The human Na+/H+ exchanger 1 is a membrane scaffold protein for extracellular signal-regulated kinase 2

Ruth Hendus-Altenburger; Elena Pedraz Cuesta; Christina Wilkens Olesen; Elena Papaleo; Jeff Alexander Schnell; Jonathan T. S. Hopper; Carol V. Robinson; Stine Helene Falsig Pedersen; Birthe Brandt Kragelund

doi:10.1186/s12915-016-0252-7

The human Na⁺/H⁺ exchanger 1 is a membrane scaffold protein for extracellular signal-regulated kinase 2

Ruth Hendus-Altenburger, Elena Pedraz Cuesta, Christina Wilkens Olesen, Elena Papaleo, Jeff Alexander Schnell, Jonathan T. S. Hopper, Carol V. Robinson, Stine Helene Falsig Pedersen, Birthe Brandt Kragelund

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Abstract

BACKGROUND: 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.

METHODS AND RESULTS: 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.

CONCLUSIONS: 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 language	English
Article number	31
Journal	BMC Biology
Volume	14
Number of pages	17
ISSN	1741-7007
DOIs	https://doi.org/10.1186/s12915-016-0252-7
Publication status	Published - 15 Apr 2016

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@article{c1c880c5b67e43bea086d8d185a54d3d,

title = "The human Na+/H+ exchanger 1 is a membrane scaffold protein for extracellular signal-regulated kinase 2",

abstract = "BACKGROUND: 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.METHODS AND RESULTS: 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.CONCLUSIONS: 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.",

author = "Ruth Hendus-Altenburger and {Pedraz Cuesta}, Elena and Olesen, {Christina Wilkens} and Elena Papaleo and Schnell, {Jeff Alexander} and Hopper, {Jonathan T. S.} and Robinson, {Carol V.} and Pedersen, {Stine Helene Falsig} and Kragelund, {Birthe Brandt}",

year = "2016",

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doi = "10.1186/s12915-016-0252-7",

language = "English",

volume = "14",

journal = "B M C Biology",

issn = "1741-7007",

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TY - JOUR

T1 - The human Na+/H+ exchanger 1 is a membrane scaffold protein for extracellular signal-regulated kinase 2

AU - Hendus-Altenburger, Ruth

AU - Pedraz Cuesta, Elena

AU - Olesen, Christina Wilkens

AU - Papaleo, Elena

AU - Schnell, Jeff Alexander

AU - Hopper, Jonathan T. S.

AU - Robinson, Carol V.

AU - Pedersen, Stine Helene Falsig

AU - Kragelund, Birthe Brandt

PY - 2016/4/15

Y1 - 2016/4/15

N2 - BACKGROUND: 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.METHODS AND RESULTS: 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.CONCLUSIONS: 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.

AB - BACKGROUND: 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.METHODS AND RESULTS: 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.CONCLUSIONS: 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.

U2 - 10.1186/s12915-016-0252-7

DO - 10.1186/s12915-016-0252-7

M3 - Journal article

C2 - 27083547

SN - 1741-7007

VL - 14

JO - B M C Biology

JF - B M C Biology

M1 - 31

ER -

The human Na+/H+ exchanger 1 is a membrane scaffold protein for extracellular signal-regulated kinase 2

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The human Na⁺/H⁺ exchanger 1 is a membrane scaffold protein for extracellular signal-regulated kinase 2