Dual Phosphorylation of suppressor of fused (Sufu) by PKA and GSK3beta regulates its stability and localization in the primary cilium

Suppressor of fused (Sufu) is an essential negative regulator of the sonic hedgehog (Shh) pathway, but little is known about how Sufu itself is normally regulated. Here, we report that Sufu is phosphorylated at Ser-342 and Ser-346 by GSK3β and cAMP-dependent protein kinase A (PKA), respectively, and phosphorylation at this dual site stabilizes Sufu against Shh signaling-induced degradation. We further show that localization of Sufu in the primary cilium is induced by Shh signaling and is required for the turnover of both phosphorylated and total Sufu. Perturbing Sufu phosphorylation with PKA inhibitors or replacing Ser-346 with alanine reduced the stay and replacing Ser-342 and Ser-346 with aspartic acid prolonged the stay of Sufu in the cilia. Finally, ciliary localization of Gli2/3 also required Smo and was similarly influenced by perturbations of PKA activity or mutations at the dual Sufu phosphorylation site. Thus, Shh likely induced trafficking of phospho-Sufu into the primary cilium in a complex with Gli2/3, and dephosphorylation triggered a retrograde export, allowing Sufu to be degraded by the ubiquitin-proteasome system.     

The mammalian Nek1 kinase is involved in primary cilium formation

Recent studies implicate primary cilium (PC) proteins in the etiologies of various polycystic kidney diseases (PKD). NIMA-related kinases (NRKs) are conserved serine/threonine kinases, which are usually defined as 'mitotic kinases'. Murine mutants for the NRKs, nek1 (kat mice) suffer from PKD, suggesting that it may be involved in cilium control. We demonstrated herein that Nek1 is localized to basal body region and that Nek1 overexpression inhibits ciliogenesis in Madin-Darby canine kidney epithelial cells. The number of primary cilia is dramatically reduced in kat2J mouse embryonic fibroblasts culture. It is thus hypothesized that Nek1 links cell cycle progression and the PC cycle.     

The exocyst localizes to the primary cilium in MDCK cells

Primary cilia play a role in the maintenance of tubular epithelial differentiation and ciliary dysfunction can result in abnormal cyst formation, such as occurs in autosomal dominant polycystic kidney disease (ADPKD). We previously showed that the exocyst, an eight-protein complex involved in the biogenesis of polarity from yeast to mammals, is centrally involved in cyst formation [Mol. Biol. Cell. 11 (2000) 4259]. Here we show that the exocyst complex localizes to the primary cilium in Madin-Darby canine kidney (MDCK) tubular epithelial cells. We further show that the exocyst is overexpressed in both cell lines and primary cell cultures of ADPKD origin, suggesting that the exocyst may be involved in the pathogenesis of ADPKD.     

Co-culture of primary pulmonary cells to model alveolar injury and translocation of proteins

Summary Primary rat alveolar type II cells and early passage rat lung fibroblasts were co-cultured on opposite sides of a collagen-coated polycarbonate filter. This is an approach to “model”, in part, an alveolar wall to study mechanisms of cytotoxicity and translocation of bioactive materials from the alveolar space to the lung interstitium. Type II cells were recovered from adult rat (Fischer 344) lungs by enzyme digestion and “panning”. Lung fibroblasts were separated from the same species, cultured initially in 10% fetal bovine serum and used in the co-culture system at early passage. The type II cells formed a monolayer of defferentiated epithelium which provided a barrier on the upper side of the collagen (human type IV)-coated filter. The fibroblasts on the bottom of the filter replicated logarithmically in the presence of serum, could be rendered quiescent in defined medium and then returned to rapid growth phase with the reintroduction of serum. The intact epithelial monolayer excluded trypan blue, albumin, platelet-derived growth factor, and alpha₂-macroglobulin from the lower compartment of the culture chamber. Altering the integrity of the monolayer by a variety of means allowed translocation of these materials through the collagen-coated filters. Particularly interesting was the effect of taurine chloramine which caused subtle changes in the alveolar epithelium and allowed subsequent translocation of albumin. In addition, we showed that rat alveolar macrophages remain viable with some spreading on the surface of the epithelial monolayer. This co-culture system will have future application in the study of how reactive oxygen species might affect the epithelial barrier, and whether macrophage-derived growth factors can influence fibroblast proliferation if the monolayer is intact or injured.     

The primary cilium: keeper of the key to cell division

Assembly of the nonmotile primary cilium of vertebrate cells requires one of the centrioles of the centrosome. A cluster of new studies, including one in this issue of Cell by Pugacheva et al. (2007), reveal that ciliary assembly proteins influence cell-cycle progression and that a centrosomal "mitotic kinase" promotes ciliary disassembly. The link between the cell cycle and the primary cilium may reflect a requirement for liberation of the ciliary centriole to allow the centrosome to form the mitotic spindle.     

Dynamics of the primary cilium in shear flow

In this work, the equilibrium shape and dynamics of a primary cilium under flow are investigated by using both theoretical modeling and experiment. The cilium is modeled as an elastic beam that may undergo large deflection due to the hydrodynamic load. Equilibrium results show that the anchoring effects of the basal body on the cilium axoneme behave as a nonlinear rotational spring. Details of the rotational spring are elucidated by coupling the elastic beam with an elastic shell. We further study the dynamics of cilium under shear flow with the cilium base angle determined from the nonlinear rotational spring, and obtain good agreement in cilium bending and relaxing dynamics when comparing between modeling and experimental results. These results potentially shed light on the physics underlying the mechanosensitive ion channel transport through the ciliary membrane.     

Polypeptide-binding proteins mediate completion of co-translational protein translocation into the mammalian endoplasmic reticulum

The first step in the secretion of most mammalian proteins is their transport into the lumen of the endoplasmic reticulum (ER). Transport of pre-secretory proteins into the mammalian ER requires signal peptides in the precursor proteins and a protein translocase in the ER membrane. In addition, hitherto unidentified lumenal ER proteins have been shown to be required for vectorial protein translocation. This requirement was confirmed in this study by using proteoliposomes that were made from microsomal detergent extracts and contained either low or high concentrations of lumenal ER proteins. Furthermore, immunoglobulin-heavy-chain-binding protein (BiP) was shown to be able to substitute for the full set of lumenal proteins and, in the case of biotinylated precursor proteins, avidin was found to be able to substitute for lumenal proteins. Thus, the polypeptide-chain-binding protein BiP was identified as one lumenal protein that is involved in efficient vectorial protein translocation into the mammalian ER.     

Cooperative requirement of the Gli proteins in neurogenesis

The Gli proteins are critical components of multiple processes in development, homeostasis and disease, including neurogenesis and tumorigenesis. However, it is unclear how the Gli code, the sum of their combinatorial positive and negative functions, dictates cell fate and behavior. Using an antisense approach to knockdown gene function in vivo, we find that each of the three Gli proteins is required for the induction of all primary neurons in the amphibian neural plate and regulates the bHLH/Notch neurogenic cascade. Analyses of endogenous Gli function in Gli-mediated neurogenesis and tumorigenesis, and in animal cap assays, reveal specific requirements that are context specific. Nuclear colocalization and binding studies suggest the formation of complexes, with the first two zinc fingers of the Gli five zinc-finger domain acting as a protein-protein interaction site. The Gli proteins therefore appear to form a dynamic physical network that underlies cooperative function, greatly extending the combinatorial possibilities of the Gli code, which may be further fine-tuned in cell fate specification by co-factor function.     

The importance of a single primary cilium

The centrosome is the main microtubule-organizing center in animal cells, and helps to influence the morphology of the microtubule cytoskeleton in interphase and mitosis. The centrosome also templates the assembly of the primary cilium, and together they serve as a nexus of cell signaling that provide cells with diverse organization, motility, and sensory functions. The majority of cells in the human body contain a solitary centrosome and cilium, and cells have evolved regulatory mechanisms to precisely control the numbers of these essential organelles. Defects in the structure and function of cilia lead to a variety of complex disease phenotypes termed ciliopathies, while dysregulation of centrosome number has long been proposed to induce genome instability and tumor formation. Here, we review recent findings that link centrosome amplification to changes in cilium number and signaling capacity, and discuss how supernumerary centrosomes may be an important aspect of a set of cilia-related disease phenotypes.