Abstract
NIK, a recently identified Nck-interacting kinase, acts upstream of the MEK kinase MEKK1 to activate the c-Jun N-terminal kinase JNK. We now show that NIK binds to and divergently activates the plasma membrane Na(+)-H(+) exchanger NHE1. In a genetic screen, NHE1 interacted with NIK at a site N-terminal (amino acids 407-502) to the Nck-binding domain, and this site is critical for its association with NHE1 in vivo. NIK also phosphorylates NHE1; however, the phosphorylation sites, which are distal to amino acid 638, are distinct from the NIK-binding site on NHE1 (amino acids 538-638). Expression of wild-type, but not a kinase-inactive, NIK in fibroblasts increased NHE1 phosphorylation and activity. The kinase domain of NIK, however, was not sufficient for this response in vivo. Full phosphorylation and activation of NHE1 required both the kinase and the NHE1-binding domains of NIK, suggesting that the NHE1-binding site functions as a targeting signal. The functional significance of an interaction between NIK and NHE1 was confirmed by the ability of a kinase-inactive NIK to selectively inhibit activation of NHE1 by platelet-derived growth factor but not by thrombin. Moreover, although NIK activates JNK through a mechanism dependent on MEKK1, it phosphorylated and activated NHE1 independently of MEKK1. These findings indicate that NIK acts downstream of platelet-derived growth factor receptors to phosphorylate and activate NHE1 divergently of its activation of JNK.
Highlights
Mitogen-activated protein kinases are components of signaling modules that are evolutionarily conserved
We found that NHE1 is a substrate for NIK kinase activity; an NHE1-binding site C-terminal to the kinase domain of NIK and a NIK-binding site N-terminal to the phosphorylation domain of NHE1 are required for this response, indicating that the kinase activity of NIK is not sufficient, but that a direct binding between these proteins is required
Immunoblotting confirmed that equivalent amounts of NHE1 were present in the immunoprecipitate for all conditions (Fig. 4B) and that the expression of NIK constructs in total cell lysates was comparable (Fig. 4C). These findings indicate that the N-terminal kinase domain of NIK is not sufficient to achieve full phosphorylation of NHE1, and they suggest that the NIK-binding site on NHE1 might function as a targeting signal to sequester NIK to the plasma membrane
Summary
Mitogen-activated protein kinases are components of signaling modules that are evolutionarily conserved. Lysates prepared from HEK293 cells transiently expressing full-length Myc-NIK were incubated with GST fusion proteins containing the full or partial C-terminal domain of NHE1.
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