title: ULK2 Ser 1027 Phosphorylation by PKA Regulates Its Nuclear Localization Occurring through Karyopherin Beta 2 Recognition of a PY-NLS Motif.
abstract: 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 ((774)gpgfgssppGaeaapslRyvPY(795)) in the S/P space domain of ULK2, which is similar to the consensus PY-NLS motif (R/K/H)X(2-5)PY. 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.
authors.affiliation: Department of Biology Education, Chungbuk National University, 1, Chungdae-ro, Seowon-gu, Cheongju, Chungbuk, Republic of Korea.
authors.forename: Sung Hwa
authors.initials: SH
authors.lastname: Shin
authors.affiliation: Department of Internal Medicine, Chungbuk National University, 1, Chungdae-ro, Seowon-gu, Cheongju, Chungbuk, Republic of Korea.
authors.forename: Eun Jeoung
authors.initials: EJ
authors.lastname: Lee
authors.affiliation: Department of Biology Education, Korea National University of Education, Taeseongtabyeon-ro, Heungdeok-gu, Cheongju, Chungbuk, Republic of Korea.
authors.forename: Jaesun
authors.initials: J
authors.lastname: Chun
authors.affiliation: Department of Biomedical Laboratory Science, Eulji University, Daejeon, Republic of Korea.
authors.forename: Sunghee
authors.initials: S
authors.lastname: Hyun
authors.affiliation: Department of Biology Education, Chungbuk National University, 1, Chungdae-ro, Seowon-gu, Cheongju, Chungbuk, Republic of Korea.
authors.forename: Sang Sun
authors.initials: SS
authors.lastname: Kang
mesh_terms: D048868:Adaptor Proteins, Signal Transducing / Q000378:metabolism; D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D001343:Autophagy; D000071183:Autophagy-Related Proteins; D002467:Cell Nucleus / Q000378:metabolism*; D002470:Cell Survival; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism*; D057809:HEK293 Cells; D006801:Humans; D008954:Models, Biological; D008969:Molecular Sequence Data; D050505:Mutant Proteins / Q000378:metabolism; D019913:Nuclear Localization Signals / Q000378:metabolism; D010766:Phosphorylation; D010768:Phosphoserine / Q000378:metabolism*; D011485:Protein Binding; D017346:Protein Serine-Threonine Kinases / Q000737:chemistry* / Q000378:metabolism*; D021381:Protein Transport; D011505:Protein-Tyrosine Kinases / Q000378:metabolism; D013347:Subcellular Fractions / Q000378:metabolism; D028961:beta Karyopherins / Q000378:metabolism*
chemical_list: C534291:ATG13 protein, human; D048868:Adaptor Proteins, Signal Transducing; D000071183:Autophagy-Related Proteins; D050505:Mutant Proteins; D019913:Nuclear Localization Signals; C407704:RB1CC1 protein, human; C108121:TNPO1 protein, human; D028961:beta Karyopherins; D010768:Phosphoserine; D011505:Protein-Tyrosine Kinases; D017346:Protein Serine-Threonine Kinases; C541703:Ulk2 protein, human; D017868:Cyclic AMP-Dependent Protein Kinases