and function are tightly regulated by various extracellular factors and intracellular signaling pathways (23, 24, 34). In β-cells, insulin receptor substrate 2 (IRS2) controls maintenance and expansion of islet mass (29, 31, 42). In fact, IRS2-deficient mice are insulin resistant, show β-cell failure and hyperglycemia, and finally develop diabetes (26, 42). In contrast, deficiency of IRS1 only causes insulin resistance without the development of diabetes due to a compensatory increase in functional β-cell mass (1, 38). These observations indicated that IRS2, but not IRS1, is necessary for maintenance and compensatory increase of β-cell mass. Furthermore, experiments with isolated islets revealed that overexpression of IRS2, but not of IRS1, can increase β-cell proliferation and protect cells against high-glucose-induced apoptosis (29). Downstream of IRS2, phosphoinositide 3-kinase (PI3K)-protein kinase B (PKB) signaling is considered to be the critical pathway for the regulation of β-cell mass and function (12, 15, 16, 27). The serine-threonine kinase PKB, also known as Akt, is required for various cellular processes, from the regulation of cell cycle, survival, and growth to glucose and protein metabolism. In mammals, three PKB/Akt isoforms have been characterized and named PKBα/Akt1, PKBβ/Akt2, and PKBγ/Akt3. Although encoded by different genes on different chromosomes, the three isoforms display high homology at the protein level with 80 to 85% identical residues and the same structural organization (43). However, they differ in terms of tissue-specific expression. PKBα is expressed in most tissues and PKBβ is highly expressed in insulin-responsive tissues, whereas PKBγ expression is prominent in the brain and testes (17). All three isoforms are expressed in β-cells (30, 37). The roles of PKB in different tissues have been studied in transgenic-mouse models. While Pkbα^−/− and Pkbγ^−/− mice show impaired fetal growth and brain development, respectively, glucose homeostasis is unaffected in both models (9, 11, 14, 39, 46). In contrast, Pkbβ^−/− mice are insulin resistant and mildly glucose intolerant and have less adipose tissue. Depending on the strain and gender, these mice show either late loss of β-cells followed by the development of diabetes and mild growth deficiency or compensatory increase of β-cell mass without age-dependent progression into overt hyperglycemia (10, 17). These studies suggested that PKBβ is the only isoform playing a role in the regulation of energy homeostasis. On the other hand, constitutive activation of PKBα in β-cells is sufficient to increase growth and proliferation (5, 40), and in INS1 cells it prevents free fatty acid (FFA)-induced apoptosis (44). Furthermore, antagonizing total PKB activity in β-cells by ectopic expression of a kinase-dead mutant causes defects in insulin secretion (4), suggesting that in islets PKB is required mainly for normal function of the β-cells. Although these data support the notion that PKB must play a role in pancreatic β-cells, they are not in line with the stronger metabolic phenotype displayed by IRS2-deficient mice. In fact, PKBα and PKBγ appear not to be required to regulate glucose homeostasis (9, 11, 39), and in the case of Pkbβ^−/− mice, even though glucose homeostasis is impaired due to strong peripheral insulin resistance, the overall metabolic phenotype is far less severe than in Irs2^−/− mice (10), indicating that the capacity for β-cell compensation is retained in the absence of PKBβ.The aim of this study was to clarify the role of PKB in the regulation of islet mass and to define the relevance of PKB isoforms for IRS2 action in β-cells. Although it had been shown that PKBα is dispensable for the regulation of glucose homeostasis (9, 11), we found lower blood glucose concentrations in Pkbα^−/− mice. Based on this observation, we assessed in more detail the metabolic and the endocrine pancreatic phenotypes of Pkbα-, Pkbβ-, or Pkbγ-deficient mice. In addition, glucose uptake into fat cells, insulin secretion, and islet cell proliferation were investigated. Contrary to previous assumptions implying that PKBβ is the only (or at least the main) isoform playing a role in the regulation of glucose metabolism, we present evidence that both PKBα and PKBβ isoforms are required in the periphery for regulation of glucose homeostasis. While we confirmed that Pkbβ^−/− mice are insulin resistant and glucose intolerant with compensatory increase of β-cell mass, Pkbα^−/− mice showed lower blood glucose levels, were more insulin sensitive, and revealed higher serum glucagon concentrations accompanied by a mild increase in α-cell mass and proliferation. Moreover, our in vitro experiments showed that PKBα is specifically activated by IRS2 in β-cells and that its activation is required for IRS2-induced proliferation in islets.     

Polo-like kinase 4 kinase activity limits centrosome overduplication by autoregulating its own stability

Accurate control of the number of centrosomes, the major microtubule-organizing centers of animal cells, is critical for the maintenance of genome integrity. Abnormalities in centrosome number can promote errors in spindle formation that lead to subsequent chromosome missegregation, and extra centrosomes are found in many cancers. Centrosomes are comprised of a pair of centrioles surrounded by amorphous pericentriolar material, and centrosome duplication is controlled by centriole replication. Polo-like kinase 4 (Plk4) plays a key role in initiating centriole duplication, and overexpression of Plk4 promotes centriole overduplication and the formation of extra centrosomes. Using chemical genetics, we show that kinase-active Plk4 is inherently unstable and targeted for degradation. Plk4 is shown to multiply self-phosphorylate within a 24–amino acid phosphodegron. Phosphorylation of multiple sites is required for Plk4 instability, indicating a requirement for a threshold level of Plk4 kinase activity to promote its own destruction. We propose that kinase-mediated, autoregulated instability of Plk4 self-limits Plk4 activity so as to prevent centrosome amplification.     

Quantitative analysis of gap-junctional intercellular communication in precision-cut mouse liver slices

Direct intercellular communication through gap junction channels is involved in the maintenance of tissue homeostasis and suppression of carcinogenesis. Gap-junctional communication is often altered in tumor cells but it can also be modulated in response to tumor promotors or inflammatory signals. In order to evaluate the effect of nongenotoxic compounds, suggested to be involved in tumor promotion, on gap junctional intercellular communication in the liver, we have developed a direct dye transfer method. The fluorescent dye Alexa Fluor 488 was iontophoretically injected into hepatocytes of freshly prepared, precision-cut mouse liver slices (250 microm). The area of dye spreading was monitored and quantified by microscopy. Comparison of dye spreading in connexin-32-deficient versus wild-type liver revealed a 96% decrease in connexin-32-deficient tissue. Induction of an acute phase response in connexin-32-deficient mice by intraperitoneal injection of lipopolysaccharide increased dye coupling by 33%, probably due to upregulation of connexin-26-containing gap junction channels.     

Deoxynivalenol in commercial beer — screening for the toxin with an indirect competitive ELISA

Antibodies specific for deoxynivalenol (DON) were prepared by immunizing rabbits with a deoxynivalenol-hemiglutaryl-HSA conjugate. The antibody showed cross-reactivity with DON, 15a-acetyl-DON (15-Ac DON), and 3a-acotyl-DON (3-Ac DON) of 100%, 216%, and 260%, respectively. No cross-reactivity was observed against 20 further trichothecenes of the A-, B-, and C-type. An indirect competitive ELISA procedure was set up for the detection of DON in liquid matrices occurring in the brewing process, Including beer. ELISA was sensitive down to 3.8 ng/mL. Detection limit for DON in tenfold diluted beer was 50 ppb. Recovery of the toxin in spiked beer was 45%, 77%, and 83% at toxin levels of 50; 500; and 1,000 ppb, respectively. ELISA results corresponded quite well with HPLC data. 196 commercial beers from several German breweries, including 36 gushing-positive samples, were assayed for DON. Screening revealed toxin levels from not detectable to 569 ppb. 69% of the gushing beers had marked levels of DON. 37% of the gushing-negative beer samples contained DON in detectable amounts with a maximum content of 172 ppb. Wheat beers had significantly higher DON-contents than beers derived from barley. Concentration of the toxin was significantly higher in gushing beers than in nongushing beers from both cereal sources.