Overexpression of Dd PK2 protein kinase causes rapid development and affects the intracellular cAMP pathway of Dictyostelium discoideum

Development ◽  
1992 ◽  
Vol 115 (3) ◽  
pp. 785-790 ◽  
Author(s):  
C. Anjard ◽  
S. Pinaud ◽  
R.R. Kay ◽  
C.D. Reymond
Keyword(s):  
Protein Kinase ◽  
Protein Kinase A ◽  
Rapid Development ◽  
Physiological Role ◽  
Intracellular Camp ◽  
Developmentally Regulated ◽  
K Cells ◽  
Frame Shift ◽  
Camp Levels ◽  
Kinase A

The Dd PK2 gene codes for a putative protein of 648 amino acids with a C-terminal half sharing high homology with protein kinase A catalytic subunits from other organisms. In order to find out more about the physiological role of the Dd PK2 kinase, its gene, and a version having a frame shift mutation in the middle of the catalytic region, were overexpressed in developing Dictyostelium cells. Both the intact gene (K-) and the frame shift mutant (Kdel-) caused rapid development with spores formed in 16–18 hours compared to the 24 hours required by their parent. This result was confirmed by the pattern of expression of some developmentally regulated genes. Other rapid developing strains (rde) are activated in the cAMP second messenger system. Both K- and Kdel-containing strains have lower cAMP levels than the parental strain during late development, thus resembling rdeC mutants. K-cells (but not Kdel-cells) produced bizarre fruiting bodies with many prostrate forms. The parallel with rde mutants was confirmed by demonstrating that K-cells are able to form spores in submerged monolayer culture. Furthermore, K-cells have about four times more protein kinase A (cAPK) activity than wild-type cells. These results indicate that the N-terminal domain of Dd PK2 is sufficient to influence cAMP levels and to provoke rapid development, whereas kinase activity seems to be required for the sporogenous phenotype. The association between elevated cAPK and Dd PK2 overexpression phenotype further indicates a role for cAPK in the formation of spores.

B-Raf-dependent regulation of the MEK-1/mitogen-activated protein kinase pathway in PC12 cells and regulation by cyclic AMP

1994 ◽  
Vol 14 (10) ◽  
pp. 6522-6530
Author(s):  
R R Vaillancourt ◽  
A M Gardner ◽  
G L Johnson
Keyword(s):  
Growth Factor ◽  
Protein Kinase ◽  
Map Kinase ◽  
Protein Kinase A ◽  
Pc12 Cells ◽  
Kinase Activation ◽  
Mitogen Activated Protein ◽  
Epidermal Growth ◽  
Camp Levels ◽  
Kinase A

Growth factor receptor tyrosine kinase regulation of the sequential phosphorylation reactions leading to mitogen-activated protein (MAP) kinase activation in PC12 cells has been investigated. In response to epidermal growth factor, nerve growth factor, and platelet-derived growth factor, B-Raf and Raf-1 are activated, phosphorylate recombinant kinase-inactive MEK-1, and activate wild-type MEK-1. MEK-1 is the dual-specificity protein kinase that selectively phosphorylates MAP kinase on tyrosine and threonine, resulting in MAP kinase activation. B-Raf and Raf-1 are growth factor-regulated Raf family members which regulate MEK-1 and MAP kinase activity in PC12 cells. Protein kinase A activation in response to elevated cyclic AMP (cAMP) levels inhibited B-Raf and Raf-1 stimulation in response to growth factors. Ras.GTP loading in response to epidermal growth factor, nerve growth factor, or platelet-derived growth factor was unaffected by protein kinase A activation. Even though elevated cAMP levels inhibited Raf activation, the growth factor activation of MEK-1 and MAP kinase was unaffected in PC12 cells. The results demonstrate that tyrosine kinase receptor activation of MEK-1 and MAP kinase in PC12 cells is regulated by B-Raf and Raf-1, whose activation is inhibited by protein kinase A, and MEK activators, whose activation is independent of cAMP regulation.

Interleukin-1 alpha stimulates dopamine release by activating type II protein kinase A in PC-12 cells

1995 ◽  
Vol 269 (6) ◽  
pp. E1083-E1088
Author(s):  
A. Joseph ◽  
A. Kumar ◽  
N. A. O'Connell ◽  
R. K. Agarwal ◽  
A. R. Gwosdow
Keyword(s):  
Protein Kinase ◽  
Protein Kinase A ◽  
Dopamine Release ◽  
Interleukin 1 ◽  
Intracellular Camp ◽  
Type I ◽  
Camp Accumulation ◽  
Type Ii ◽  
Pc 12 Cells ◽  
Kinase A

A recent study from this laboratory [A. R. Gwosdow, N. A. O'Connell, and A. B. Abou-Samra. Am. J. Physiol. 263 (Endocrinol. Metab. 26): E461-E466, 1992] showed that the inflammatory mediator interleukin-1 alpha (IL-1 alpha) stimulates catecholamine release from primary cultures of rat adrenal cells. The present studies were conducted to determine whether 1) IL-1 alpha stimulates catecholamine/dopamine release from the adrenal medullary cell line PC-12 and 2) the adenosine 3',5'-cyclic monophosphate (cAMP)-protein kinase A (PKA) pathway is involved in IL-1 alpha-induced dopamine release from PC-12 cells. The results indicate that IL-1 alpha significantly (P < 0.05) elevated dopamine release after a 24-h incubation period. IL-1 alpha did not stimulate cAMP accumulation at any time period between 5 min and 2 h. In contrast, forskolin-treated cells elevated (P < 0.05) intracellular cAMP levels and increased dopamine release. Because IL-1 alpha did not affect cAMP accumulation, the effect of IL-1 alpha on PKA activity was investigated. IL-1 alpha increased (P < 0.05) PKA activity at 15 and 30 min and returned to control levels by 1 h. Forskolin also increased (P < 0.05) PKA activity. The type of PKA activated (P < 0.05) by IL-1 alpha was type II PKA. In contrast, forskolin activated (P < 0.05) type I and type II PKA. Inhibition of PKA with the PKA inhibitor H-8 blocked PKA activity and dopamine secretion by both IL-1 alpha and forskolin in PC-12 cells. These observations demonstrate that 1) IL-1 alpha stimulated dopamine release from PC-12 cells by activating PKA, 2) the mechanism of IL-1 alpha activation of PKA does not involve detectable increases in intracellular cAMP accumulation, and 3) IL-1 alpha activates type II PKA, which is used by IL-1 alpha to stimulate dopamine secretion from PC-12 cells.

scholarly journals Regulation of transforming growth factor β-induced responses by protein kinase A in pancreatic acinar cells

2008 ◽  
Vol 295 (1) ◽  
pp. G170-G178 ◽  
Author(s):  
Huibin Yang ◽  
Cheong J. Lee ◽  
Lizhi Zhang ◽  
Maria Dolors Sans ◽  
Diane M. Simeone
Keyword(s):  
Protein Kinase ◽  
Protein Kinase A ◽  
Transforming Growth Factor ◽  
Acinar Cells ◽  
Lung Epithelial Cells ◽  
Pancreatic Acinar Cells ◽  
Intracellular Camp ◽  
Physical Interaction ◽  
Pancreatic Acini ◽  
Kinase A

TGF-β is an important regulator of growth and differentiation in the pancreas and has been implicated in pancreatic tumorigenesis. We have recently demonstrated that TGF-β can activate protein kinase A (PKA) in mink lung epithelial cells (Zhang L, Duan C, Binkley C, Li G, Uhler M, Logsdon C, Simeone D. Mol Cell Biol 24: 2169–2180, 2004). In this study, we sought to determine whether TGF-β activates PKA in pancreatic acinar cells, the mechanism by which PKA is activated, and PKA's role in TGF-β-mediated growth regulatory responses. TGF-β rapidly activated PKA in pancreatic acini while having no effect on intracellular cAMP levels. Coimmunoprecipitation experiments demonstrated a physical interaction between a Smad3/Smad4 complex and the regulatory subunits of PKA. TGF-β also induced activation of the PKA-dependent transcription factor CREB. Both the specific PKA inhibitor H89 and PKI peptide significantly blocked TGF-β's ability to activate PKA and CREB. TGF-β-mediated growth inhibition and TGF-β-induced p21 and SnoN expression in pancreatic acinar cells were blocked by H89 and PKI peptide. This study demonstrates that this novel cross talk between TGF-β and PKA signaling pathways may play an important role in regulating TGF-β signaling in the pancreas.

cAMP-positive regulation of angiotensinogen gene expression and protein secretion in rat adipose tissue

2004 ◽  
Vol 286 (3) ◽  
pp. E434-E438 ◽  
Author(s):  
Valérie Serazin ◽  
Marie-Noelle Dieudonné ◽  
Mireille Morot ◽  
Philippe de Mazancourt ◽  
Yves Giudicelli
Keyword(s):  
Adipose Tissue ◽  
Protein Kinase ◽  
Protein Kinase A ◽  
Protein Secretion ◽  
Intracellular Camp ◽  
Pathological State ◽  
Mrna Levels ◽  
Adrenergic Stimulation ◽  
Rat Adipose Tissue ◽  
Kinase A

The adipose renin-angiotensin system (RAS) has been assigned to participate in the control of adipose tissue development and in the pathogenesis of obesity-related hypertension. In adipose cells, the biological responses to β-adrenergic stimulation are mediated by an increase in intracellular cAMP. Because cAMP is known to promote adipogenesis and because an association exists between body fat mass, hypertension, and increased sympathetic stimulation, we examined the influence of cAMP on angiotensinogen (ATG) expression and secretion in rat adipose tissue. Exposure of primary cultured differentiated preadipocytes to the cAMP analog 8-bromoadenosine 3′,5′-cyclic monophosphate (8-BrcAMP) or cAMP-stimulating agents (forskolin and IBMX) results in a significant increase in ATG mRNA levels. In adipose tissue fragments, 8-BrcAMP also increases ATG mRNA levels and protein secretion, but not in the presence of the protein kinase A inhibitor H89. The addition of isoproterenol, known to stimulate the synthesis of intracellular cAMP via β-adrenoreceptors, had the same stimulatory effect on ATG expression and secretion. These results indicate that cAMP in vitro upregulates ATG expression and secretion in rat adipose tissue via the protein kinase A-dependent pathway. Further studies are required to determine whether this regulatory pathway is activated in human obesity, where increased sympathetic tone is frequently observed, and to elucidate the importance of adipose ATG to the elevated blood pressure observed in this pathological state.

scholarly journals B-Raf-dependent regulation of the MEK-1/mitogen-activated protein kinase pathway in PC12 cells and regulation by cyclic AMP.

1994 ◽  
Vol 14 (10) ◽  
pp. 6522-6530 ◽  
Author(s):  
R R Vaillancourt ◽  
A M Gardner ◽  
G L Johnson
Keyword(s):  
Growth Factor ◽  
Protein Kinase ◽  
Map Kinase ◽  
Protein Kinase A ◽  
Pc12 Cells ◽  
Kinase Activation ◽  
Mitogen Activated Protein ◽  
Epidermal Growth ◽  
Camp Levels ◽  
Kinase A

Growth factor receptor tyrosine kinase regulation of the sequential phosphorylation reactions leading to mitogen-activated protein (MAP) kinase activation in PC12 cells has been investigated. In response to epidermal growth factor, nerve growth factor, and platelet-derived growth factor, B-Raf and Raf-1 are activated, phosphorylate recombinant kinase-inactive MEK-1, and activate wild-type MEK-1. MEK-1 is the dual-specificity protein kinase that selectively phosphorylates MAP kinase on tyrosine and threonine, resulting in MAP kinase activation. B-Raf and Raf-1 are growth factor-regulated Raf family members which regulate MEK-1 and MAP kinase activity in PC12 cells. Protein kinase A activation in response to elevated cyclic AMP (cAMP) levels inhibited B-Raf and Raf-1 stimulation in response to growth factors. Ras.GTP loading in response to epidermal growth factor, nerve growth factor, or platelet-derived growth factor was unaffected by protein kinase A activation. Even though elevated cAMP levels inhibited Raf activation, the growth factor activation of MEK-1 and MAP kinase was unaffected in PC12 cells. The results demonstrate that tyrosine kinase receptor activation of MEK-1 and MAP kinase in PC12 cells is regulated by B-Raf and Raf-1, whose activation is inhibited by protein kinase A, and MEK activators, whose activation is independent of cAMP regulation.

scholarly journals Pde1 Phosphodiesterase Modulates Cyclic AMP Levels through a Protein Kinase A-Mediated Negative Feedback Loop in Cryptococcus neoformans

2005 ◽  
Vol 4 (12) ◽  
pp. 1971-1981 ◽  
Author(s):  
Julie K. Hicks ◽  
Yong-Sun Bahn ◽  
Joseph Heitman
Keyword(s):  
Protein Kinase ◽  
Cyclic Amp ◽  
Protein Kinase A ◽  
Cryptococcus Neoformans ◽  
Mutant Strain ◽  
Negative Feedback ◽  
Feedback Loop ◽  
Negative Feedback Loop ◽  
Camp Levels ◽  
Kinase A

ABSTRACT The virulence of the human pathogenic fungus Cryptococcus neoformans is regulated by a cyclic AMP (cAMP)-dependent protein kinase A (PKA) signaling cascade that promotes mating and the production of melanin and capsule. In this study, genes encoding homologs of the Saccharomyces cerevisiae low- and high-affinity phosphodiesterases, PDE1 and PDE2, respectively, were deleted in serotype A strains of C. neoformans. The resulting mutants exhibited moderately elevated levels of melanin and capsule production relative to the wild type. Epistasis experiments indicate that Pde1 functions downstream of the Gα subunit Gpa1, which initiates cAMP-dependent signaling in response to an extracellular signal. Previous work has shown that the PKA catalytic subunit Pka1 governs cAMP levels via a negative feedback loop. Here we show that a pde1Δ pka1Δ mutant strain exhibits cAMP levels that are dramatically increased (∼15-fold) relative to those in a pka1Δ single mutant strain and that a site-directed mutation in a consensus PKA phosphorylation site reduces Pde1 function. These data provide evidence that fluctuations in cAMP levels are modulated by both Pka1-dependent regulation of Pde1 and another target that comprise a robust negative feedback loop to tightly constrain intracellular cAMP levels.

NO releases bombesin-like immunoreactivity from enteric synaptosomes by cross-activation of protein kinase A

1999 ◽  
Vol 276 (6) ◽  
pp. G1521-G1530 ◽  
Author(s):  
M. Kurjak ◽  
R. Fritsch ◽  
D. Saur ◽  
V. Schusdziarra ◽  
H. D. Allescher
Keyword(s):  
Protein Kinase ◽  
Protein Kinase A ◽  
Dependent Protein Kinase ◽  
No Donor ◽  
Camp Dependent Protein Kinase ◽  
Dependent Protein ◽  
Camp Levels ◽  
No Releases ◽  
Kinase A

The effect of nitric oxide (NO) on the release of bombesin-like immunoreactivity (BLI) was examined in synaptosomes of rat small intestine. The NO donor S-nitroso- N-acetylpenicillamine (SNAP; 10−7 to 10−4 M) significantly stimulated BLI release. In the presence of the NO scavenger oxyhemoglobin (10−3 M) or the guanylate cyclase inhibitor ODQ (10−5 M), SNAP-induced BLI release was antagonized. In addition, SNAP increased the synaptosomal cGMP content and elevation of cGMP levels by zaprinast (3 × 10−5 M), an inhibitor of the cGMP-specific phosphodiesterase (PDE) type 5, and increased basal and SNAP-induced BLI release. NO-induced BLI release was blocked by Rp-adenosine 3′,5′-cyclic monophosphorothioate (3 × 10−5 M and 10−4 M), an inhibitor of the cAMP-dependent protein kinase A, whereas KT-5823 (3 × 10−6 M) and Rp-8-(4-chlorophenylthio)-cGMP (5 × 10−5M), inhibitors of the cGMP-dependent protein kinase G, had no effect. Because cGMP inhibits the cAMP-specific PDE3, thereby increasing cAMP levels, the role of PDE3 was investigated. Trequinsin (10−8 M), a specific blocker of PDE3, stimulated basal BLI release but had no additive effect on NO-induced release, suggesting a similar mechanism of action. These data demonstrate that because of a cross-activation of cAMP-dependent protein kinase A by endogenous cGMP BLI can be released by NO from enteric synaptosomes.

scholarly journals Phosphorylation of cAMP-specific PDE4A5 (phosphodiesterase-4A5) by MK2 (MAPKAPK2) attenuates its activation through protein kinase A phosphorylation

2011 ◽  
Vol 435 (3) ◽  
pp. 755-769 ◽  
Author(s):  
Kirsty F. MacKenzie ◽  
Derek A. Wallace ◽  
Elaine V. Hill ◽  
Diana F. Anthony ◽  
David J. P. Henderson ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Protein Kinase A ◽  
P38 Mapk ◽  
Stress Responses ◽  
Mammalian Cells ◽  
Signal Transduction Pathway ◽  
Mitogen Activated Protein Kinase ◽  
Intracellular Camp ◽  
Camp Accumulation ◽  
Kinase A

cAMP-specific PDE (phosphodiesterase) 4 isoforms underpin compartmentalized cAMP signalling in mammalian cells through targeting to specific signalling complexes. Their importance is apparent as PDE4 selective inhibitors exert profound anti-inflammatory effects and act as cognitive enhancers. The p38 MAPK (mitogen-activated protein kinase) signalling cascade is a key signal transduction pathway involved in the control of cellular immune, inflammatory and stress responses. In the present study, we show that PDE4A5 is phosphorylated at Ser147, within the regulatory UCR1 (ultraconserved region 1) domain conserved among PDE4 long isoforms, by MK2 (MAPK-activated protein kinase 2, also called MAPKAPK2). Phosphorylation by MK2, although not altering PDE4A5 activity, markedly attenuates PDE4A5 activation through phosphorylation by protein kinase A. This modification confers the amplification of intracellular cAMP accumulation in response to adenylate cyclase activation by attenuating a major desensitization system to cAMP. Such reprogramming of cAMP accumulation is recapitulated in wild-type primary macrophages, but not MK2/3-null macrophages. Phosphorylation by MK2 also triggers a conformational change in PDE4A5 that attenuates PDE4A5 interaction with proteins whose binding involves UCR2, such as DISC1 (disrupted in schizophrenia 1) and AIP (aryl hydrocarbon receptor-interacting protein), but not the UCR2-independent interacting scaffold protein β-arrestin. Long PDE4 isoforms thus provide a novel node for cross-talk between the cAMP and p38 MAPK signalling systems at the level of MK2.

scholarly journals TSH Regulates Pendrin Membrane Abundance and Enhances Iodide Efflux in Thyroid Cells

Endocrinology ◽  
2012 ◽  
Vol 153 (1) ◽  
pp. 512-521 ◽  
Author(s):  
Liuska Pesce ◽  
Aigerim Bizhanova ◽  
Juan Carlos Caraballo ◽  
Whitney Westphal ◽  
Maria L. Butti ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Protein Kinase ◽  
Protein Kinase A ◽  
Phosphorylation Site ◽  
Physiological Role ◽  
Thyroid Cells ◽  
Hormone Synthesis ◽  
Iodide Transport ◽  
Rat Thyroid ◽  
Kinase A

Thyroid hormones are essential for normal development and metabolism. Their synthesis requires transport of iodide into thyroid follicles. The mechanisms involving the apical efflux of iodide into the follicular lumen are poorly elucidated. The discovery of mutations in the SLC26A4 gene in patients with Pendred syndrome (congenital deafness, goiter, and defective iodide organification) suggested a possible role for the encoded protein, pendrin, as an apical iodide transporter. We determined whether TSH regulates pendrin abundance at the plasma membrane and whether this influences iodide efflux. Results of immunoblot and immunofluorescence experiments reveal that TSH and forskolin rapidly increase pendrin abundance at the plasma membrane through the protein kinase A pathway in PCCL-3 rat thyroid cells. The increase in pendrin membrane abundance correlates with a decrease in intracellular iodide as determined by measuring intracellular 125iodide and can be inhibited by specific blocking of pendrin. Elimination of the putative protein kinase A phosphorylation site T717A results in a diminished translocation to the membrane in response to forskolin. These results demonstrate that pendrin translocates to the membrane in response to TSH and suggest that it may have a physiological role in apical iodide transport and thyroid hormone synthesis.

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