Regulation of PKA binding to AKAPs in the heart: alterations in human heart failure.

BACKGROUND cAMP-dependent protein kinase (PKA) regulates a broad range of cellular responses in the cardiac myocyte. Downstream regulation of the PKA pathway is mediated by a class of scaffolding proteins called A-kinase anchoring proteins (AKAPs), which sequester PKA to specific subcellular locations through binding to its regulatory subunit (R). However, the effect of RII autophosphorylation on AKAP binding and the degree of RII autophosphorylation in failing and nonfailing human hearts remains unknown. METHODS AND RESULTS We investigated AKAP-RII binding by overlay analysis and surface plasmon resonance spectroscopy and measured RII autophosphorylation in human hearts by backphosphorylation. Binding of Ht31 peptide (representing the RII-binding region of AKAPs) to cardiac RII was increased approximately 145% (P<0.01) for autophosphorylated RII relative to unphosphorylated control. By surface plasmon resonance, RII autophosphorylation significantly increased binding affinity to Ht31 by approximately 200% (P<0.01). Baseline PKA-dependent phosphorylation of RII was significantly decreased approximately 30% (P<0.05) in human hearts with dilated cardiomyopathy compared with nonfailing controls. CONCLUSIONS These results suggest that AKAP binding of PKA in the heart is regulated by RII autophosphorylation. Therefore AKAP targeting of PKA may be reduced in patients with end-stage heart failure. This mechanism may be responsible for the decreased cAMP-dependent phosphorylation of proteins in dilated cardiomyopathy that we and others have previously observed.

[1]  J. Scott,et al.  mAKAP: an A-kinase anchoring protein targeted to the nuclear membrane of differentiated myocytes. , 1999, Journal of cell science.

[2]  S. Taylor,et al.  The consequences of introducing an autophosphorylation site into the type I regulatory subunit of cAMP-dependent protein kinase. , 1989, The Journal of biological chemistry.

[3]  L. Langeberg,et al.  Cloning and Characterization of A-kinase Anchor Protein 100 (AKAP100) , 1995, The Journal of Biological Chemistry.

[4]  O. Rosen,et al.  Mechanism of self-phosphorylation of adenosine 3':5'-monophosphate-dependent protein kinase from bovine cardiac muscle. , 1976, The Journal of biological chemistry.

[5]  J. Scott,et al.  Protein Kinase A Anchoring* , 1997, The Journal of Biological Chemistry.

[6]  S. Green,et al.  cAMP-Dependent Regulation of Cardiac L-Type Ca2+ Channels Requires Membrane Targeting of PKA and Phosphorylation of Channel Subunits , 1997, Neuron.

[7]  D. Johnson,et al.  Regulation of cAMP-dependent protein kinase: enzyme activation without dissociation. , 1995, Biochemistry.

[8]  J. Scott,et al.  Mutational Analysis of the A-Kinase Anchoring Protein (AKAP)-binding Site on RII , 1996, The Journal of Biological Chemistry.

[9]  M. Bond,et al.  Troponin I phosphorylation and myofilament calcium sensitivity during decompensated cardiac hypertrophy. , 1998, American journal of physiology. Heart and circulatory physiology.

[10]  D. Walsh,et al.  Fluorescence resonance energy transfer within a heterochromatic cAMP-dependent protein kinase holoenzyme under equilibrium conditions: new insights into the conformational changes that result in cAMP-dependent activation. , 1993, Biochemistry.

[11]  E. Krebs,et al.  Primary structure of the regulatory subunit of type II cAMP-dependent protein kinase from bovine cardiac muscle. , 1982, Proceedings of the National Academy of Sciences of the United States of America.

[12]  O. Rosen,et al.  Effect of cAMP and ATP on the reassociation of phosphorylated and nonphosphorylated subunits of the cAMP-dependent protein kinase from bovine cardiac muscle. , 1977, The Journal of biological chemistry.

[13]  Susan S. Taylor,et al.  cAMP-dependent protein kinase: framework for a diverse family of regulatory enzymes. , 1990, Annual review of biochemistry.

[14]  A. Mildvan,et al.  Magnetic resonance studies of the effect of the regulatory subunit on metal and substrate binding to the catalytic subunit of bovine heart protein kinase. , 1980, The Journal of biological chemistry.

[15]  I. Fraser,et al.  Association of the type II cAMP-dependent protein kinase with a human thyroid RII-anchoring protein. Cloning and characterization of the RII-binding domain. , 1992, The Journal of biological chemistry.

[16]  F. Schoen,et al.  Deficient production of cyclic AMP: pharmacologic evidence of an important cause of contractile dysfunction in patients with end-stage heart failure. , 1987, Circulation.

[17]  M. Bond,et al.  Protein kinase A (PKA)-dependent troponin-I phosphorylation and PKA regulatory subunits are decreased in human dilated cardiomyopathy. , 1999, Circulation.

[18]  J. Drazba,et al.  A-kinase Anchoring Protein 100 (AKAP100) is Localized in Multiple Subcellular Compartments in the Adult Rat Heart , 1998, The Journal of cell biology.

[19]  D. Dransfield,et al.  Ezrin is a cyclic AMP‐dependent protein kinase anchoring protein , 1997, The EMBO journal.

[20]  S. Taylor,et al.  The regulatory subunit of neural cAMP-dependent protein kinase II represents a unique gene product. , 1985, The Journal of biological chemistry.

[21]  J. Scott,et al.  Type II regulatory subunits are not required for the anchoring-dependent modulation of Ca2+ channel activity by cAMP-dependent protein kinase. , 1997, Proceedings of the National Academy of Sciences of the United States of America.

[22]  D C Harrison,et al.  Decreased catecholamine sensitivity and beta-adrenergic-receptor density in failing human hearts. , 1982, The New England journal of medicine.

[23]  M. Bond,et al.  Troponin I phosphorylation in spontaneously hypertensive rat heart: effect of beta-adrenergic stimulation. , 1997, The American journal of physiology.

[24]  P. Allen,et al.  Troponin I phosphorylation in the normal and failing adult human heart. , 1997, Circulation.