The PKARIα Subunit of Protein Kinase A Modulates the Activation of p90RSK1 and Its Function

Abstract

Cyclic AMP-dependent protein kinase (PKA) 3 plays a pivotal role in manifesting an array of biological actions ranging from cell proliferation and tumorigenesis to increased inotropic and chronotropic effects in the heart as well as regulation of long term potentiation and memory. The PKA holoenzyme is a heterotetramer and consists of two catalytic (PKAc) subunits bound to a dimer of regulatory subunits. To date, four isoforms of the PKAc (PKAc␣, PKAc␤, PKAc␥, and PKAc␦) and four isoforms of the regulatory subunits (RI␣, RI␤, RII␣, and RII␤) have been described (1). The various isoforms of PKA subunits are expressed differently in a tissue-and cell-specific manner (2). In addition to binding and inhibiting the activity of PKAc via their pseudo substrate region (3-6), the R subunits also interact with PKA-anchoring proteins (AKAPs) and facilitate the localization of PKA in specific subcellular compartments (7,8). More than 50 AKAP family members have been described, and although most of these have a higher affinity for the RII subunits (9), certain AKAPs such as D-AKAP1 and D-AKAP2 preferentially bind the PKARI␣ subunit (10 -12). Because the AKAPs also bind other signaling molecules such as phosphatases (PP2B) and kinases (protein kinase C), they act as scaffolds to organize and integrate specific signaling events within specific compartments in the cells (7,8,13,14).We have shown that the PKARI␣ and PKAc␣ subunits of PKA interact with the inactive and active forms of p90 RSK1 (RSK1), respectively (15). Binding of inactive RSK1 to PKARI␣ decreases the interactions between PKARI␣ and PKAc, whereas the association of active RSK1 with PKAc increases interactions between PKARI␣ and PKAc such that larger amounts of cAMP are required to activate PKAc in the presence of active RSK1 (15). Moreover, the indirect (via subunits of PKA) interaction of RSK1 with AKAPs is required for the nuclear localization of active RSK1 (15), and disruption of the interactions of RSK1⅐PKA complex from AKAPs results in increased cytoplasmic distribution of active RSK1 with a concomitant increase in phosphorylation of its cytosolic substrates such as BAD and reduced cellular apoptosis (15). These findings show the functional and biological significance of RSK1⅐ PKA⅐AKAP interactions.Besides inhibiting PKAc activity, the physiological role of PKARI␣ is underscored by the findings that mutations in the PKAR1A gene that result in haploinsufficiency of PKARI␣ are

p90 Ribosomal S6 Kinase 1 (RSK1) and the Catalytic Subunit of Protein Kinase A (PKA) Compete for Binding the Pseudosubstrate Region of PKAR1α

“…Our findings demonstrate that the NTK domain of RSK1 is the PKARI␣ binding site, and consistent with our previous reports that inactive RSK1 interacts with PKARI␣ (20,21), substitution of Ser-221 in the activation loop of RSK1 with negatively charged Asp residue abrogated this interaction. Additionally, our data show that the pseudosubstrate domain of PKARI␣ comprising the sequence ( 93 RRRRGAI 99 ) is necessary for association with RSK1; Ala-98 represents the phosphorylation site (P site) if it were Thr or Ser.…”

“…Moreover, RSK1 competed with PKAc for binding to PKARI␣ and modulated endogenous PKAc activity. Akin to our recent report showing that silencing of PKARI␣ activates RSK1 (21), the dissociation of the PKARI␣/RSK1 interaction by a cell-permeable peptide corresponding to the pseudosubstrate region of PKARI␣ mimicked the silencing of PKARI␣ and, by increasing the amount of active RSK1, augmented the anti-apoptotic actions of RSK1. These data demonstrate that the catalytic cleft of the inactive NTK interacts with the pseudosubstrate region of PKARI␣ and that this mode of association is necessary for regulating the activation of RSK1 and its biological actions as well as regulating endogenous PKAc activity.…”

“…The catalytic cleft residues in PKAc also contact the pseudosubstrate site on PKARI␣ and the cognate inhibitory region on PKARII with a resultant inhibition of PKAc activity (7,8,28). In the case of RSK1, however, the inactive, but not the active form of RSK1 associates with PKARI␣ (20,21), and substitution of Ser-221 with Asp abrogates this interaction, suggesting that the Ser-221 phosphorylation or a negative charge at this site alters the orientation of the residues in the NTK catalytic cleft, which interacts with the pseudosubstrate site on PKARI␣. This explains why active RSK1 does not interact with PKARI␣ and also explains the inability of PKARI␣ to inhibit the catalytic activity of active RSK1 (see the supplemental data in Chaturvedi et al (20)).…”

“…Recently, using a variety of approaches including silencing of PKARI␣, we have shown that the PKARI␣/RSK1 interaction is necessary to bring RSK1 in proximity of PP2Ac on AKAPs such as D-AKAP1 (21). By bringing RSK1 in proximity of PP2Ac, PKARI␣ ensures that the RSK1 is dephosphorylated and maintained in its inactive form in the absence of any growth factors (21). Therefore, we reasoned that the cell-permeable, palmitoylated peptide Wt-PS that competes with PKARI␣ for association with RSK1 (Fig.…”

“…anti-apoptotic actions) of RSK1 (20). Recently, we have also shown that the indirect association of RSK1 with D-AKAP1 brings the RSK1 in proximity of the catalytic subunit of protein phosphatase 2A (PP2Ac) that is bound to D-AKAP1, and this permits PP2Ac to dephosphorylate and regulate RSK1 activation (21). Moreover, we have recently shown that the last 13 residues on RSK1 form the PKAc binding site and that autophosphorylation of Ser-732 by NTK of RSK1 within this region is required for interactions with PKAc (22).…”