Smad5 acts as an intracellular pH messenger and maintains bioenergetic homeostasis

Abstract

Both environmental cues and intracellular bioenergetic states profoundly affect intracellular pH (pHi). How a cell responds to pHi changes to maintain bioenergetic homeostasis remains elusive. Here we show that Smad5, a well-characterized downstream component of bone morphogenetic protein (BMP) signaling responds to pHi changes. Cold, basic or hypertonic conditions increase pHi, which in turn dissociates protons from the charged amino acid clusters within the MH1 domain of Smad5, prompting its relocation from the nucleus to the cytoplasm. On the other hand, heat, acidic or hypotonic conditions decrease pHi, blocking the nuclear export of Smad5, and thus causing its nuclear accumulation. Active nucleocytoplasmic shuttling of Smad5 induced by environmental changes and pHi fluctuation is independent of BMP signaling, carboxyl terminus phosphorylation and Smad4. In addition, ablation of Smad5 causes chronic and irreversible dysregulation of cellular bioenergetic homeostasis and disrupted normal neural developmental processes as identified in a differentiation model of human pluripotent stem cells. Importantly, these metabolic and developmental deficits in Smad5-deficient cells could be rescued only by cytoplasmic Smad5. Cytoplasmic Smad5 physically interacts with hexokinase 1 and accelerates glycolysis. Together, our findings indicate that Smad5 acts as a pHi messenger and maintains the bioenergetic homeostasis of cells by regulating cytoplasmic metabolic machinery.

Genetic Engineering of Human Embryonic Stem Cells for Precise Cell Fate Tracing during Human Lineage Development

“…22212) ( Hockemeyer et al., 2009 ). AAVS1-LSLm1-GFP, AAVS1-LSLm2-GFP, AAVS1-LSLm3-GFP, and AAVS1-LSLm4-GFP were obtained by replacing the 5′ LoxP site within the AAVS1-LSL-GFP donor vector with the mutant LoxP sequence shown in Figure 5 A. gRNAs were constructed as described previously ( Fang et al., 2017 , Mali et al., 2013 ), and gRNA targeting sequences for PAX6 and FOXA2 are shown in Figure S1 . PAX6-P2A-Cre, FOXA2-P2A-Cre, PAX6-Cre ERT2 , and FOXA2-Cre ERT2 donor plasmids were constructed by ligating the left and right homologous arms with Cre or Cre ERT2 cassettes PCR amplified from pCAG-Cre-ERT2 (Addgene, no.…”

Roles of pH in control of cell proliferation

“…). More recently, histones, (see below), as well as Smad5, whose nuclear‐cytoplasmic translocation is regulated by direct proton binding, have also been proposed to serve as cellular sensors of acid‐base status, although additional work seems warranted before a functionally relevant role of these proteins in acid‐base‐sensing can be established.…”

Cancer and pH Dynamics: Transcriptional Regulation, Proteostasis, and the Need for New Molecular Tools

“…In addition to extracellular pH, pHi has been shown to control the subcellular localization of the transcriptional effector, Smad5 [ 58 ]. Fang and colleagues showed that alkalinized pHi induced cytoplasmic Smad5 accumulation and accelerated glycolytic flux whereas acidic pHi induced nuclear localization and expression of Smad5 target genes [ 58 ].…”

“…In addition to extracellular pH, pHi has been shown to control the subcellular localization of the transcriptional effector, Smad5 [ 58 ]. Fang and colleagues showed that alkalinized pHi induced cytoplasmic Smad5 accumulation and accelerated glycolytic flux whereas acidic pHi induced nuclear localization and expression of Smad5 target genes [ 58 ]. Additionally, it was also shown that Smad5 plays a crucial role in maintaining the cellular bioenergetic homeostasis by regulating hexokinase 1 (HK1) [ 58 ], a rate-limiting enzyme in glycolysis [ 4 ].…”