Glycogen Synthase Kinase 3β Interaction Protein Functions as an A-kinase Anchoring Protein

Abstract

A-kinase anchoring proteins (AKAPs) include a family of scaffolding proteins that target protein kinase A (PKA) and other signaling proteins to cellular compartments and thereby confine the activities of the associated proteins to distinct regions within cells. AKAPs bind PKA directly. The interaction is mediated by the dimerization and docking domain of regulatory subunits of PKA and the PKA-binding domain of AKAPs. Analysis of the interactions between the dimerization and docking domain and various PKA-binding domains yielded a generalized motif allowing the identification of AKAPs. Our bioinformatics and peptide array screening approaches based on this signature motif identified GSKIP (glycogen synthase kinase 3␤ interaction protein) as an AKAP. GSKIP directly interacts with PKA and GSK3␤ (glycogen synthase kinase 3␤). It is widely expressed and facilitates phosphorylation and thus inactivation of GSK3␤ by PKA. GSKIP contains the evolutionarily conserved domain of unknown function 727. We show here that this domain of GSKIP and its vertebrate orthologues binds both PKA and GSK3␤ and thereby provides a mechanism for the integration of PKA and GSK3␤ signaling pathways. A-kinase anchoring proteins (AKAPs)3 are a family of scaffoldingproteinscharacterizedbytheabilitytobindcAMPdependent protein kinase (protein kinase A (PKA)). They tether PKA in the vicinity of its substrates, thereby facilitating their phosphorylation. In addition, AKAPs bind further signaling molecules, including other protein kinases (e.g. protein kinase C and protein kinase D), phosphodiesterases (e.g. PDE4D), and protein phosphatases (e.g. PP1 and PP2B/calcineurin). A few AKAPs possess catalytic activity. For example, AKAP-Lbc is a Rho guanine nucleotide exchange factor (1-3). Thus, AKAPs assemble multiprotein complexes and thereby coordinate cellular signaling. AKAPs are required for many cellular processes, including vasopressin-mediated water reabsorption in renal principal cells and ␤-adrenoreceptor-dependent increases of cardiac myocyte contractility (2, 4, 5).The PKA holoenzyme consists of a dimer of regulatory RI or RII subunits and two catalytic subunits, each bound to one R subunit. Upon binding of two molecules of cAMP to each R subunit, the catalytic subunits dissociate and phosphorylate their substrates (6, 7). The interaction of AKAPs with PKA is mediated by the PKA-anchoring domain of AKAPs and the dimerization and docking (DD) domain of R subunit dimers. Because most AKAPs preferentially anchor RII subunits, PKAanchoring domains are termed RII-binding domains (RIIBD). These domains are structurally conserved amphipathic helices, 14 -18 amino acid residues in length (8, 9). Based on recently described determinants of the RIIBD/DD domain interaction (8 -10), we developed a bioinformatics and peptide array screening approach to identify new AKAPs. For one of the discovered proteins, GSKIP (GSK3␤ interaction protein), we show that it functions as an AKAP.Mammalian cells express two isoforms of glycogen synthase kinase-3 (GSK3), GSK...

Pharmacological Interference With Protein-protein Interactions of Akinase Anchoring Proteins as a Strategy for the Treatment of Disease

“…This AKAP signature motif was used to screen protein databases for new AKAPs. This approach identified GSK3β interaction protein (GSKIP) as an AKAP [223]. Recently, another bioinformatics tool for the mapping of PKA binding domains and the prediction of novel AKAPs, THAHIT (the AKAP/amphipathic helix identification tool), was introduced and a list of new RIα and RIIα binding domains for existing AKAPs was published suggesting that various AKAPs are D--AKAPs [224].…”

“…The β--sheet, in turn, is followed by an α--helix that binds GSK3β (amino acids 116--139). GSKIP facilitates the phosphorylation of GSK3β at Serine 9 by PKA and thereby the inactivation of GSK3β [223].…”

“…However, this is relevant as AKAP--PKA interactions are apparently regulated by AKAP--inherent mechanisms. The β--sheet covering the AKB of GSKIP may have to be displaced prior to R subunit binding or for modulating the affinity of the interaction [223]. The binding affinity of AKAP18δ for PKA is influenced by its N terminus; a N--terminally truncated version lacking the first 123 amino acids binds RII subunits of PKA with higher affinity than the full--length protein: K D = 9 nM for AKAP18δ (124--353) and K D = 31 nM for full--length AKAP18δ [68,225].…”

“…A consensus sequence of polar and hydrophobic amino acids in conserved positions defines an AKAP signature motif: [222,223]. This AKAP signature motif was used to screen protein databases for new AKAPs.…”

Screening for Small Molecule Disruptors of AKAP–PKA Interactions

“…The α-helices dock with their hydrophobic phases into the D/D domains of the regulatory subunits of PKA. The conserved RII-binding domains have been used to identify novel AKAPs such as GSKIP [7] and were the basis for uncovering the involvement of AKAP-PKA interactions in defined processes. The latter has been achieved with peptides for disruption of the interactions.…”