Phosphatases Control the Duration and Range of cAMP/PKA Microdomains

CUTL1, also known as CDP (CCAAT Displacement Protein), Cut, or Cux-1, is a homeodomain transcription factor known to play an essential role in development and cell cycle progression. Previously, we identified CUTL1 as modulator of cell motility and invasiveness. Here we report that protein kinase A (PKA), known to inhibit tumor progression in various tumor types, directly phosphorylates CUTL1 at serine 1215 in NIH3T3 fibroblasts. The PKA-induced phosphorylation results in decreased DNA binding affinity of CUTL1 and diminished CUTL1-mediated cell cycle progression and cell motility. Furthermore, the expression of several CUTL1 target genes involved in proliferation and migration, such as DNA polymerase A and DKK2, was modulated by PKA-induced phosphorylation. These data identify CUTL1 as a novel target of PKA through which this protein kinase can modulate tumor cell motility and tumor progression.

A-Kinase anchor proteins (AKAPs) immobilize and concentrate protein kinase A (PKA) isoforms at specific subcellular compartments. Intracellular targeting of PKA holoenzyme elicits rapid and efficient phosphorylation of target proteins, thereby increasing sensitivity of downstream effectors to cAMP action. AKAP121 targets PKA to the cytoplasmic surface of mitochondria. Here we show that conditional expression of AKAP121 in PC12 cells selectively enhances cAMP.PKA signaling to mitochondria. AKAP121 induction stimulates PKA-dependent phosphorylation of the proapoptotic protein BAD at Ser(155), inhibits release of cytochrome c from mitochondria, and protects cells from apoptosis. An AKAP121 derivative mutant that localizes on mitochondria but does not bind PKA down-regulates PKA signaling to the mitochondria and promotes apoptosis. These findings indicate that PKA anchored by AKAP121 transduces cAMP signals to the mitochondria, and it may play an important role in mitochondrial physiology.

Background Phosphorylation of L-type calcium channels (LTCC) by cAMP-dependent protein kinase A (PKA) increases calcium current (ICa). However, it is unclear if PKA-dependent regulation of ICa is impaired in heart failure (HF) despite evidence for impaired β-adrenergic signaling. Recently, a novel PKA activation pathway by oxidation of regulatory subunit I (RI) has been identified. Objective We investigated the impact of redox-activated PKA for regulation of ICa, intracellular calcium (Ca) handling and contractile function in a pressure overload heart failure mouse model. Methods Knock-in mice (KI) that lack redox-dependent PKA activation (exchange of cysteine 17 of RI with serine) were compared to wild-type (WT) at baseline, 7 days and 6 weeks after transverse aortic constriction (TAC). Mouse echocardiography was performed to evaluate in-vivo cardiac function. ICa was measured by whole-cell patch clamp, PKA activity, cAMP levels, and protein levels of central Ca handling proteins were assessed at different time points in vitro. Results At baseline, no alterations of left ventricular (LV) function (echocardiography) were observed between WT and KI. TAC induced a significant RI oxidation in WT but not KI mice. Despite this difference, at 7 days after TAC, development of LV hypertrophy and impairment of systolic LV function in vivo were similar between WT and KI. Compared to baseline, 7 days after TAC a significant stimulation of peak ICa was observed in WT. In contrast to WT, the stimulation of peak ICa was absent in KI mice at 7 days after TAC. This impairment in peak ICa occurred despite a comparable increase in global PKA activity (ELISA), which was most likely due to increased cAMP levels in our TAC model (assessed by FLIM-FRET). Notably, cAMP levels were comparably increased in between groups (ELISA). Importantly, at 6 weeks after TAC, WT mice showed a mild additional deterioration of systolic LV function in vivo. In contrast, LV function was significantly more impaired in KI mice 6 weeks after TAC, which was accompanied by a significant increase in KI mice mortality. Comparing 6 weeks to 7 days after TAC, there was no stimulation of peak ICa in WT and even a significant decrease in peak ICa in KI mice. In accordance, PKA-dependent LTCC phosphorylation was absent in KI mice 6 weeks after TAC (western blotting). Conclusion Redox-activated PKA seems to exert a protective role by stimulation of ICa during pressure overload. Funding Acknowledgement Type of funding source: Public grant(s) – National budget only. Main funding source(s): Deutsche Forschungsgemeinschaft

Although adenosine 3′,5′-monophosphate (cAMP) is a small diffusible second messenger, its activity is localized through the actions of scaffolds such as A kinase anchoring proteins (AKAPs), which interact with protein kinase A (PKA), the major downstream effector of cAMP, and other proteins involved in cAMP signaling. Zhang et al. report the development through protein engineering of a reversible A-kinase reporter protein, AKAR2. This reporter was then used to show that chronic insulin treatment of 3T3-L1 adipocytes led to a delay in the PKA response to the β-adrenergic receptor (β-AR) agonist isoproteronol. This was surprising given that chronic insulin exposure leads to the production of higher concentrations of cAMP in response to β-AR stimulation than those observed in cells not treated with insulin. Activation of the downstream endogenous PKA target CREB (cAMP response element-binding protein) was also delayed. Chronic insulin treatment did not inhibit phosphorylation of AKAR2 by stimuli that bypassed the β-AR, which suggested that PKA was not globally inhibited. Insulin treatment decreased the interaction between β-AR and the regulatory subunit of PKA in response to subsequent exposure to isoproteronol, which suggested that insulin altered the AKAP function, which led to desensitization of the β-AR response. Injection of a peptide that blocked the interaction between AKAP and the regulatory subunit of PKA also blocked activation of the AKAR2 reporter by isoproteronol. These results provide evidence for another mechanism of cross talk between signaling pathways, through disruption of the scaffolding protein functions. J. Zhang, C. J. Hupfeld, S. S. Taylor, J. M. Olefsky, R. Y. Tsien, Insulin disrupts β-adrenergic signalling to protein kinase A in adipocytes. Nature 437 , 569-573 (2005). [Online Journal]

Protein Implicated in Nonsyndromic Mental Retardation Regulates Protein Kinase A (PKA) Activity

Cyclic AMP response element-binding protein-binding protein (CBP) functions as a transcriptional coactivator through interactions with a number of cellular and viral transcription factors. It has been suggested to play a central integrative role in gene regulation. However, little is known about signal cascades that can regulate CBP activity. Here we show that either nerve growth factor (NGF) or cAMP treatment led to enhanced activity of CBP in PC12 cells. The C-terminal glutamine-rich activation domain of CBP was shown to be responsible for induction by NGF and cAMP. NGF-induced enhancement of CBP activity was also observed in protein kinase A (PKA)-deficient PC12 cells, whereas cAMP failed to increase the transcriptional activity of CBP in these cells. Moreover, the specific PKA inhibitor H-89 blocked cAMP-induced but not NGF-induced up-regulation of CBP activity. The up-regulation of CBP transcriptional activity in response to NGF was, however, prevented by the specific inhibitor of mitogen-activated protein kinase (p42/44(MAPK)) activation, PD98059, which had no effect on the up-regulation induced by cyclic AMP, indicating that activation of the mitogen-activated protein kinase signal pathway is specifically involved in the NGF-induced activation of CBP. In addition, expression of a dominant-negative interfering mutant of p42/44(MAPK) can prevent the NGF-mediated induction of the CBP activity, whereas expression of a p42/44(MAPK) constitutively active mutant can enhance the transcriptional activity of CBP. These data indicate that activation of the p42/p44(MAPK) cascade mediates the up-regulation of the transcriptional activity of CBP by NGF, whereas the similar up-regulation induced by cyclic AMP is mediated by PKA activation.

Protein kinase A and/or C inhibitors potentiate isoproterenol-induced cyclic AMP accumulation in intact human lymphocytes

Histamine Induces Melanogenesis and Morphologic Changes by Protein Kinase A Activation via H2 Receptors in Human Normal Melanocytes

Spatial organisation of AKAP18 and PDE4 isoforms in renal collecting duct principal cells

A most versatile kinase: The catalytic subunit of PKA in T-cell biology.

Although Cystic fibrosis transmembrane conductance regulator (CFTR) has been shown to regulate the activity of NHE3, the potential reciprocal interaction of NHE3 to modulate the protein kinase A (PKA)-dependent regulation of CFTR in epithelial cells is still unknown. In the present work, we describe experiments to define the interactions between CFTR and NHE3 with the regulatory, scaffolding protein, NHERF that organize their PKA-dependent regulation in a renal epithelial cell line that expresses endogenous CFTR. The expression of rat NHE3 significantly decreased PKA-dependent activation of CFTR without altering CFTR expression, and this decrease was prevented by mutation of either of the two rat NHE3 PKA target serines to alanine (S552A or S605A). Inhibition of CFTR expression by antisense treatment resulted in an acute decrease in PKA-dependent regulation of NHE3 activity. CFTR, NHE3, and ezrin were recognized by NHERF-2 but not NHERF-1 in glutathione S-transferase pull-down experiments. Ezrin may function as a protein kinase A anchoring protein (AKAP) in this signaling complex, because blocking the binding of PKA to an AKAP by incubation with the S-Ht31 peptide inhibited the PKA-dependent regulation of CFTR in the absence of NHE3. In the A6-NHE3 cells S-Ht31 blocked the PKA regulation of NHE3 whereas it now failed to affect the regulation of CFTR. We conclude that CFTR and NHE3 reciprocally interact via a shared regulatory complex comprised of NHERF-2, ezrin, and PKA.

Drug resistance poses a significant threat to ongoing malaria control efforts. Coupled with lack of a malaria vaccine, there is an urgent need for the development of new antimalarials with novel mechanisms of action and low susceptibility to parasite drug resistance. Protein Kinase A (PKA) has been implicated as a critical regulator of pathogenesis in malaria. Therefore, we sought to investigate the effects of disrupted PKA signaling as a possible strategy for inhibition of parasite replication. Host PKA activity is partly regulated by a class of proteins called A Kinase Anchoring Proteins (AKAPs), and interaction between HsPKA and AKAP can be inhibited by the stapled peptide Stapled AKAP Disruptor 2 (STAD-2). STAD-2 was tested for permeability to and activity against Plasmodium falciparum blood stage parasites in vitro. The compound was selectively permeable only to infected red blood cells (iRBC) and demonstrated rapid antiplasmodial activity, possibly via iRBC lysis (IC50 ≈ 1 μM). STAD-2 localized within the parasite almost immediately post-treatment but showed no evidence of direct association with PKA, indicating that STAD-2 acts via a PKA-independent mechanism. Furosemide-insensitive parasite permeability pathways in the iRBC were largely responsible for uptake of STAD-2. Further, peptide import was highly specific to STAD-2 as evidenced by low permeability of control stapled peptides. Selective uptake and antiplasmodial activity of STAD-2 provides important groundwork for the development of stapled peptides as potential antimalarials. Such peptides may also offer an alternative strategy for studying protein-protein interactions critical to parasite development and pathogenesis.

β‐adrenergic receptor (β‐AR) signaling is the primary mechanism to increase cardiac contractility. However, chronic β‐AR stimulation, which occurs in HF, results in impairment of the β‐adrenergic pathway, leading to reduced contractility due in part by desensitization of these receptors. To induce contractility, β‐ARs signal through protein kinase A (PKA)‐dependent phosphorylation, which affects calcium homeostasis. PKA binds to A‐kinase anchoring proteins (AKAPs), which localize PKA and its substrates to defined intracellular locations. Gravin, an AKAP that targets PKA, protein kinase C (PKC) and protein phosphatase 2B (PP2B) to β2‐ARs, is involved in the modulation of the desensitization / resensitization cycle of β2‐ARs by facilitating PKA‐dependent phosphorylation and scaffolding of the β2‐AR complex. Thus, disruption of gravin's scaffolding action might prove beneficial to restore cardiac contractility during heart failure. In our studies, we used isoproterenol to chronically stimulate the β‐ARs to induce heart failure. We found that gravin‐t/t mice, harboring the truncated non‐functional gravin, leads to restriction of receptor desensitization during chronic β‐AR stimulation, due to loss in the assembly of the desensitization complex mediated by gravin's scaffolding. The β‐AR density is significantly higher in gravin‐t/t mice than wild‐type animals after 2 weeks of isoproterenol treatment. This decrease in the desensitization leads to the availability of more receptors on the heart to mediate chronotropy, ionotropy and lusitropy. Together, gravin‐t/t mice exhibited enhanced cardiac function in the presence of chronic β‐AR stimulation. PKA‐dependent substrates including cardiac troponin I (cTnI), phospholamban (PLB), and cardiac myosin binding protein C (cMyBPC), which show aberration activation patterns during heart failure, had normal phosphorylation levels in the gravin‐t/t mice, compared to the wild‐type mice. Gravin‐t/t mice also displayed enhanced myofilaments sensitivity to calcium compared to the wild‐type mice. Our results indicate that gravin plays an important role in regulating the β‐ AR pathway and disruption of its scaffolding augments cardiac function. Thus, blocking of gravin's scaffolding can be further explored as a potential therapeutic target for heart failure.Support or Funding InformationResearch reported in this poster was supported by the NHLBI institute of NIH under Award Number R01HL085487 (to B.K.M.) and R15HL124458 (to B.K.M.). This project was also supported by the Mouse Phenotyping Core at Baylor College of Medicine with funding from the NIH (U54 HG006348).

Inflammatory mediators not only activate "pain-"sensing neurons, the nociceptors, to trigger acute pain sensations, more important, they increase nociceptor responsiveness to produce inflammatory hyperalgesia. For example, prostaglandins activate G(s)-protein-coupled receptors and initiate cAMP- and protein kinase A (PKA)-mediated processes. We demonstrate for the first time at the cellular level that heat-activated ionic currents were potentiated after exposure to the cAMP activator forskolin in rat nociceptive neurons. The potentiation was prevented in the presence of the selective PKA inhibitor PKI(14-22), suggesting PKA-mediated phosphorylation of the heat transducer protein. PKA regulatory subunits were found in close vicinity to the plasma membrane in these neurons, and PKA catalytic subunits only translocated to the cell periphery when activated. The translocation and the current potentiation were abolished in the presence of an A-kinase anchoring protein (AKAP) inhibitor. Similar current changes after PKA activation were obtained from human embryonic kidney 293t cells transfected with the wild-type heat transducer protein vanilloid receptor 1 (VR-1). The forskolin-induced current potentiation was greatly reduced in cells transfected with VR-1 mutants carrying point mutations at the predicted PKA phosphorylation sites. The heat transducer VR-1 is therefore suggested as the molecular target of PKA phosphorylation, and potentiation of current responses to heat depends on phosphorylation at predicted PKA consensus sites. Thus, the PKA/AKAP/VR-1 module presents as the molecular correlate of G(s)-mediated inflammatory hyperalgesia.

Most studies indicate that failing human hearts have greater baseline myofibrillar Ca2+ sensitivity of tension development than nonfailing hearts. Phosphorylation of cardiac troponin I (TnI) by cAMP-dependent protein kinase (PKA) decreases the affinity of the troponin complex for Ca2+, thus altering the Ca2+ sensitivity of force production. We tested the hypothesis that PKA-dependent TnI phosphorylation is altered in the failing human heart and investigated changes in PKA regulatory subunits as a potential mechanism. Using in vitro back-phosphorylation with [gamma-32P]ATP, we demonstrated a significant (P<0.05) approximately 25% reduction in baseline PKA-dependent TnI phosphorylation in human hearts with dilated cardiomyopathy (DCM) compared with nonfailing (NF) human hearts. There was no significant difference in cAMP content or maximal PKA activity between DCM and NF hearts, but expression of the regulatory subunits of PKA-I (RI) and PKA-II (RII) was significantly decreased in DCM versus NF hearts (RI by approximately 40%, P<0.05; RII by approximately 30%, P<0.01). PKA activity is regulated at the substrate level through interactions of PKA regulatory subunits with A-kinase anchoring proteins. The reduced baseline PKA-dependent phosphorylation of TnI in DCM may be due to decreased expression of RI and RII and consequently reduced anchoring of PKA holoenzyme. These findings provide new evidence of deficiencies in downstream regulation of the beta-adrenergic pathway in the failing human heart and may account for increased baseline myofibrillar Ca2+ sensitivity.