Abstract
Na+,K+-ATPase (NKA) is essential for maintenance of cellular and whole-body water and ion homeostasis. In the kidney, a major site of ion transport, NKA consumes ~ 50% of ATP, indicating a tight coordination of NKA and energy metabolism. AMP-activated protein kinase (AMPK), a cellular energy sensor, regulates NKA by modulating serine phosphorylation of the α1-subunit, but whether it modulates other important regulatory phosphosites, such as Tyr10, is unknown. Using human kidney (HK-2) cells, we determined that the phosphorylation of Tyr10 was stimulated by the epidermal growth factor (EGF), which was opposed by inhibitors of Src kinases (PP2), tyrosine kinases (genistein), and EGF receptor (EGFR, gefitinib). AMPK activators AICAR and A-769662 suppressed the EGF-stimulated phosphorylation of EGFR (Tyr1173) and NKAα1 at Tyr10. The phosphorylation of Src (Tyr416) was unaltered by AICAR and increased by A-769662. Conversely, ouabain (100 nM), a pharmacological NKA inhibitor and a putative adrenocortical hormone, enhanced the EGF-stimulated Tyr10 phosphorylation without altering the phosphorylation of EGFR (Tyr1173) or Src (Tyr416). Ouabain (100–1000 nM) increased the ADP:ATP ratio, while it suppressed the lactate production and the oxygen consumption rate in a dose-dependent manner. Treatment with ouabain or gene silencing of NKAα1 or NKAα3 subunit did not activate AMPK. In summary, AMPK activators and ouabain had antagonistic effects on the phosphorylation of NKAα1 at Tyr10 in cultured HK-2 cells, which implicates a role for Tyr10 in coordinated regulation of NKA-mediated ion transport and energy metabolism.Graphical
Highlights
Na+,K+-ATPase (NKA), known as Na–K pump, is essential for the cellular as well as the whole-body water and ion homeostasis (Rossier et al 2015; Skou 1957, 1998).NKA, which comprises a catalytic α-subunit and a glycoprotein β-subunit (Blanco and Mercer 1998; Craig and Kyte 1980; Kyte 1971; Morth et al 2007), consumes one ATP to pump two K+ ions into and three N a+ ions out of the cell (Post and Jolly 1957; Sen and Post 1964), maintaining ion concentrations, cell excitability, as well as the driving force for the N a+-driven secondary transport of nutrients, such as glucose
We examined whether and how epidermal growth factor (EGF), AMPK, and ouabain modulated the phosphorylation of NKA α1-subunit (NKAα1) at Tyr10 in HK-2 cells, an epithelial cell line obtained from the proximal tubules of normal adult human kidney (Ryan et al 1994)
While aminoimidazole-4-carboxamide 1-β-d-ribofuranoside (AICAR) suppressed the phosphorylation of ERK1/2 in the absence of EGF, A-769662 stimulated it (Fig. 5L). These results suggested that pharmacological AMPK activation opposed the EGF-stimulated phosphorylation of EGF receptor (EGFR) (Tyr1173) as well as NKAα1 (Tyr10), AICAR and A-769662 produced divergent downstream signalling events
Summary
Na+,K+-ATPase (NKA), known as Na–K pump, is essential for the cellular as well as the whole-body water and ion homeostasis (Rossier et al 2015; Skou 1957, 1998).NKA, which comprises a catalytic α-subunit (isoforms α1–4) and a glycoprotein β-subunit (isoforms β1–3) (Blanco and Mercer 1998; Craig and Kyte 1980; Kyte 1971; Morth et al 2007), consumes one ATP to pump two K+ ions into and three N a+ ions out of the cell (Post and Jolly 1957; Sen and Post 1964), maintaining ion concentrations, cell excitability, as well as the driving force for the N a+-driven secondary transport of nutrients, such as glucose. Na+,K+-ATPase (NKA), known as Na–K pump, is essential for the cellular as well as the whole-body water and ion homeostasis (Rossier et al 2015; Skou 1957, 1998). The kidney consumes ~ 50% of ATP for energizing NKA (Clausen et al 1991; Rolfe and Brown 1997), not least in the proximal tubules, which represent ~ 12% of the total ATP consumption in the kidney (Ferrannini 2017). In diabetes mellitus the proximal tubules reabsorb a markedly increased load of glucose and other solutes, which further increases ATP consumption by NKA (Ferrannini 2017). A coordinated regulation of NKA and energy metabolism is required to maintain homeostasis
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