DLG5 connects cell polarity and Hippo signaling protein networks by linking PAR-1 with MST1/2.

Disruption of apical-basal polarity is implicated in developmental disorders and cancer; however, the mechanisms connecting cell polarity proteins with intracellular signaling pathways are largely unknown. We determined previously that membrane-associated guanylate kinase (MAGUK) protein discs large homolog 5 (DLG5) functions in cell polarity and regulates cellular proliferation and differentiation via undefined mechanisms. We report here that DLG5 functions as an evolutionarily conserved scaffold and negative regulator of Hippo signaling, which controls organ size through the modulation of cell proliferation and differentiation. Affinity purification/mass spectrometry revealed a critical role of DLG5 in the formation of protein assemblies containing core Hippo kinases mammalian ste20 homologs 1/2 (MST1/2) and Par-1 polarity proteins microtubule affinity-regulating kinases 1/2/3 (MARK1/2/3). Consistent with this finding, Hippo signaling is markedly hyperactive in mammalian Dlg5-/- tissues and cells in vivo and ex vivo and in Drosophila upon dlg5 knockdown. Conditional deletion of Mst1/2 fully rescued the phenotypes of brain-specific Dlg5 knockout mice. Dlg5 also interacts genetically with Hippo effectors Yap1/Taz Mechanistically, we show that DLG5 inhibits the association between MST1/2 and large tumor suppressor homologs 1/2 (LATS1/2), uses its scaffolding function to link MST1/2 with MARK3, and inhibits MST1/2 kinase activity. These data reveal a direct connection between cell polarity proteins and Hippo, which is essential for proper development of multicellular organisms.

Distribution of stanniocalcin binding sites in the lamina terminalis of the rat.

Stanniocalcin (STC-1), a 50 kDa glycoprotein hormone that regulates calcium/phosphate homeostasis in bony fish and mammals, has been shown to be expressed in central neurons and choroid plexus, and to exert a protective effect against hypercalcemic and hypoxic damage to neurons. Circumventricular organs are known to function in the regulation of ion and body fluid balance. Therefore, the possibility exists that STC-1 may be involved in the regulation of calcium/phosphate and fluid homeostasis through its actions on these central sites. In the present study, the distribution of STC-1 binding sites in forebrain circumventricular organs of the rat were investigated by in situ ligand binding using a stanniocalcin-alkaline phosphatase (STC-AP) fusion protein. Cells exhibiting STC-1 binding sites were found throughout the lamina terminalis. Dense cytoplasmic staining was observed predominantly within ependymal cells lining the anterior third ventricle region (AV3V), as well as cells of the choroid plexus. Additionally, neurons of the organum vasculosum of the lamina terminalis, the dorsal and ventral components of the median preoptic nucleus and the rostral aspects of the subfornical organ exhibited dense STC-1 cytoplasmic staining. STC-1 binding sites were also found in cells of the supraoptic nucleus, suprachiasmatic nucleus and anteroventral preoptic nucleus. These data suggest that STC-1 binding sites localized on the ependymal cells of the AV3V region and neurons of circumventricular organs may be associated with neuronal pathways involved in calcium/phosphate and fluid homeostasis.

The respiratory effects of stanniocalcin-1 (STC-1) on intact mitochondria and cells: STC-1 uncouples oxidative phosphorylation and its actions are modulated by nucleotide triphosphates.

Stanniocalcin-1 (STC-1) is one of only a handful of hormones that are targeted to mitochondria. High affinity receptors for STC-1 are present on cytoplasmic membranes and both the outer and inner mitochondrial membranes of nephron cells and hepatocytes. In both cell types, STC-1 is also present within the mitochondrial matrix and receptors presumably enable its sequestration. Furthermore, studies in bovine heart sub-mitochondrial particles have shown that STC-1 has concentration-dependent stimulatory effects on electron transport chain activity. The aim of the present study was to determine if the same effects could be demonstrated in intact, respiring mitochondria. At the same time, we also sought to demonstrate the functionality, if any, of an ATP binding cassette that has only recently been identified within the N-terminus of STC-1 by Prosite analysis. Intact, respiring mitochondria were isolated from rat muscle and liver and exposed to increasing concentrations of recombinant human STC-1 (STC-1). Following a 1h exposure to 500 nM STC-1, mitochondria from both organs displayed significant increases in respiration rate as compared to controls. Moreover, STC-1 uncoupled oxidative phosphorylation as ADP:O ratios were significantly reduced in mitochondria from both tissues. The resulting uncoupling was correlated with enhanced mitochondrial (45)Ca uptake in the presence of hormone. Respiratory studies were also conducted on a mouse inner medullary collecting cell line, where STC-1 had time and concentration-dependent stimulatory effects within the physiological range. In the presence of nucleotide triphosphates such as ATP and GTP (5mM) the respiratory effects of STC-1 were attenuated or abolished. Receptor binding studies revealed that this was due to a four-fold decrease in binding affinity (KD) between ligand and receptor. The results suggest that STC-1 stimulates mitochondrial electron transport chain activity and calcium transport, and that these effects are negatively modulated by nucleotide triphosphates.

In vivo interaction proteomics reveal a novel p38 mitogen-activated protein kinase/Rack1 pathway regulating proteostasis in Drosophila muscle.

Several recent studies suggest that systemic aging in metazoans is differentially affected by functional decline in specific tissues, such as skeletal muscle. In Drosophila, longevity appears to be tightly linked to myoproteostasis, and the formation of misfolded protein aggregates is a hallmark of senescence in aging muscle. Similarly, defective myoproteostasis is described as an important contributor to the pathology of several age-related degenerative muscle diseases in humans, e.g., inclusion body myositis. p38 mitogen-activated protein kinase (MAPK) plays a central role in a conserved signaling pathway activated by a variety of stressful stimuli. Aging p38 MAPK mutant flies display accelerated motor function decline, concomitant with an enhanced accumulation of detergent-insoluble protein aggregates in thoracic muscles. Chemical genetic experiments suggest that p38-mediated regulation of myoproteostasis is not limited to the control of reactive oxygen species production or the protein degradation pathways but also involves upstream turnover pathways, e.g., translation. Using affinity purification and mass spectrometry, we identified Rack1 as a novel substrate of p38 MAPK in aging muscle and showed that the genetic interaction between p38b and Rack1 controls muscle aggregate formation, locomotor function, and longevity. Biochemical analyses of Rack1 in aging and stressed muscle suggest a model whereby p38 MAPK signaling causes a redistribution of Rack1 between a ribosome-bound pool and a putative translational repressor complex.