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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.Here, 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.This 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.

Original publication

DOI

10.1186/s12915-016-0252-7

Type

Journal article

Journal

BMC biology

Publication Date

15/04/2016

Volume

14

Pages

31 - 31

Addresses

Structural Biology and NMR Laboratory, Department of Biology, University of Copenhagen, Ole Maaløes Vej 5, DK-2200, Copenhagen N, Denmark.

Keywords

Cell Line, Humans, Mitogen-Activated Protein Kinase 1, Mitogen-Activated Protein Kinase 3, Sodium-Hydrogen Antiporter, Cation Transport Proteins, Enzyme Activation, Amino Acid Sequence, Protein Structure, Tertiary, Protein Folding, Phosphorylation, Models, Molecular, Molecular Sequence Data, Protein Interaction Maps, Intrinsically Disordered Proteins