by
Yohei Matsunaga;
Hyundoo Hwang;
Barbara Franke;
Rhys Williams;
McKenna Penley;
Hiroshi Qadota;
Hong Yi;
Levi Morran;
Hang Lu;
Olga Mayans;
Guy Benian
Background: In this study, we investigated the mechanism(s) by which delta opioids induce their potent activation of extracellular signal-regulated protein kinases (ERKs) in different cell lines expressing the cloned δ-opioid receptor (δ-OR). While it has been known for some time that OR stimulation leads to the phosphorylation of both ERK isoforms, the exact progression of events has remained elusive. Results: Our results indicate that the transphosphorylation of an endogenous epidermal growth factor receptor (EGFR) in the human embryonic kidney (HEK-293) cell line does not occur when co-expressed δ-ORs are stimulated by the δ-opioid agonist, D-Ser-Leu-enkephalin-Thr (DSLET). Moreover, neither pre-incubation of cultures with the selective EGFR antagonist, AG1478, nor down-regulation of the EGFR to a point where EGF could no longer activate ERKs had an inhibitory effect on ERK activation by DSLET. These results appear to rule out any structural or catalytic role for the EGFR in the δ-opioid-mediated MAPK cascade. To confirm these results, we used C6 glioma cells, a cell line devoid of the EGFR. In δ-OR-expressing C6 glioma cells, opioids produce a robust phosphorylation of ERK 1 and 2, whereas EGF has no stimulatory effect. Furthermore, antagonists to the RTKs that are endogenously expressed in C6 glioma cells (insulin receptor (IR) and platelet-derived growth factor receptor (PDGFR)) were unable to reduce opioid-mediated ERK activation. Conclusion: Taken together, these data suggest that the transactivation of resident RTKs does not appear to be required for OR-mediated ERK phosphorylation and that the tyrosine-phosphorylated δ-OR, itself, is likely to act as its own signalling scaffold.
Bud formation by Saccharomyces cerevisiae must be coordinated with the nuclear cycle to enable successful proliferation. Many environmental stresses temporarily disrupt bud formation, and in such circumstances, the morphogenesis checkpoint halts nuclear division until bud formation can resume. Bud emergence is essential for degradation of the mitotic inhibitor, Swe1. Swe1 is localized to the septin cytoskeleton at the bud neck by the Swe1-binding protein Hsl7. Neck localization of Swe1 is required for Swe1 degradation. Although septins form a ring at the presumptive bud site before bud emergence, Hsl7 is not recruited to the septins until after bud emergence, suggesting that septins and/or Hsl7 respond to a "bud sensor." Here we show that recruitment of Hsl7 to the septin ring depends on a combination of two septin-binding kinases: Hsl1 and Elm1. We elucidate which domains of these kinases are needed and show that artificial targeting of those domains suffices to recruit Hsl7 to septin rings even in unbudded cells. Moreover, recruitment of Elm1 is responsive to bud emergence. Our findings suggest that Elm1 plays a key role in sensing bud emergence.
by
Nikita Pozdeyev;
Carla Taylor;
Rashidul Haque;
Shyam S. Chaurasia;
Amy Visser;
Aamera Thazyeen;
Yuhong Du;
Haian Fu;
Joan Weller;
David C. Klein;
P Michael Iuvone
14-3-3 proteins are a ubiquitous, highly conserved family of chaperone proteins involved in signal transduction, regulation of cell cycle, intracellular trafficking/targeting, cytoskeletal structure, and transcription. Although 14-3-3 proteins are among the most abundant proteins in the CNS, very little is known about their functional roles in the vertebrate retina. In the present study, we demonstrated that photoreceptors express 14-3-3 protein(s) and identified a 14-3-3 binding partner in photoreceptor cells, the melatonin-synthesizing enzyme arylalkylamine N-acetyltransferase (AANAT). Importantly, our data demonstrate that the binding of 14-3-3 to AANAT is regulated by light, with dramatic functional consequences. During the night in darkness, retinal AANAT is phosphorylated and forms a complex with 14-3-3 proteins with an apparent molecular weight of ∼90 kDa. Phosphorylation of AANAT facilitates the binding of enzyme to 14-3-3 proteins. Within the complex, AANAT is catalytically activated and protected from dephosphorylation and degradation. Light disrupts the AANAT/14-3-3 complex, leading to catalytic inactivation, dephosphorylation, and proteolytic degradation of the enzyme. In the presence of the proteasome inhibitor, lactacystin, light results in the formation of a high molecular weight complex (>150 kDa), which may represent an intermediate in the AANAT degradation process. These findings provide new insight into the roles of 14-3-3 proteins in photoreceptor cells and to the mechanisms controlling melatonin synthesis in the vertebrate retina.
The actin cytoskeleton is critical for form and function of vascular cells, serving mechanical, organizational and signaling roles. Because many cytoskeletal proteins are sensitive to reactive oxygen species, redox regulation has emerged as a pivotal modulator of the actin cytoskeleton and its associated proteins. Here, we summarize work implicating oxidants in altering actin cytoskeletal proteins and focus on how these alterations affect cell migration, proliferation and contraction of vascular cells. Finally, we discuss the role of oxidative modification of the actin cytoskeleton in vivo and highlight its importance for vascular diseases.
CBX7 is a polycomb group protein, and despite being implicated in many diseases, its role in cell proliferation has been controversial: some groups described its pro-proliferative properties, but others illustrated its inhibitory effects on cell growth. To date, the reason for the divergent observations remains unknown. While several isoforms for CBX7 were reported, no studies investigated whether the divergent roles of CBX7 could be due to distinct functions of CBX7 isoforms. In this study, we newly identified mouse CBX7 transcript variant 1 (mCbx7v1), which is homologous to the human CBX7 gene (hCBX7v1) and is expressed in various mouse organs.
We revealed that mCbx7v1 and hCBX7v1 encode a 36 kDa protein (p36CBX7) whereas mCbx7 and hCBX7v3 encode a 22 kDa protein (p22CBX7). This study further demonstrated that p36CBX7 was localized to the nucleus and endogenously expressed in proliferating cells whereas p22CBX7 was localized to the cytoplasm, induced by serum starvation in both human and mouse cells, and inhibited cell proliferation. Together, these data indicate that CBX7 isoforms are localized in different locations in a cell and play differing roles in cell proliferation. This varying function of CBX7 isoforms may help us understand the distinct function of CBX7 in various studies.
Mammalian target of rapamycin complex 1 (mTORC1) is a key regulator of cell growth, proliferation and metabolism. mTORC1 regulates protein synthesis positively and autophagy negatively. Autophagy is a major system to manage bulk degradation and recycling of cytoplasmic components and organelles. Tuberous sclerosis complex (TSC) 1 and 2 form a heterodimeric complex and inactivate Ras homolog enriched in brain, resulting in inhibition of mTORC1. Here, we investigated the effects of hyperactivation of mTORC1 on cardiac function and structure using cardiac-specific TSC2-deficient (TSC2-/-) mice. TSC2-/- mice were born normally at the expected Mendelian ratio. However, the median life span of TSC2-/- mice was approximately 10 months and significantly shorter than that of control mice. TSC2-/- mice showed cardiac dysfunction and cardiomyocyte hypertrophy without considerable fibrosis, cell infiltration or apoptotic cardiomyocyte death. Ultrastructural analysis of TSC2-/- hearts revealed misalignment, aggregation and a decrease in the size and an increase in the number of mitochondria, but the mitochondrial function was maintained. Autophagic flux was inhibited, while the phosphorylation level of S6 or eukaryotic initiation factor 4E -binding protein 1, downstream of mTORC1, was increased. The upregulation of autophagic flux by trehalose treatment attenuated the cardiac phenotypes such as cardiac dysfunction and structural abnormalities of mitochondria in TSC2-/- hearts. The results suggest that autophagy via the TSC2-mTORC1 signaling pathway plays an important role in maintenance of cardiac function and mitochondrial quantity and size in the heart and could be a therapeutic target to maintain mitochondrial homeostasis in failing hearts.
Haspin, a highly conserved kinase in eukaryotes, has been shown to be responsible for phosphorylation of histone H3 at threonine 3 (H3T3ph) during mitosis, in mammals and yeast. Here we report that haspin is the kinase that phosphorylates H3T3 in Drosophila melanogaster and it is involved in sister chromatid cohesion during mitosis. Our data reveal that haspin also phosphorylates H3T3 in interphase. H3T3ph localizes in broad silenced domains at heterochromatin and lamin-enriched euchromatic regions. Loss of haspin compromises insulator activity in enhancer-blocking assays and triggers a decrease in nuclear size that is accompanied by changes in nuclear envelope morphology. We show that haspin is a suppressor of position-effect variegation involved in heterochromatin organization. Our results also demonstrate that haspin is necessary for pairing-sensitive silencing and it is required for robust Polycomb-dependent homeotic gene silencing. Haspin associates with the cohesin complex in interphase, mediates Pds5 binding to chromatin and cooperates with Pds5-cohesin to modify Polycomb-dependent homeotic transformations. Therefore, this study uncovers an unanticipated role for haspin kinase in genome organization of interphase cells and demonstrates that haspin is required for homeotic gene regulation.
Two urea transporters, UT-A1 and UT-A3, are expressed in the kidney terminal inner medullary collecting duct (IMCD) and are important for the production of concentrated urine. UT-A1, as the largest isoform of all UT-A urea transporters, has gained much attention and been extensively studied; however, the role and the regulation of UT-A3 are less explored. In this study, we investigated UT-A3 regulation by glycosylation modification. A site-directed mutagenesis verified a single glycosylation site in UT-A3 at Asn279. Loss of the glycosylation reduced forskolin-stimulated UT-A3 cell membrane expression and urea transport activity. UT-A3 has two glycosylation forms, 45 and 65 kDa. Using sugar-specific binding lectins, the UT-A3 glycosylation profile was examined. The 45-kDa form was pulled down by lectin concanavalin A (Con A) and Galant husnivalis lectin (GNL), indicating an immature glycan with a high amount of mannose (Man), whereas the 65-kDa form is a mature glycan composed of acetylglucosamine (GlcNAc) and poly-N-acetyllactosame (poly-LacNAc) that was pulled down by wheat germ agglutinin (WGA) and tomato lectin, respectively. Interestingly, the mature form of UT-A3 glycan contains significant amounts of sialic acid. We explored the enzymes responsible for directing UT-A3 sialylation. Sialyltransferase ST6GalI, but not ST3GalIV, catabolizes UT-A3 α2,6-sialylation. Activation of protein kinase C (PKC) by PDB treatment promoted UT-A3 glycan sialylation and membrane surface expression. The PKC inhibitor chelerythrine blocks ST6GalI-induced UT-A3 sialylation. Increased sialylation by ST6GalI increased UT-A3 protein stability and urea transport activity. Collectively, our study reveals a novel mechanism of UT-A3 regulation by ST6GalI-mediated sialylation modification that may play an important role in kidney urea reabsorption and the urinary concentrating mechanism.