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 (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.

Tip60 Tumor Suppressor Requires Its NLS Motif to Interact with Importin <i>α</i>

“…Tip60 NLS mutant which could not be self-acetylated was mainly found in the cytoplasm based on confocal analysis (Figure 2(A), Figure 2(D)). The blockade of nuclear localization of NLS mutant might be due to the inhibition of importin α binding on its middle domain by point mutation, not by self-acetylation (on Lys109, Lys113, Lys222, or Lys360) or its HAT activity [30]. Because Lys222 of Tip60 is overlapped with the putative NLS, self-acetylation may affect binding between importin α and Tip60 (Figure 1(A)).…”

SGK1 Inhibits ULK2 Autophagy Activity

“…Unlike ULK1 that is predominantly found in the cytosol, ULK2 is mainly located in the nucleus, although it is also found in the cytosol. Recently, the mechanism by which ULK2 is localized to the nucleus has been reported [11].…”

“…ULK2 does not have any recognizable short, basic, classic import, or export sequences. It contains PY-NLS sequences which contribute to its nuclear localization [11]. This character is a distinguishable feature from ULK1.…”

“…Thus, its localization is likely to be indirectly regulated through association with binding proteins. It has been suggested that a binding protein can regulate the subcellular localization of ULK2 by inhibiting its nuclear export [11].…”

Dissecting the Physiological Roles of ULK1/2 in the Mouse Brain

“…Interestingly, ULK2 has also been shown to localize to the nucleus with the aid of an importer receptor-Kapβ2, even under basal conditions in HEK293 cells. The functional significance of the nuclear ULK2 has not yet been elucidated (Shin et al, 2015).…”