Human heart failure is accompanied by altered protein kinase A subunit expression and post-translational state

β-Adrenergic receptor blockade reduces total mortality and all-cause hospitalizations in patients with heart failure (HF). Nonetheless, β-blockade does not halt disease progression, suggesting that cAMP-dependent protein kinase (PKA) signaling downstream of β-adrenergic receptor activation may persist through unique post-translational states. In this study, human myocardial tissue was used to examine the state of PKA subunits. As expected, total myosin binding protein-C phosphorylation and Ser23/24 troponin I phosphorylation significantly decreased in HF. Examination of PKA subunits demonstrated no change in type II regulatory (RIIα) or catalytic (Cα) subunit expression, although site specific RIIα (Ser96) and Cα (Thr197) phosphorylation were increased in HF. Further, the expression of type I regulatory subunit (RI) was increased in HF. Isoelectric focusing of RIα demonstrated up to three variants, consistent with reports that Ser77 and Ser83 are in vivo phosphorylation sites. Western blots with site-specific monoclonal antibodies showed increased Ser83 phosphorylation in HF. 8-fluo-cAMP binding by wild type and phosphomimic Ser77 and Ser83 mutant RIα proteins demonstrated reduced Kd for the double mutant as compared to WT RIα. Therefore, failing myocardium displays altered expression and post-translational modification of PKA subunits that may impact downstream signaling.     

Overexpression of the type II regulatory subunit of the cAMP-dependent protein kinase eliminates the type I holoenzyme in mouse cells

Mammalian tissues and cell lines express two major types of cAMP-dependent protein kinase, PKA-I and PKA-II, which can be distinguished at the molecular level by the presence of either type I or type II regulatory subunits in the holoenzyme. An expression vector for the mouse type II regulatory subunit (RII alpha) was transfected into ras-transformed NIH3T3 (R3T3) cells, which contain approximately equal amounts of both holoenzymes, PKA-I and PKA-II. In RII alpha-overexpressing R3T3 cells, PKA-II levels were increased, and the level of PKA-I declined. The decrease in PKA-I was dependent on the amount of RII alpha expressed, and at high levels of RII alpha expression, PKA-I was completely eliminated. In contrast, overexpression of the type I regulatory subunit (RI alpha) did not alter PKA isozyme levels. We propose that competition between RII alpha and RI alpha for a limited pool of catalytic subunit results in preferential assembly of PKA-II and that significant amounts of PKA-I are formed only if catalytic subunit is present in excess of the RII alpha subunit. The PKA-I isozyme, which is absent in untransformed 3T3 cells, is not essential for the transformed phenotype of R3T3 cells. RII alpha-overexpressing R3T3 cells that are devoid of PKA-I continued to exhibit a transformed phenotype including anchorage-independent growth. Overexpression of RII alpha provides a genetic approach that may prove useful in demonstrating specific functions for the two PKA isozymes in cAMP-dependent signal transduction pathways.     

Serotonin regulates the secretion and autocrine action of a neuropeptide to activate MAPK required for long-term facilitation in Aplysia

In Aplysia, long-term facilitation (LTF) of sensory neuron synapses requires activation of both protein kinase A (PKA) and mitogen-activated protein kinase (MAPK). We find that 5-HT through activation of PKA regulates secretion of the sensory neuron-specific neuropeptide sensorin, which binds autoreceptors to activate MAPK. Anti-sensorin antibody blocked LTF and MAPK activation produced by 5-HT and LTF produced by medium containing sensorin that was secreted from sensory neurons after 5-HT treatment. A single application of 5-HT followed by a 2 hr incubation with sensorin produced protein synthesis-dependent LTF, growth of new presynaptic varicosities, and activation of MAPK and its translocation into sensory neuron nuclei. Inhibiting PKA during 5-HT applications and inhibiting receptor tyrosine kinase or MAPK during sensorin application blocked both LTF and MAPK activation and translocation. Thus, long-term synaptic plasticity is produced when stimuli activate kinases in a specific sequence by regulating the secretion and autocrine action of a neuropeptide.     

Differential binding of cAMP-dependent protein kinase regulatory subunit isoforms Ialpha and IIbeta to the catalytic subunit

Limited trypsin digestion of type I cAMP-dependent protein kinase holoenzyme results in a proteolytic-resistant Delta(1-72) regulatory subunit core, indicating that interaction between the regulatory and catalytic subunits extends beyond the autoinhibitory site in the R subunit at the NH(2) terminus. Sequence alignment of the two R subunit isoforms, RI and RII, reveals a significantly sequence diversity at this specific region. To determine whether this sequence diversity is functionally important for interaction with the catalytic subunit, specific mutations, R133A and D328A, are introduced into sites adjacent to the active site cleft in the catalytic subunit. While replacing Arg(133) with Ala decreases binding affinity for RII, interaction between the catalytic subunit and RI is not affected. In contrast, mutant C(D328A) showed a decrease in affinity for binding RI while maintaining similar affinities for RII as compared with the wild-type catalytic subunit. These results suggest that sequence immediately NH(2)-terminal to the consensus inhibition site in RI and RII interacts with different sites at the proximal region of the active site cleft in the catalytic subunit. These isoform-specific differences would dictate a significantly different domain organization in the type I and type II holoenzymes.     

Mechanisms for generating the autonomous cAMP-dependent protein kinase required for long-term facilitation in Aplysia

The formation of a persistently active cAMP-dependent protein kinase (PKA) is critical for establishing long-term synaptic facilitation (LTF) in Aplysia. The injection of bovine catalytic (C) subunits into sensory neurons is sufficient to produce protein synthesis-dependent LTF. Early in the LTF induced by serotonin (5-HT), an autonomous PKA is generated through the ubiquitin-proteasome-mediated proteolysis of regulatory (R) subunits. The degradation of R occurs during an early time window and appears to be a key function of proteasomes in LTF. Lactacystin, a specific proteasome inhibitor, blocks the facilitation induced by 5-HT, and this block is rescued by injecting C subunits. R is degraded through an allosteric mechanism requiring an elevation of cAMP coincident with the induction of a ubiquitin carboxy-terminal hydrolase.     

Binding of 5,5'-bis[8-(phenylamino)-1-naphthalenesulfonate] by the regulatory subunits of adenosine cyclic 3',5'-phosphate dependent protein kinase

Binding to the regulatory subunits of types I and II adenosine cyclic 3',5'-phosphate (cAMP) dependent protein kinase (RI and RII, respectively) produces large distinctive increases in fluorescence and optical activity of 5,5'-bis[8-(phenylamino)-1-naphthalenesulfonate] [bis(ANS)]. Both specific and nonspecific interactions are involved. Association of the regulatory subunits with either the catalytic subunit or cAMP results in dissociation of a major portion of the bound bis(ANS) as detected by changes in fluorescence and circular dichroism. The results are consistent with the accepted cAMP binding properties of RI and RII, showing cooperativity in case of RI and two heterologous binding sites for RII. cGMP has the same overall effect on bis(ANS) binding as cAMP. However, very high concentrations are required for complete dissociation of bis(ANS) from RII, consistent with the observation that cGMP is inefficient in bringing about the dissociation of the type II holoenzyme. Magnesium binding to sites having dissociation constants of ca. 12 mM increases the interaction of bis(ANS) with both of the isolated regulatory subunits. Experiments involving the 37 000-dalton fragment of RII indicate that the limited proteolytic cleavage was heterogeneous, with only 24-39% of the resulting population interacting strongly with the catalytic subunit.     

Substrate enhances the sensitivity of type I protein kinase a to cAMP

The functional significance of the presence of two major (types I and II) isoforms of the cAMP-dependent protein kinase (PKA) is still enigmatic. The present study showed that peptide substrate enhanced the activation of PKA type I at low, physiologically relevant concentrations of cAMP through competitive displacement of the regulatory RI subunit. The effect was similar whether the substrate was a short peptide or the physiological 60-kDa protein tyrosine hydroxylase. In contrast, substrate failed to affect the cAMP-sensitivity of PKA type II. Size exclusion chromatography confirmed that substrate acted to physically enhance the dissociation of the RIalpha and Calpha subunits of PKA type I, but not the RIIalpha and Calpha subunits of PKA type II. Substrate availability can therefore fine-tune the activation of PKA type I by cAMP, but not PKA type II. The cAMP-dissociated RII and C subunits of PKA type II reassociated much faster than the PKA type I subunits in the presence of substrate peptide. This suggests that only PKA type II is able to rapidly reverse its activation after a burst of cAMP when exposed to high substrate concentration. We propose this as a possible reason why PKA type II is preferentially found in complexes with substrates undergoing rapid phosphorylation cycles.     

R-subunit isoform specificity in protein kinase A: distinct features of protein interfaces in PKA types I and II by amide H/2H exchange mass spectrometry

The two isoforms (RI and RII) of the regulatory (R) subunit of cAMP-dependent protein kinase or protein kinase A (PKA) are similar in sequence yet have different biochemical properties and physiological functions. To further understand the molecular basis for R-isoform-specificity, the interactions of the RIIβ isoform with the PKA catalytic (C) subunit were analyzed by amide H/²H exchange mass spectrometry to compare solvent accessibility of RIIβ and the C subunit in their free and complexed states. Direct mapping of the RIIβ-C interface revealed important differences between the intersubunit interfaces in the type I and type II holoenzyme complexes. These differences are seen in both the R-subunits as well as the C-subunit. Unlike the type I isoform, the type II isoform complexes require both cAMP-binding domains, and ATP is not obligatory for high affinity interactions with the C-subunit. Surprisingly, the C-subunit mediates distinct, overlapping surfaces of interaction with the two R-isoforms despite a strong homology in sequence and similarity in domain organization. Identification of a remote allosteric site on the C-subunit that is essential for interactions with RII, but not RI subunits, further highlights the considerable diversity in interfaces found in higher order protein complexes mediated by the C-subunit of PKA.     

Characterization of a cyclic AMP-resistant Chinese hamster ovary cell mutant containing both wild-type and mutant species of type I regulatory subunit of cyclic AMP-dependent protein kinase

We have characterized a cyclic AMP-resistant Chinese hamster ovary (CHO) cell mutant in which one of two major species of type I regulatory subunit (RI) of cyclic AMP-dependent protein kinase is altered. Wild-type CHO cell extracts contain two cyclic AMP-dependent protein kinase activities. As shown by DEAE-cellulose chromatography, there is a peak of type I protein kinase activity in mutant extracts, but the type II protein kinase activity is considerably reduced even though free type II regulatory subunit (RII) is present. The type I kinase from the mutant has an altered RI (RI*) whose KD for the binding of 8-N3[32P] cAMP (KD = 1.3 X 10(-5) M) is increased by more than 200-fold compared to RI from the wild-type enzyme (KD = 5.5 X 10(-8) M). No differences were found between the catalytic subunits from the wild-type and mutant type I kinases. A large portion of RI in mutant and wild-type extracts is present in the free form. The RI* derived from mutant type I protein kinase shows altered labeling by 8-N3[32P]cAMP (KD = 1.3 X 10(-5) M) whereas the free RI from the mutant is labeled normally by the photoaffinity label (KD = 7.2 X 10(-8) M), suggesting that the RI* which binds to the catalytic subunit is functionally different from the free form of RI. The decreased amount of type II kinase activity in the mutant appears to be due to competition of RI* with RII for binding to the catalytic subunit. Translation of mRNA from wild-type CHO cells results in the synthesis of two different charge forms of RI, providing biochemical confirmation of two different species of RI in CHO cells. Additional biochemical evidence based on isoelectric focusing behavior of 8-N3[32P]cAMP-labeled RI species and [35S]methionine-labeled RI from mutant and wild-type extracts confirms the charge heterogeneity of RI species in CHO cells. These genetic and biochemical data taken together are consistent with the conclusion that there are at least two different species of RI present in CHO cells and that one of these species is altered in the mutant analyzed in this work.     

Molecular basis for isoform-specific autoregulation of protein kinase A

Protein kinase A (PKA) isozymes are distinguishable by the inhibitory pattern of their regulatory (R) subunits with RI subunits containing a pseudophosphorylation P(0)-site and RII subunits being a substrate. Under physiological conditions, RII does not inhibit PrKX, the human X chromosome encoded PKA catalytic (C) subunit. Using a live cell Bioluminescence Resonance Energy Transfer (BRET) assay, Surface Plasmon Resonance (SPR) and kinase activity assays, we identified the P(0)-position of the R subunits as the determinant of PrKX autoinhibition. Holoenzyme formation only takes place with an alanine at position P(0), whereas RI subunits containing serine, phosphoserine or aspartate do not bind PrKX. Surprisingly, PrKX reversibly associates with RII when changing P(0) from serine to alanine. In contrast, PKA-Calpha forms holoenzyme complexes with all wildtype and mutant R subunits; however, holoenzyme re-activation by cAMP is severely affected. Only PKA type II or mutant PKA type I holoenzymes (P(0): Ser or Asp) are able to dissociate fully upon maximally elevated intracellular cAMP. The data are of particular significance for understanding PKA isoform-specific activation patterns in living cells.     

Phosphorylation of the cAMP-dependent protein kinase (PKA) regulatory subunit modulates PKA-AKAP interaction, substrate phosphorylation, and calcium signaling in cardiac cells

Subcellular compartmentalization of the cAMP-dependent protein kinase (PKA) by protein kinase A-anchoring proteins (AKAPs) facilitates local protein phosphorylation. However, little is known about how PKA targeting to AKAPs is regulated in the intact cell. PKA binds to an amphipathic helical region of AKAPs via an N-terminal domain of the regulatory subunit. In vitro studies showed that autophosphorylation of type II regulatory subunit (RII) can alter its affinity for AKAPs and the catalytic subunit (PKA(cat)). We now investigate whether phosphorylation of serine 96 on RII regulates PKA targeting to AKAPs, downstream substrate phosphorylation and calcium cycling in primary cultured cardiomyocytes. We demonstrated that, whereas there is basal phosphorylation of RII subunits, persistent maximal activation of PKA results in a phosphatase-dependent loss of RII phosphorylation. To investigate the functional effects of RII phosphorylation, we constructed adenoviral vectors incorporating mutants which mimic phosphorylated (RIIS96D), nonphosphorylated (RIIS96A) RII, or wild-type (WT) RII and performed adenoviral infection of neonatal rat cardiomyocytes. Coimmunoprecipitation showed that more AKAP15/18 was pulled down by the phosphomimic, RIIS96D, than RIIS96A. Phosphorylation of phospholamban and ryanodine receptor was significantly increased in cells expressing RIIS96D versus RIIS96A. Expression of recombinant RII constructs showed significant effects on cytosolic calcium transients. We propose a model illustrating a central role of RII phosphorylation in the regulation of local PKA activity. We conclude that RII phosphorylation regulates PKA-dependent substrate phosphorylation and may have significant implications for modulation of cardiac function.     

Reconstitution of types I and II adenosine cyclic 3',5'-phosphate dependent protein kinase

Fluorescence intensity and anisotropy measurements using the fluorescent adenosine cyclic 3',5'-phosphate (cAMP) analogue 1,N6-ethenoadenosine cyclic 3',5'-phosphate (epsilon-cAMP) are sensitive to the dissociation of epsilon-cAMP which occurs when either the type I or the type II regulatory subunit (RI or RII) of cAMP-dependent protein kinase associates with the catalytic subunit. Studies using epsilon-cAMP show that MgATP has opposite effects on the reconstitution of both types of protein kinase: MgATP strongly stabilizes the type I holoenzyme while it slightly destabilizes the type II holoenzyme. The synthetic substrate Kemptide has a small inhibitory effect on the reconstitution of both holoenzymes when tested at 10 microM concentration. The protein kinase inhibitor has a larger effect which is especially pronounced in the reassociation of the type I enzyme. The diminished relative ability of the type I regulatory subunit to compete with the protein kinase inhibitor suggests that the combined effects of the two opposing equilibria (epsilon-cAMP and catalytic subunit binding) are different for the two types of regulatory subunits. Displacement experiments show that cAMP and epsilon-cAMP bind about equally well to the type I subunit. Slow conformational changes accompanying the binding of epsilon-cAMP by both regulatory subunits are greatly accelerated with the holoenzymes, suggesting that dissociation of the holoenzymes occurs via ternary complexes. The time courses of epsilon-cAMP binding also show the heterogeneity of binding characteristics of RII. The 37 000-dalton fragment of type II subunit retains the epsilon-cAMP binding properties of the native subunit. However, only a fraction of the fragment preparation (approximately 32% estimated from sedimentation measurements) binds the catalytic subunit well, suggesting heterogeneity of cleavage.     

Localization of cAMP-dependent signal transducers in early rat liver carcinogenesis

 Cyclic AMP (cAMP) is an important regulator of liver growth and differentiation. The main intracellular cAMP receptor, cAMP-dependent protein kinase (PKA), consists of two regulatory (R) and two catalytic (C) subunits. There are two classes, RI and RII, of the regulatory subunit, giving rise to type I (RI₂C₂) and type II (RII₂C₂) PKA. The RI/RII ratio generally decreases during organ development, and increases during carcinogenesis. Alterations in this ratio have been implicated as an important factor in experimental and clinical carcinogenesis. We have studied the expression of RIα, RIIα, Cα, and an important substrate of PKA, the cAMP-response element binding protein, during rat liver carcinogenesis. Two-color immunofluorescence and confocal laser scan microscopy were used to characterize localization of the cAMP-dependent signal transducers in hepatocytes, bile ducts, oval cells, and preneoplastic lesions. We found that bile ducts and oval cells (putative liver stem cells) contained a higher RI/RII ratio than hepatocytes and preneoplastic lesions. Thus, an altered RI/RII ratio was not detected during early rat liver carcinogenesis, but may contribute to differentiation of putative liver stem cells to hepatocytes. Accepted: 19 August 1997     

Type II regulatory subunit dimerization determines the subcellular localization of the cAMP-dependent protein kinase

The type II cAMP-dependent protein kinase (PKA) is localized to specific subcellular environments through binding of dimeric regulatory subunits (RII) to anchoring proteins. Cytoskeletal localization occurs through RII dimer interaction with the PKA substrate molecule microtubule-associated protein 2 (MAP2). RII alpha deletion mutants and RII alpha/endonexin chimeras retained MAP2 binding activity if they contained the first 79 residues of the molecule. Disruption of RII alpha dimerization always prevented MAP2 interaction because 1) RII delta 1-14 (an amino-terminal deletion mutant lacking residues 1-14) was unable to bind MAP2 or form dimers, and 2) a modified RII alpha monomer including residues 1-14 did not bind MAP2. Chimeric proteins containing the first 30 residues of RII alpha fused to endonexin II formed dimers but did not bind MAP2. This suggested other side-chains between residues 30-79 also participate in MAP2 interaction. Peptide studies indicate additional contact with MAP2 may occur through an acidic region (residues 68-82) close to the RII autoinhibitor domain. Therefore, anchored PKA holoenzyme topology may position the catalytic subunit and MAP2 as to allow its preferential phosphorylation upon kinase activation.     

Association of protein kinase A type I with detergent-resistant structures of mammalian sperm cells

The finding that flagellar movement in detergent-permeabilized sperm cells is restored when Mg ATP and cAMP are added implicated detergent-resistant protein kinase A (PKA) in the regulation of sperm motility. It is widely believed that only the PKA regulatory subunit RII can associate with the cytoskeleton and/or organelles. In this paper we used monoclonal antibodies against the PKA catalytic subunit and RI subunit and demonstrated that PKA type I is also associated with the sperm cytoskeleton. To our knowledge, this is the first report showing anchored PKA type I. This association was found in sperm of nonrodent mammalian species and, to a lesser extent, also in mouse sperm. The PKA catalytic subunit is bound to the cytoskeleton secondarily via its complex with the regulatory subunit. The detergent-resistant complexes of RI and catalytic subunits localize predominantly to the flagellum. Ultrastructural immunogold labeling revealed the association of detergent-resistant PKA type I with outer dense fibers (ODF) and the fibrous sheath (FS) but not with microtubules. This location is consistent with a proposed role of PKA in regulation of FS sliding on underlying ODF. Mol. Reprod. Dev. 50:79–85, 1998. © 1998 Wiley-Liss, Inc.