Dual function of UNC-51-like kinase 3 (Ulk3) in the Sonic hedgehog signaling pathway.

The Sonic hedgehog (Shh) signaling pathway controls a variety of developmental processes and is implicated in tissue homeostasis maintenance and neurogenesis in adults. Recently, we identified Ulk3 as an active kinase able to positively regulate Gli proteins, mediators of the Shh signaling in mammals. Here, we provide several lines of evidence that Ulk3 participates in the transduction of the Shh signal also independently of its kinase activity. We demonstrate that Ulk3 through its kinase domain interacts with Suppressor of Fused (Sufu), a protein required for negative regulation of Gli proteins. Sufu blocks Ulk3 autophosphorylation and abolishes its ability to phosphorylate and positively regulate Gli proteins. We show that Shh signaling destabilizes the Sufu-Ulk3 complex and induces the release of Ulk3. We demonstrate that the Sufu-Ulk3 complex, when co-expressed with Gli2, promotes generation of the Gli2 repressor form, and that reduction of the Ulk3 mRNA level in Shh-responsive cells results in higher potency of the cells to transmit the Shh signal. Our data suggests a dual function of Ulk3 in the Shh signal transduction pathway and propose an additional way of regulating Gli proteins by Sufu, through binding to and suppression of Ulk3.

Identification of a novel serine/threonine kinase ULK3 as a positive regulator of Hedgehog pathway.

The Hedgehog (Hh) signaling pathway plays crucial roles in embryonic development and is implicated in tissue homeostasis maintenance and neurogenesis in adults. Aberrant activation of Hh signaling is associated with various developmental abnormalities and several types of cancer. Genetic and biochemical studies ascertain serine/threonine kinase Fused (Fu) as a protein involved in Hh signaling in Drosophila. However, the role of Fu is not fully conserved in mammals suggesting involvement of other kinases in the mammalian Hh signaling pathway. In search of potential homologues to Drosophila and human Fu, we have cloned human serine/threonine kinase ULK3 and assessed its ability to regulate GLI transcription factors, mediators of SHH signaling. We demonstrate that ULK3 enhances endogenous and over-expressed GLI1 and GLI2 transcriptional activity in cultured cells, as assessed by GLI-luciferase reporter assay. Besides that, ULK3 alters subcellular localization of GLI1, as assessed by immunofluorescent staining and immunoblotting assays. We show that ULK3 is an autophosphorylated kinase and phosphorylates GLI proteins in vitro. We also demonstrate that ULK3 catalytical activity is crucial for its function in SHH pathway. We show that ULK3 is widely expressed and its expression is higher in a number of tissues where Shh signaling is known to be active. Our data suggest that serine/threonine kinase ULK3 is involved in the SHH pathway as a positive regulator of GLI proteins.

The toxicity and fate of phenolic pollutants in the contaminated soils associated with the oil-shale industry.

Phenol, cresols, dimethylphenols and resorcinols considered major pollutants in the oil-shale semi-coke dump leachates (up to 380 mg phenols/L) that contaminate the surrounding soils and pose a threat to the groundwater in the North-East of Estonia. However; despite high residual concentrations of polyaromatic hydrocarbons (PAHs) and oil products in these soils, the concentration of phenols (especially their water-extractable fraction) was low, not exceeding 0.7 mg/kg dwt. The aim of the current study was to evaluate the role of biodegradation and aging on the decrease of hazard caused by phenolic pollution. The extractability of phenols (phenol, cresols, dimethylphenols and resorcinols) and their biodegradability by the microbial population was studied in the 13 soils sampled from the Estonian oil-shale region, territories of former gas stations, and from presumably non-polluted areas. Phenol, 5-methylresorcinol, p-cresol and resorcinol could be considered easily degradable in the soils as the microbial populations from majority of the soils studied were able to grow on mineral medium supplemented with these phenols as a single source of carbon. 2,3- and 2,4- and 3,4-dimethylphenols could be considered less easily biodegradable. The semi-coke dump leachate polluted soil (containing no dibasic phenols, 43 mg of monobasic phenols, 1348 mg of oil products and 35 mg of PAHs per g dwt) was analyzed chemically (HPLC) and toxicologically (Flash-Assay using Vibrio fischeri) for the leaching of phenols during shaking of soil-water slurries for 24 h. Only 5.8% of the total concentration of phenols was water-extractable, whereas about 50% of the leached amount was biodegraded by the soil microorganisms. Phenol and cresols were biodegraded by 80%, but the concentration of dimethylphenols practically did not change. The pollutants (measured as total water-extractable toxicity) were desorbed from the soil particles by the 8th h of extraction, whereas the toxicity of the aqueous phase continued to increase, probably due to the formation of toxic metabolites. The concentration of water-extractable phenols was too low to explain the toxicity of the extract. Also the impact of PAHs and oil products was excluded. Thus, the relatively low concentration of phenols in the oil-shale region soils is most probably the reflection of both natural attenuation and pollution aging. Therefore, the impact of phenolic compounds to the net bioavailable hazard is probably not so remarkable as has been considered. The actual pollutants causing the soils from the oil-shale region, however, need to be elucidated.

Mammalian homologues of Drosophila fused kinase.

Sonic Hedgehog (Shh) signaling pathway is implicated in various developmental and postnatal processes. Much of the current knowledge about the mechanisms of Shh signal transduction in vertebrates comes from the investigations of the respective pathway in fruit fly Drosophila melanogaster. In Drosophila, serine/threonine kinase fused is involved in all aspects of regulation of the Hh-dependent transcription factor cubitus interruptus possessing both catalytic and regulatory functions. Two proteins, Stk36 and Ulk3, share similarity with fu and have been suggested as mammalian fu homologues. However, in vivo data clarify that Stk36 is not required for embryonic development in mice and participates in Shh-independent genesis of motile cilia. Even if Stk36 is associated with any pathological or physiological aspect of postnatal Shh signaling in mammals, it has perhaps only regulatory functions since its catalytic activity seems to be lost during evolution. In contrast to Stk36, Ulk3 is an active kinase. In non-stimulated cells, Ulk3 catalytic activity is blocked, and it is involved in negative control of Gli proteins, mediators of Shh signaling. In response to Shh, Ulk3 positively regulates Gli proteins by directly phosphorylating them. Thus, Ulk3 is able to recapitulate both positive and negative roles of fu in vitro. However, Ulk3 functioning in vivo remains to be investigated.

Smoothened agonist augments proliferation and survival of neural cells.

Sonic hedgehog signaling pathway is important in developmental processes like dorsoventral neural tube patterning, neural stem cell proliferation and neuronal and glial cell survival. Shh is also implicated in the regulation of the adult hippocampal neurogenesis. Recently, nonpeptidyl Smoothened activators of the Shh pathway have been identified. The aim of this study was to investigate the effects of chlorobenzothiophene-containing molecule, Smo agonist (SAG), which has been shown to activate Shh signaling pathway, in neurogenesis and neuronal survival in in vitro and in vivo models. Our in vitro experiments showed that SAG induces increased expression of Gli1 mRNA, transcriptional target and mediator of Shh signal. In vitro experiments also demonstrated that SAG in low-nanomolar concentrations induces proliferation of neuronal and glial precursors without affecting the differentiation pattern of newly produced cells. In contrast to Shh, SAG did not induce neurotoxicity in neuronal cultures. The SAG and Shh treatment also promoted the survival of newly generated neural cells in the dentate gyrus after their intracerebroventricular administration to adult rats. We propose that SAG and similar compounds represent attractive molecules to be developed for treatment of disorders where stimulation of the generation and survival of new neural cells would be beneficial.