Abstract
Protein kinase A-anchoring proteins (AKAPs) influence fundamental cellular processes by directing the cAMP-dependent protein kinase (PKA) toward its intended substrates. In this report we describe the identification and characterization of a ternary complex of AKAP220, the PKA holoenzyme, and the IQ domain GTPase-activating protein 2 isoform (IQGAP2) that is enriched at cortical regions of the cell. Formation of an IQGAP2-AKAP220 core complex initiates a subsequent phase of protein recruitment that includes the small GTPase Rac. Biochemical and molecular biology approaches reveal that PKA phosphorylation of Thr-716 on IQGAP2 enhances association with the active form of the Rac GTPase. Cell-based experiments indicate that overexpression of an IQGAP2 phosphomimetic mutant (IQGAP2 T716D) enhances the formation of actin-rich membrane ruffles at the periphery of HEK 293 cells. In contrast, expression of a nonphosphorylatable IQGAP2 T716A mutant or gene silencing of AKAP220 suppresses formation of membrane ruffles. These findings imply that IQGAP2 and AKAP220 act synergistically to sustain PKA-mediated recruitment of effectors such as Rac GTPases that impact the actin cytoskeleton.
Highlights
JUNE 24, 2011 VOLUME 286 NUMBER 25 rified with RII subunits on cAMP-agarose affinity columns [7]
We show that IQ domain GTPase-activating protein 2 (IQGAP2) and AKAP220 form a core complex that favors PKAdependent recruitment of the small GTPase Rac to augment certain aspects of actin remodeling
The detection of peptides derived from AKAP220 and the regulatory (R) and catalytic (C) subunits of protein kinase (PKA) served as internal controls
Summary
JUNE 24, 2011 VOLUME 286 NUMBER 25 rified with RII subunits on cAMP-agarose affinity columns [7]. Immunoprecipitation of endogenous AKAP220 from HEK 293 cells resulted in the co-fractionation of IQGAP2 and C subunit of PKA as assessed by immunoblotting (Fig. 1A, top and middle panels, lane 2). Depletion of the anchoring protein resulted in 3.8-fold (n ϭ 4) reduction in IQGAP2-associated PKA activity compared with scrambled siRNA controls (Fig. 1F, compare columns 1 and 3).
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