ULK2 Ser 1027 Phosphorylation by PKA Regulates Its Nuclear Localization Occurring through Karyopherin Beta 2 Recognition of a PY-NLS Motif

Uncoordinated 51-like kinase 2 (ULK2), a member of the serine/threonine kinase family, plays an essential role in the regulation of autophagy in mammalian cells. Given the role of autophagy in normal cellular homeostasis and in multiple diseases, improved mechanistic insight into this process may result in the development of novel therapeutic approaches. Here, we present evidence that ULK2 associates with karyopherin beta 2 (Kapβ2) for its transportation into the nucleus. We identify a potential PY-NLS motif (⁷⁷⁴gpgfgssppGaeaapslRyvPY⁷⁹⁵) in the S/P space domain of ULK2, which is similar to the consensus PY-NLS motif (R/K/H)X _2–5PY. Using a pull-down approach, we observe that ULK2 interacts physically with Kapβ2 both in vitro and in vivo. Confocal microscopy confirmed the co-localization of ULK2 and Kapβ2. Localization of ULK2 to the nuclear region was disrupted by mutations in the putative Kapβ2-binding motif (P794A). Furthermore, in transient transfection assays, the presence of the Kapβ2 binding site mutant (the cytoplasmic localization form) was associated with a substantial increase in autophagy activity (but a decrease in the in vitro serine-phosphorylation) compared with the wild type ULK2. Mutational analysis showed that the phosphorylation on the Ser1027 residue of ULK2 by Protein Kinase A (PKA) is the regulatory point for its functional dissociation from Atg13 and FIP 200, nuclear localization, and autophagy. Taken together, our observations indicate that Kapβ2 interacts with ULK2 through ULK2’s putative PY-NLS motif, and facilitates transport from the cytoplasm to the nucleus, depending on its Ser1027 residue phosphorylation by PKA, thereby reducing autophagic activity.     

Surface Plasmon Resonance Imaging-Based Protein Array Chip System for Monitoring a Hexahistidine-Tagged Protein during Expression and Purification

A surface plasmon resonance imaging-based Ni²⁺-iminodiacetic acid-coated gold chip system was developed to enable specific detection of a hexahistidine-tagged recombinant protein in crude extracts or in column chromatography fractions. This system is especially advantageous for high-throughput analysis of multiple proteins.     

Examining axial diffusion of nitric oxide in the lungs using heliox and breath hold.

Exhaled nitric oxide (NO) is highly dependent on exhalation flow; thus exchange dynamics of NO have been described by multicompartment models and a series of flow-independent parameters that describe airway and alveolar exchange. Because the flow-independent NO airway parameters characterize features of the airway tissue (e.g., wall concentration), they should also be independent of the physical properties of the insufflating gas. We measured the total mass of NO exhaled (A(I,II)) from the airways after five different breath-hold times (5-30 s) in healthy adults (21-38 yr, n = 9) using air and heliox as the insufflating gas, and then modeled A(I,II) as a function of breath-hold time to determine airway NO exchange parameters. Increasing breath-hold time results in an increase in A(I,II) for both air and heliox, but A(I,II) is reduced by a mean (SD) of 31% (SD 6) (P < 0.04) in the presence of heliox, independent of breath-hold time. However, mean (SD) values (air, heliox) for the airway wall diffusing capacity [3.70 (SD 4.18), 3.56 pl.s(-1).ppb(-1) (SD 3.20)], the airway wall concentration [1,439 (SD 487), 1,503 ppb (SD 644>)], and the maximum airway wall flux [4,156 (SD 2,502), 4,412 pl/s (SD 2,906)] using a single-path trumpet-shaped airway model that considers axial diffusion were independent of the insufflating gas (P > 0.55). We conclude that a single-path trumpet model that considers axial diffusion captures the essential features of airway wall NO exchange and confirm earlier reports that the airway wall concentration in healthy adults exceeds 1 ppm and thus approaches physiological concentrations capable of modulating smooth muscle tone.     

Growth medium sterilization using decomposition of peracetic acid for more cost-efficient production of omega-3 fatty acids by Aurantiochytrium

Bioprocess and Biosystems Engineering - Aurantiochytrium can produce significant amounts of omega-3 fatty acids, specifically docosahexaenoic acid and docosapentaenoic acid. Use of a glucose-based...     

Purification and characterization of an inhibitor protein with cytochalasin-like acitvity from bovine adrenal medulla

A protein preparation with cytochalasin-like activity has been obtained from bovine adrenal medulla. Analysis by electrophoresis in SDS-polyacrylamide gels and chromatography in a Sephacryl S-200 column indicated that the inhibitor activity coincided with a 90 000 dalton polypeptide. The inhibitor decreased high-affinity binding of [³H]cytochalasin B to actin nuclei, apparently by competing with the drug for thesame binding site. At substoichometric levels, the inhibitor had a potent effect on actin filament elongation and on actin-dependent gelation of cell extracts in vitro. These results suggest that the inhibitor may be involved in the control of actin filament assembly and interaction in the adrenal medulla.