Protein Kinase A Opposes the Phosphorylation-dependent Recruitment of Glycogen Synthase Kinase 3ß to A-kinase Anchoring Protein 220.

The proximity of an enzyme to its substrate can influence rate and magnitude of catalysis. A-kinase anchoring protein 220 (AKAP220) is a multivalent anchoring protein that can sequester a variety of signal transduction enzymes. These include protein kinase A (PKA) and glycogen synthase kinase 3ß (GSK3ß). Using a combination of molecular and cellular approaches we show that GSK3ß phosphorylation of Thr-1132 on AKAP220 initiates recruitment of this kinase into the enzyme scaffold. We also find that AKAP220 anchors GSK3ß and its substrate ß-catenin in membrane ruffles. Interestingly, GSK3ß can be released from the multienzyme complex in response to PKA phosphorylation on serine 9, which suppresses GSK3ß activity. The signaling scaffold may enhance this regulatory mechanism, as AKAP220 has the capacity to anchor two PKA holoenzymes. Site 1 on AKAP220 (residues 610-623) preferentially interacts with RII, whereas site 2 (residues 1633-1646) exhibits a dual specificity for RI and RII. In vitro affinity measurements revealed that site 2 on AKAP220 binds RII with ∼10-fold higher affinity than site 1. Occupancy of both R subunit binding sites on AKAP220 could provide a mechanism to amplify local cAMP responses and enable cross-talk between PKA and GSK3ß.

Anchored phosphatases modulate glucose homeostasis.

Endocrine release of insulin principally controls glucose homeostasis. Nutrient-induced exocytosis of insulin granules from pancreatic ß-cells involves ion channels and mobilization of Ca(2+) and cyclic AMP (cAMP) signalling pathways. Whole-animal physiology, islet studies and live-ß-cell imaging approaches reveal that ablation of the kinase/phosphatase anchoring protein AKAP150 impairs insulin secretion in mice. Loss of AKAP150 impacts L-type Ca(2+) currents, and attenuates cytoplasmic accumulation of Ca(2+) and cAMP in ß-cells. Yet surprisingly AKAP150 null animals display improved glucose handling and heightened insulin sensitivity in skeletal muscle. More refined analyses of AKAP150 knock-in mice unable to anchor protein kinase A or protein phosphatase 2B uncover an unexpected observation that tethering of phosphatases to a seven-residue sequence of the anchoring protein is the predominant molecular event underlying these metabolic phenotypes. Thus anchored signalling events that facilitate insulin secretion and glucose homeostasis may be set by AKAP150 associated phosphatase activity.

Phosphorylation-dependent regulation of cyclin D1 and cyclin A gene transcription by TFIID subunits TAF1 and TAF7.

The largest transcription factor IID (TFIID) subunit, TBP-associated factor 1 (TAF1), possesses protein kinase and histone acetyltransferase (HAT) activities. Both enzymatic activities are essential for transcription from a subset of genes and G(1) progression in mammalian cells. TAF7, another TFIID subunit, binds TAF1 and inhibits TAF1 HAT activity. Here we present data demonstrating that disruption of the TAF1/TAF7 interaction within TFIID by protein phosphorylation leads to activation of TAF1 HAT activity and stimulation of cyclin D1 and cyclin A gene transcription. Overexpression and small interfering RNA knockdown experiments confirmed that TAF7 functions as a transcriptional repressor at these promoters. Release of TAF7 from TFIID by TAF1 phosphorylation of TAF7 increased TAF1 HAT activity and elevated histone H3 acetylation levels at the cyclin D1 and cyclin A promoters. Serine-264 of TAF7 was identified as a substrate for TAF1 kinase activity. Using TAF7 S264A and S264D phosphomutants, we determined that the phosphorylation state of TAF7 at S264 influences the levels of cyclin D1 and cyclin A gene transcription and promoter histone H3 acetylation. Our studies have uncovered a novel function for the TFIID subunit TAF7 as a phosphorylation-dependent regulator of TAF1-catalyzed histone H3 acetylation at the cyclin D1 and cyclin A promoters.

AKAP220 protein organizes signaling elements that impact cell migration.

Cell movement requires the coordinated reception, integration, and processing of intracellular signals. We have discovered that the protein kinase A anchoring protein AKAP220 interacts with the cytoskeletal scaffolding protein IQGAP1 to influence cell motility. AKAP220/IQGAP1 networks receive and integrate calcium and cAMP second messenger signals and position signaling enzymes near their intended substrates at leading edges of migrating cells. IQGAP1 supports calcium/calmodulin-dependent association of factors that modulate microtubule dynamics. AKAP220 suppresses GSK-3ß and positions this kinase to allow recruitment of the plus-end microtubule tracking protein CLASP2. Gene silencing of AKAP220 alters the rate of microtubule polymerization and the lateral tracking of growing microtubules and retards cell migration in metastatic human cancer cells. This reveals an unappreciated role for this anchored kinase/microtubule effector protein network in the propagation of cell motility.

Anchored protein kinase A recruitment of active Rac GTPase.

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.