cAMP-stimulated Protein Phosphatase 2A Activity Associated with Muscle A Kinase-anchoring Protein (mAKAP) Signaling Complexes Inhibits the Phosphorylation and Activity of the cAMP-specific Phosphodiesterase PDE4D3

Abstract

The concentration of the second messenger cAMP is tightly controlled in cells by the activity of phosphodiesterases. We have previously described how the protein kinase A-anchoring protein mAKAP serves as a scaffold for the cAMP-dependent protein kinase PKA and the cAMP-specific phosphodiesterase PDE4D3 in cardiac myocytes. PKA and PDE4D3 constitute a negative feedback loop whereby PKA-catalyzed phosphorylation and activation of PDE4D3 attenuate local cAMP levels. We now show that protein phosphatase 2A (PP2A) associated with mAKAP complexes is responsible for reversing the activation of PDE4D3 by catalyzing the dephosphorylation of PDE4D3 serine residue 54. Mapping studies reveal that a C-terminal mAKAP domain (residues 2085-2319) binds PP2A. Binding to mAKAP is required for PP2A function, such that deletion of the C-terminal domain enhances both base-line and forskolin-stimulated PDE4D3 activity. Interestingly, PP2A holoenzyme associated with mAKAP complexes in the heart contains the PP2A targeting subunit B56␦. Like PDE4D3, B56␦ is a PKA substrate, and PKA phosphorylation of mAKAP-bound B56␦ enhances phosphatase activity 2-fold in the complex. Accordingly, expression of a B56␦ mutant that cannot be phosphorylated by PKA results in increased PDE4D3 phosphorylation. Taken together, our findings demonstrate that PP2A associated with mAKAP complexes promotes PDE4D3 dephosphorylation, serving both to inhibit PDE4D3 in unstimulated cells and also to mediate a cAMP-induced positive feedback loop following adenylyl cyclase activation and B56␦ phosphorylation. In general, PKA⅐PP2A⅐mAKAP complexes exemplify how protein kinases and phosphatases may participate in molecular signaling complexes to dynamically regulate localized intracellular signaling.

A-kinase anchoring proteins: scaffolding proteins in the heart

“…Dephosphorylation of PDE4D3 is mediated by the phosphatase PP2A that is also associated with mAKAP␤ ( Fig. 3A) (18). Collectively, these findings suggest that the mAKAP complex generates cyclic pulses of PKA activity, a hypothesis that was confirmed experimentally by live cell imaging studies (19).…”

B56δ-related protein phosphatase 2A dysfunction identified in patients with intellectual disability

“…Such dysphosphorylation may have far-reaching consequences for regulation of localized signaling. One example could be the signaling complex scaffolded by the neural variant of the cAMP-dependent PKA anchoring protein mAKAP, that binds B56δ and several other phosphatases (PP1, PP2B) and kinases (PKA, PDK1, RSK3, ERK5) (26,27). Dephosphorylation of PP2A-B56 substrates may also be hindered, e.g., the transcription factor and PKA-substrate HAND (28), the neural cyclin-dependent kinase 5 (CDK5) activator CDK5R1 (21), and DARPP-32 (24,25).…”

The Large Isoforms of A-Kinase Anchoring Protein 18 Mediate the Phosphorylation of Inhibitor-1 by Protein Kinase A and the Inhibition of Protein Phosphatase 1 Activity

“…For example, the direct association between AKAP220 and PP1 inhibits phosphatase activity (Schillace et al, 2001). In addition, mAKAP binds to PP2A and mediates a cAMP-induced increase of phosphatase activity through PKA phosphorylation of the PP2A-B56␦ subunit (Dodge-Kafka et al, 2010). In contrast to these other complexes, PP1 bound to AKAP18 is active, and PKA phosphorylation of the phosphatase's regulator results in its inhibition.…”

“…As a control, I-1 did not associate with mAKAP, another muscle-enriched AKAP involved in the induction of cardiac hypertrophy (Supplementary Fig. 1) (Dodge-Kafka et al, 2010). The molecular mass of the intense, 45-kDa band implied that the major isoform pulled down by GST-I-1 was either AKAP18␦ or AKAP18␥.…”