An Adenylyl Cyclase-mAKAPβ Signaling Complex Regulates cAMP Levels in Cardiac Myocytes

Michael S Kapiloff,Leslie A Piggott,Rachna Sadana,Jinliang Li,Lorena A Heredia,Edward Henson,Riad Efendiev,Carmen W Dessauer

doi:10.1074/jbc.m109.030072

Abstract

Protein kinase A-anchoring proteins (AKAPs) play important roles in the compartmentation of cAMP signaling, anchoring protein kinase A (PKA) to specific cellular organelles and serving as scaffolds that assemble localized signaling cascades. Although AKAPs have been recently shown to bind adenylyl cyclase (AC), the functional significance of this association has not been studied. In cardiac myocytes, the muscle protein kinase A-anchoring protein beta (mAKAPbeta) coordinates cAMP-dependent, calcium, and MAP kinase pathways and is important for cellular hypertrophy. We now show that mAKAPbeta selectively binds type 5 AC in the heart and that mAKAPbeta-associated AC activity is absent in AC5 knock-out hearts. Consistent with its known inhibition by PKA phosphorylation, AC5 is inhibited by association with mAKAPbeta-PKA complexes. AC5 binds to a unique N-terminal site on mAKAP-(245-340), and expression of this peptide disrupts endogenous mAKAPbeta-AC association. Accordingly, disruption of mAKAPbeta-AC5 complexes in neonatal cardiac myocytes results in increased cAMP and hypertrophy in the absence of agonist stimulation. Taken together, these results show that the association of AC5 with the mAKAPbeta complex is required for the regulation of cAMP second messenger controlling cardiac myocyte hypertrophy.

Highlights

MAKAP␤, expressed in striated myocytes, is one of two known splice variants encoded by the single mAKAP (AKAP6) gene [7]
MAKAP␤ serves as the scaffold for a multimolecular signaling complex that in addition to protein kinase A (PKA) includes the ryanodine receptor (RyR2), the protein phosphatases PP2A and calcineurin, phosphodiesterase 4D3 (PDE4D3), exchange protein activated by cAMP (Epac1), ERK5, and MEK5 mitogen-activated protein kinases, molecules implicated in the regulation of cardiac hypertrophy [4, 7,8,9,10,11,12,13]. mAKAP␤ complexes facilitate crosstalk between MAP kinase, calcium, and cAMP signaling pathways, permitting feedback inhibition of cAMP levels and the dynamic regulation of PKA and ERK5 activity (4, 9 –13)
We show that adenylyl cyclase (AC) binds directly to mAKAP␤ in cardiac myocytes, establishing mAKAP␤ as the first identified AKAP to bind directly all of the components required for cAMP signaling, including enzymes for cAMP synthesis (AC), downstream signaling (PKA), and degradation (PDE4D3). (Epac1 binds mAKAP␤ indirectly though PDE4D3 [11].) AC binding does not appear to compete with the other components of mAKAP complexes for which binding sites have been mapped, as AC [275– 340], nesprin-1␣ (1065–1286) [4], PDE4D3 (1286 –1831) [10], PKA (2055–2072)(8),andphosphatidyldependent kinase 1 (PDK1) in brain [227–232] [7] bind distinct mAKAP domains

Summary

MATERIALS AND METHODS

Reagents—Antibodies were obtained as described under supplemental methods. The mAKAP␤ isoform is identical to residues 245–2314 of mAKAP␣. All mutants are numbered according to mAKAP␣. The mammalian expression plasmids for wild type and ⌬PKA (deletion of residues 2053–2073) mAKAP␤ are as previously described [4]. Adenoviruses that express C-terminally Myc-tagged mAKAP 1–587, 245–340 (Myc-AC-BD), 245–587, 1286 –1833, and 1835–2312 fragments were generated using the pTRE shuttle vector and the Adeno-X Tet-off System (Clontech) as previously described for

Cardiac myocytes or transfected

Samples were washed three times

Findings

DISCUSSION