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AKAP Functions for Rac1

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Science Signaling  28 May 2013:
Vol. 6, Issue 277, pp. ec124
DOI: 10.1126/scisignal.2004368

The guanosine triphosphatase (GTPase) Rac1 and its effector kinases of the p21-activated kinase (PAK) family activate the extracellular signal–regulated kinase (ERK) pathway to stimulate cellular proliferation. Bachmann et al. identified the RIIβ regulatory subunits of protein kinase A (PKA), which dissociate from the catalytic PKAc subunits upon cyclic adenosine monophosphate (cAMP) binding, in a screen for Rac1-interacting proteins. The interaction was confirmed with a fluorescent protein complementation assay in transfected cells and a cAMP affinity purification assay with mouse brain lysates and by in vitro assays with recombinant proteins. Mutational analysis identified two binding sites on Rac1 that were important for binding to RIIβ, and one of these, BD2, was similar to the RII-binding domain found in PKA anchoring proteins (AKAPs). The same region of RII that interacts with AKAPs was involved in the Rac1 interaction. Mutation of the BD2 domain in the context of a constitutively active Rac1 mutant (Q61L-P) disrupted the interaction with RIIβ. Overexpression of the constitutively active Rac1 mutant (Q61L) increased the abundance of the PKA holoenzyme, which was detected with a protein complement assay composed of labeled regulatory and PKAc subunits. Although application of forskolin to increase cAMP in HEK 293 cells did not affect ERK phosphorylation or activity, in cells overexpressing constitutively active Rac1, forskolin enhanced ERK activity. Overexpression of either Rac1 Q61L or Rac1 Q61L-A1 (a mutant with reduced RIIβ interaction) enhanced basal PAK4 phosphorylation, but only the Rac1 Q61L–expressing cells exhibited enhanced cAMP-mediated ERK and PAK4 phosphorylation. An ovarian cancer cell line, OVCAR3, exhibited increased PAK and ERK phosphorylation in response to either forskolin or a β-adrenergic receptor agonist, and these responses were blocked by a PKA inhibitor or membrane-permeable peptides that disrupt AKAP-PKA interactions. Thus, the authors propose that by anchoring PKA and the downstream target PAK, Rac1 may enable cAMP-mediated enhancement of ERK signaling.

V. A. Bachmann, A. Riml, R. G. Huber, G. S. Baillie, K. R. Liedl, T. Valovka, E. Stefan, Reciprocal regulation of PKA and Rac signaling. Proc. Natl. Acad. Sci. U.S.A. 110, 8531–8536 (2013). [Abstract] [Full Text]