Dynamics of cAMP-Dependent Protein Kinase

Johnson, David A.; Akamine, Pearl; Radzio‐Andzelm, Elzbieta; Madhusudan, M; Taylor, Susan S.

doi:10.1021/cr000226k

Cited by 376 publications

(474 citation statements)

References 173 publications

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“…Generally, a protein segment subject to phosphorylation interacts with the active site of the kinase in an extended conformation (48) and thus the phosphorylation motif is located in a flexible loop, or at the N or C terminus of a protein (48). Therefore if we assume Ser-38 is a phosphorylation site, this segment of SERCA2 must be surface exposed and mobile.…”

Section: Discussionmentioning

confidence: 99%

Critical Evaluation of Cardiac Ca2+-ATPase Phosphorylation on Serine 38 Using a Phosphorylation Site-specific Antibody

Rodríguez¹,

Jackson²,

Colyer

2004

Journal of Biological Chemistry

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The phosphorylation of the cardiac muscle isoform of the sarcoplasmic reticulum (SR) Ca 2؉ -ATPase (SERCA2a) on serine 38 has been described as a regulatory event capable of very significant enhancement of enzyme activity (Hawkins, C., Xu, A., and Narayanan, N. (1994) J. Biol. Chem. 269, 31198 -31206). Independent confirmation of these observations has not been forthcoming. This study has utilized a polyclonal antibody specific for the phosphorylated serine 38 epitope on the Ca 2؉ -ATPase to evaluate the phosphorylation of SERCA2a in isolated sarcoplasmic reticulum vesicles and isolated rat ventricular myocytes. A quantitative Western blot approach failed to detect serine 38-phosphorylated Ca 2؉ -ATPase in either kinase-treated sarcoplasmic reticulum vesicles or suitably stimulated cardiac myocytes. Calibration standards confirmed that the detection sensitivity of assays was adequate to detect Ser-38 phosphorylation if it occurred on at least 1% of Ca 2؉ -ATPase molecules in SR vesicle experiments or on at least 0.1% of Ca 2؉ -ATPase molecules in cardiac myocytes. The failure to detect a phosphorylated form of the Ca 2؉ -ATPase in either preparation (isolated myocyte, purified sarcoplasmic reticulum vesicles) suggests that Ser-38 phosphorylation of the Ca 2؉ -ATPase is not a significant regulatory feature of cardiac Ca 2؉ homeostasis.Regulation of Ca 2ϩ sequestration by cardiac sarcoplasmic reticulum has been identified as a key control point in cardiac muscle contraction (1, 2). Stimulation of the rate of Ca 2ϩ uptake occurs during exercise, or following ␤-adrenergic stimulation (3) and is associated with an enhanced force of contraction and an increased rate of relaxation. This accounts for much of the positive inotropic and positive lusitropic effects of these interventions. In contrast, Ca 2ϩ sequestration by cardiac SR 1 is abnormally slow in the muscle of individuals with heart failure (4 -6). In animal studies, normalization of the rate of Ca 2ϩ sequestration has been shown to prevent progression of heart failure (7) and thus the molecular mechanism of control of Ca 2ϩ transport into the sarcoplasmic reticulum has become a focus for research into purposeful therapies to combat human heart failure (7,8).Ca 2ϩ transport into cardiac SR is an enzymatic process performed by the (Ca 2ϩ -Mg 2ϩ )-ATPase (9) (or SERCA2a, Ref. 10), a member of the P-type ATPase family (11). The transport process involves the movement of two Ca 2ϩ from the cytoplasm into the lumen of the SR following the hydrolysis of a single molecule of ATP (12), although significantly lower coupling efficiencies have been measured empirically (13). The reaction cycle is relatively slow and thus a large number of SERCA2 molecules are expressed in cardiac muscle (up to 45% of total SR protein content, 14) to achieve the rates of Ca 2ϩ sequestration required by the kinetics of contraction and relaxation.Regulation of Ca 2ϩ transport into the SR occurs on an acute time scale (seconds to minutes) through the transient modification of the proteins inv...

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Section: Discussionmentioning

confidence: 99%

Critical Evaluation of Cardiac Ca2+-ATPase Phosphorylation on Serine 38 Using a Phosphorylation Site-specific Antibody

Rodríguez¹,

Jackson²,

Colyer

2004

Journal of Biological Chemistry

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“…In the last decade, a full appreciation for the conformational complexity of these enzymes has been developed (11,18). The essential kinase core has been crystallized in many different conformations (19)(20)(21) that are distinguished by rotations between the ATP and substrate-binding lobes and by movements in various secondary structural elements. These properties have become very important to investigate on practical grounds because many known inhibitors can induce different conformational states in the enzymes that are important for inhibitor design.…”

mentioning

confidence: 99%

“…Such observations raise the exciting possibility that conformational changes in PKA may be coupled to an event that governs the net rate of protein phosphorylation. These early studies, however, lack a useful correlation between observed conformational changes, such as those observed in H-D exchange experiments (2) and the 3-dimensional structure of the enzyme (21).…”

mentioning

confidence: 99%

Ligand-induced global transitions in the catalytic domain of protein kinase A

Hyeon

Jennings²,

Adams³

et al. 2009

Proc. Natl. Acad. Sci. U.S.A.

112

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“…The Rsubunits are composed of an N-terminal dimerization/docking domain, a flexible linker that includes an autoinhibitory segment, and two tandem CBDs (CBD-A and CBD-B; Fig. 1b) (5). The CBD-A of the isoform I␣ of the R-subunit of PKA (RI␣) contains a noncontiguous ␣-subdomain, which mediates the interactions with the C-subunit and a contiguous ␤-subdomain that forms a ␤-sandwich and contains the cAMP binding pocket (i.e., the phosphate binding cassette or PBC) ( Fig.…”

mentioning

confidence: 99%

cAMP activation of PKA defines an ancient signaling mechanism

Das

Esposito

Abu-Abed

et al. 2007

Proc. Natl. Acad. Sci. U.S.A.

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115

106

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allostery ͉ NMR ͉ cyclic nucleotide binding domain T he cAMP binding domain (CBD) and cAMP are conserved from bacteria to humans as a ubiquitous signaling mechanism to translate extracellular stress signals into appropriate biological responses (1). The major receptor for cAMP in higher eukaryotes, cAMP-dependent PKA (2), is ubiquitous in mammalian cells where it exists in two forms: the inactive tetrameric holoenzyme and the active dissociated catalytic subunit (Csubunit). In the inactive holoenzyme, two C-subunits are bound to a dimeric regulatory subunit (R-subunit) (Fig. 1a). Upon binding cAMP, the R-subunits undergo a conformational change that unleashes the active C-subunits (3, 4). The Rsubunits are composed of an N-terminal dimerization/docking domain, a flexible linker that includes an autoinhibitory segment, and two tandem CBDs (CBD-A and CBD-B; Fig. 1b) (5). The CBD-A of the isoform I␣ of the R-subunit of PKA (RI␣) contains a noncontiguous ␣-subdomain, which mediates the interactions with the C-subunit and a contiguous ␤-subdomain that forms a ␤-sandwich and contains the cAMP binding pocket (i.e., the phosphate binding cassette or PBC) ( Fig. 1 c and d) (6).Crystal structures of CBD-A of RI␣ in its cAMP-(6) and C-bound (7) states have revealed two very different conformations, highlighting the conformational plasticity of this ancient domain. Although these static crystal structures define two stable end points, questions remain about the allosteric control of the reversible shuttling between the two states. How does the signal generated by cAMP binding to the PBC (Fig. 1 c and d) propagate through a long-range allosteric network that spans both ␣-and ␤-subdomains? Previous analyses (8-16) have led to the proposal of an initial allosteric model in which the ␣-and ␤-subdomains are directly coupled to each other through a salt bridge between E200 and R241 and also possibly through a hydrophobic hinge defined by the L203, I204, and Y229 side-chain cluster (9,11,12). However, mutations (17), sequence conservation analyses (1), structurebased comparisons (1), and genetic screening (18,19) indicate that several other sites, which are not accounted for by the existing model, are also likely to play an active role in the cAMP-mediated activation of PKA. To comprehensively understand this allosteric mechanism, it is therefore necessary to elucidate at high resolution how cAMP remodels the free energy landscape of CBD-A, which serves as the central controlling unit of PKA. For this purpose, we have investigated by NMR RI␣ (residues 119-244), a construct that spans both ␣-and ␤-subdomains of CBD-A and retains high-

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Dynamics of cAMP-Dependent Protein Kinase

Cited by 376 publications

References 173 publications

Critical Evaluation of Cardiac Ca2+-ATPase Phosphorylation on Serine 38 Using a Phosphorylation Site-specific Antibody

Critical Evaluation of Cardiac Ca2+-ATPase Phosphorylation on Serine 38 Using a Phosphorylation Site-specific Antibody

Ligand-induced global transitions in the catalytic domain of protein kinase A

cAMP activation of PKA defines an ancient signaling mechanism

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