Abstract
Serum- and glucocorticoid-induced kinase 1 (SGK1) is known to have consensus sequence of phosphorylation site R-x-R-x-x-(S/T)-Φ, where Φ is any hydrophobic amino acid and arginine residues are conserved at positions −5 and −3 relative to positions of Ser/Thr residues that are phosphorylated in the presence of SGK1. UNC-21-like kinase 2 (ULK2) also harbors putative SGK1 phosphorylation sites at both Ser507 (502RsRnsSG508) and Ser750 (745RtRttSV751) residues. Thus, the objective of this study was to determine whether Ser507 and Ser750 residues of ULK2 could be phosphorylation sites of SGK1 as one of its authentic substrate proteins. Using ULK2 507 and 750 serine residue un- or phosphorylation analog (S507AS750A or 507DS750D), we observed that modification of Ser507 or Ser750 residue was required to activate the kinase activity of ULK2 and sensitize ULK2 to stress or starvation while simultaneously enhancing its active state and autophagy characteristics, suggesting that phosphorylation at Ser750 or Ser507 residue could modulate its subcellular localization and protein interaction with AMPK1α to activate ULK2. We also observed that ULK2 autophagy activity was enhanced by GSK650394 (an SGK1 inhibitor) to compensate survival capacity through increasing its association with LC3 and phosphorylation. When SGK1 known to be associated with cell survival was inhibited by GSK650394, ULK2 autophagy pathway was activated to avoid cell death alternatively. Thus, our observations indicate that phosphorylation of ULK2 by SGK1 can regulate cell survival as an alternative modulation of ULK2 functions.
Highlights
IntroductionUncoordinated 51-like kinase 2 (ULK2), like ULK1, is a member of the serine/threonine kinase protein family that plays an essential role in the regulation
Western blot assays were conducted with a rabbit UNC-21-like kinase 2 (ULK2) Ab or an anti-phosphor Ser/Thr phosphorylation specific antibody in order to confirm whether Ser507 or Ser750 residue of ULK2 could be phosphorylated by Serum- and glucocorticoid-induced kinase 1 (SGK1) in HEK293 cells [second lane] The autophagy activity of ULK2 WT or mutants was measured with the antibody against LC3 as manufacture’ recommendation
In addition to a positive effect of the phosphorylation on both Ser507 and Ser750 of ULK2, a potential effect of autophagy with LC3II binding occurred via its action at an intracellular site in the C-terminus of the kinase mediated by adenosine monophosphate-activated kinase 1α [AMPK1α] binding (Figure 3)
Summary
Uncoordinated 51-like kinase 2 (ULK2), like ULK1, is a member of the serine/threonine kinase protein family that plays an essential role in the regulation. Its role seems to be redundant with that of ULK1. ULK2 functions in many fundamental biological processes, including cell fate determination, metabolism, transcriptional control, and autophagy [4] [5] [6]. Together with ULK1, ULK2 plays a central role in the autophagy signaling pathway [7] [8]. Recent studies have suggested that the activity of ULK2 must be carefully regulated by mechanisms that are individually modulated for each substrate to avoid indiscriminate phosphorylation by ULK1 [9] [10]. UNC-21-like kinase 2 (ULK2) harbors putative phosphorylation residues Ser507and Ser750 by serum- and glucocorticoid-induced kinase 1 (SGK1)
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