Abstract
BackgroundExtracellular signal-regulated kinase 2 (ERK2) is an S/T kinase with more than 200 known substrates, and with critical roles in regulation of cell growth and differentiation and currently no membrane proteins have been linked to ERK2 scaffolding.Methods and resultsHere, we identify the human Na+/H+ exchanger 1 (hNHE1) as a membrane scaffold protein for ERK2 and show direct hNHE1-ERK1/2 interaction in cellular contexts. Using nuclear magnetic resonance (NMR) spectroscopy and immunofluorescence analysis we demonstrate that ERK2 scaffolding by hNHE1 occurs by one of three D-domains and by two non-canonical F-sites located in the disordered intracellular tail of hNHE1, mutation of which reduced cellular hNHE1-ERK1/2 co-localization, as well as reduced cellular ERK1/2 activation. Time-resolved NMR spectroscopy revealed that ERK2 phosphorylated the disordered tail of hNHE1 at six sites in vitro, in a distinct temporal order, with the phosphorylation rates at the individual sites being modulated by the docking sites in a distant dependent manner.ConclusionsThis work characterizes a new type of scaffolding complex, which we term a “shuffle complex”, between the disordered hNHE1-tail and ERK2, and provides a molecular mechanism for the important ERK2 scaffolding function of the membrane protein hNHE1, which regulates the phosphorylation of both hNHE1 and ERK2.Electronic supplementary materialThe online version of this article (doi:10.1186/s12915-016-0252-7) contains supplementary material, which is available to authorized users.
Highlights
Extracellular signal-regulated kinase 2 (ERK2) is an sites in human Na+/H+ exchanger 1 (hNHE1); (S/T) kinase with more than 200 known substrates, and with critical roles in regulation of cell growth and differentiation and currently no membrane proteins have been linked to ERK2 scaffolding
The recently suggested links between hNHE1 and ERK1/2 activation by determining relative T202/Y204 (ERK1)/2 prompted us to investigate whether ERK1/2 and NHE1 directly interact in a cellular context
The detection of multiple proximity ligation assay (PLA) puncta when cells were incubated with both NHE1 and ERK1/2 antibodies revealed the presence of hNHE1-ERK1/2 complexes in AP-1 WT hNHE1 cells (Fig. 1a), compared to a much lower signal in negative controls incubated with one antibody only (Fig. 1b)
Summary
Extracellular signal-regulated kinase 2 (ERK2) is an S/T kinase with more than 200 known substrates, and with critical roles in regulation of cell growth and differentiation and currently no membrane proteins have been linked to ERK2 scaffolding. ERK2 has been linked to more than 200 different substrates whose phosphorylation by ERK2 is orchestrated by coordination of signaling networks through common binding to so-called scaffold proteins [1]. D-domains, known as docking sites for ERK and JNK, LXL (DEJL) domains, or kinase interaction motifs (KIMs) have the canonical sequence of 2–5 basic residues (R/K), spaced by 1–6 residues to a hydrophobic motif ΦXΦ, where Φ is generally V, L, or I [8, 9]. The F-site recruitment site in ERK2 is much less studied and incompletely understood It binds to F-sites, called DEF (docking site for ERK, FXFP)-domains with the canonical FXFP sequence [12]. The only structure available of an F-site recruitment site-interacting protein is that of ERK2 in complex with the 15 kDa phosphoprotein enriched in astrocytes (PEA-15), which notably lacks any of the above-mentioned motifs [18]
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