Editors' ChoiceMAPK Signaling

Scaffolding Through Phosphatidic Acid–Enriched Domains

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Science Signaling  06 Jan 2009:
Vol. 2, Issue 52, pp. ec6
DOI: 10.1126/scisignal.252ec6

Phosphatidic acid (PA) is a plasma membrane lipid produced by enzymes such as diacylglycerol kinase in response to growth factor stimulation. Raf-1, a mitogen-activated protein kinase kinase kinase (MAPKKK), as well as the Ras guanine nucleotide exchange factor Sos, interact with PA. Kraft et al. now show that the kinase suppressor of Ras (KSR) proteins also bind PA and that this interaction appears to be important for activation of MAPK signaling. The addition of exogenous dioleoyl phosphatidic acid (DOPA) in the form of small unilamellar vesicles stimulated phosphorylation of the ERK MAPKs in several cell lines; however, DOPA vesicles failed to stimulate the activity of the MAPKKK Raf-1 or the MAPKK MEK1 in vitro. Using a tryptophan fluorescence assay, the authors showed that peptides corresponding to the PA binding region (PABR) of KSR1 or Raf-1 bound DOPA in vitro and that the Raf-1 PABR had a higher affinity for PA (78 nM) than did the KSR PABR (1.2 μM). Furthermore, binding required a polybasic motif and mutation of the basic residues abolished binding. Circular dichroism assays revealed that the PABR peptides were unstructured and that addition of DOPA micelles induced a structural change. KSR1 tagged with enhanced green fluorescent protein (EGFP) was predominantly localized to the cytosol, with a small population colocalizing near the plasma membrane. Insulin or DOPA vesicles increased the abundance of EGFP-KSR1 at the membrane. The membrane-associated form colocalized with PA, which was detected with a biotinylated form of the Raf-1-PABR. The abundance at the membrane of a double-mutant, GFP-tagged form of KSR, in which the basic residues were mutated and could not bind PA, was unchanged after the addition of DOPA or insulin. The colocalization of EGFP-KSR with Ras transiently increased after the addition of DOPA or insulin. Ras and the PA probe colocalized under unstimulated conditions; however, insulin or DOPA increased the colocalization of EGFP-KSR1 with Ras and the PA probe. The authors suggest that, when PA concentrations increase in membrane microdomains, the scaffold KSR is recruited to these regions of the membrane and, along with the other proteins that bind PA, organizes the MAPK complex to stimulate signaling.

C. A. Kraft, J. L. Garrido, E. Fluharty, L. Leiva-Vega, G. Romero, Role of phosphatidic acid in the coupling of the ERK cascade. J. Biol. Chem. 283, 36636–36645 (2008). [Abstract] [Full Text]

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