Cytoskeletal Dynamics

Stimulating H+ Efflux to Promote Reorganization

Science Signaling  29 Sep 2009:
Vol. 2, Issue 90, pp. ec322
DOI: 10.1126/scisignal.290ec322

Akt, a protein kinase activated by growth factor signaling, has been implicated in cancer initiation and metastasis. Its regulation of the actin cytoskeleton is likely pertinent to the latter. Noting that, like Akt, the sodium-hydrogen exchanger NHE1 promotes cell proliferation and survival and has been implicated in cell migration, Meima et al. investigated involvement of NHE1 in Akt-dependent regulation of cytoskeleton dynamics. After an in vitro kinase assay showed that purified Akt1 phosphorylated a fusion protein containing the NHE1 cytoplasmic regulatory domain, the authors used mass spectrometry to identify Ser648 (of rabbit NHE1), which is part of an amino acid motif favorable for Akt phosphorylation, as the phosphorylation site. This was confirmed by a combination of mutational analysis and immunoblotting. Insulin and platelet-derived growth factor (PDGF)—both of which activate Akt—stimulated phosphorylation of Ser648 of a tagged form of NHE1 stably expressed in PS120 fibroblasts lacking endogenous NHE1. Insulin and PDGF stimulated recovery from an acid load in cells containing wild-type tagged NHE1 but not in those with a form in which Ser648 was replaced with alanine (NHE1-S648A); moreover, inhibition of phosphatidylinositol 3-kinase (PI3K) or Akt blocked the ability of the growth factors to stimulate NHE1 activity. Insulin and PDGF stimulated reorganization of the actin cytoskeleton in cells containing wild-type NHE1 but failed to do so in cells with either NHE1-S648A or a mutant form of NHE1 unable to mediate H+ efflux. The authors thus conclude that Akt-dependent phosphorylation and activation of NHE1 plays a critical role in insulin and PDGF-dependent reorganization of the actin cytoskeleton. They note that NHE1 is unusual among Akt substrates in that—at least in this system—its activity is stimulated by Akt (although, in myocardial cells, Akt phosphorylation of NHE1 on Ser648 has been found to be inhibitory).

M. E. Meima, B. A. Webb, H. E. Witkowska, D. L. Barber, The sodium-hydrogen exchanger NHE1 is an Akt substrate necessary for actin filament reorganization by growth factors. J. Biol. Chem. 284, 26666–26675 (2009). [Abstract] [Full Text]