Spatio-temporal imaging of EGF-induced activation of protein kinase A by FRET in living cells

2004 ◽  
Author(s):  
Jin Jun Wang ◽  
Xiao-Chuan Chen ◽  
Da Xing
Keyword(s):  
Protein Kinase ◽  
Protein Kinase A ◽  
Living Cells ◽  
Spatio Temporal ◽  
Kinase A

scholarly journals Monitoring of cAMP Synthesis and Degradation in Living Cells

Physiology ◽  
2006 ◽  
Vol 21 (2) ◽  
pp. 86-92 ◽  
Author(s):  
Viacheslav O. Nikolaev ◽  
Martin J. Lohse
Keyword(s):  
Protein Kinase ◽  
Protein Kinase A ◽  
Cyclic Nucleotide ◽  
Living Cells ◽  
Cellular Effects ◽  
Cyclic Nucleotide Gated Channels ◽  
Camp Synthesis ◽  
Spatio Temporal ◽  
Synthesis And Degradation ◽  
Kinase A

cAMP is an important second messenger with a plethora of cellular effects and biological roles. To monitor and visualize cAMP in intact living cells, electrophysiological and fluorescent methods have been developed based on activation of all three types of cAMP effectors: protein kinase A, cyclic nucleotide-gated channels, and exchange protein directly activated by cAMP. In this review, we describe and compare these techniques in terms of their robustness, sensitivity and spatio-temporal resolution.

scholarly journals Quantitation of protein kinase A-mediated trafficking of cardiac sodium channels in living cells

2006 ◽  
Vol 72 (2) ◽  
pp. 250-261 ◽  
Author(s):  
H HALLAQ ◽  
Z YANG ◽  
P VISWANATHAN ◽  
K FUKUDA ◽  
W SHEN ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Protein Kinase A ◽  
Sodium Channels ◽  
Living Cells ◽  
Cardiac Sodium Channels ◽  
Kinase A

scholarly journals In spermatozoa, Stat1 is activated during capacitation and the acrosomal reaction

Reproduction ◽  
2007 ◽  
Vol 134 (3) ◽  
pp. 425-433 ◽  
Author(s):  
Yadira Bastián ◽  
Armando Zepeda-Bastida ◽  
Salvador Uribe ◽  
Adela Mújica
Keyword(s):  
Transcription Factors ◽  
Protein Kinase ◽  
Embryonic Development ◽  
Protein Kinase A ◽  
Signal Transducer ◽  
Acrosomal Reaction ◽  
Specific Proteins ◽  
Spatio Temporal ◽  
And Migration ◽  
Kinase A

A role for sperm-specific proteins during the early embryonic development has been suggested by a number of recent studies. However, little is known about the participation of transcription factors in that stage. Here, we show that the signal transducer and activator of transcription 1 (Stat1), but not Stat4, was phosphorylated in response to capacitation and the acrosomal reaction (AR). Moreover, Stat1 phosphorylation correlated with changes in its localization: during capacitation, Stat1 moved from the cytoplasm to the theca/flagellum fraction. During AR, Stat1 phosphorylation increased again. In addition, blocking protein kinase A (PKA) and PKC during capacitation abolished both phosphorylation and migration of Stat1. Our results show tight spatio–temporal rearrangements of Stat1, suggesting that after fertilization Stat1 participates in the first rounds of transcription within the male pronucleus.

scholarly journals A Membrane Permeable Prodrug of S223 for Selective Epac2 Activation in Living Cells

Cells ◽  
2019 ◽  
Vol 8 (12) ◽  
pp. 1589 ◽  
Author(s):  
Yunjian Xu ◽  
Frank Schwede ◽  
Hans Wienk ◽  
Anders Tengholm ◽  
Holger Rehmann
Keyword(s):  
Protein Kinase ◽  
Protein Kinase A ◽  
Cyclic Nucleotide ◽  
Adenosine Monophosphate ◽  
Living Cells ◽  
Effector Proteins ◽  
Nucleotide Exchange ◽  
Acetoxymethyl Ester ◽  
Different Types ◽  
Kinase A

Signalling by cyclic adenosine monophosphate (cAMP) occurs via various effector proteins, notably protein kinase A and the guanine nucleotide exchange factors Epac1 and Epac2. These proteins are activated by cAMP binding to conserved cyclic nucleotide binding domains. The specific roles of the effector proteins in various processes in different types of cells are still not well defined, but investigations have been facilitated by the development of cyclic nucleotide analogues with distinct selectivity profiles towards a single effector protein. A remaining challenge in the development of such analogues is the poor membrane permeability of nucleotides, which limits their applicability in intact living cells. Here, we report the synthesis and characterisation of S223-AM, a cAMP analogue designed as an acetoxymethyl ester prodrug to overcome limitations of permeability. Using total internal reflection imaging with various fluorescent reporters, we show that S223-AM selectively activates Epac2, but not Epac1 or protein kinase A, in intact insulin-secreting β-cells, and that this effect was associated with pronounced activation of the small G-protein Rap. A comparison of the effects of different cAMP analogues in pancreatic islet cells deficient in Epac1 and Epac2 demonstrates that cAMP-dependent Rap activity at the β-cell plasma membrane is exclusively dependent on Epac2. With its excellent selectivity and permeability properties, S223-AM should get broad utility in investigations of cAMP effector involvement in many different types of cells.

Predicted location and limited accessibility of protein kinase A phosphorylation site on Na-K-ATPase

2001 ◽  
Vol 280 (4) ◽  
pp. C1017-C1026 ◽  
Author(s):  
Kathleen J. Sweadner ◽  
Marina S. Feschenko
Keyword(s):  
Protein Kinase ◽  
Protein Kinase A ◽  
Phosphorylation Site ◽  
Living Cells ◽  
Catalytic Subunit ◽  
Future Research ◽  
Dependent Protein Kinase ◽  
Plasmic Reticulum ◽  
Dependent Protein ◽  
Kinase A

Regulation of Na-K-ATPase by cAMP-dependent protein kinase occurs in a variety of tissues. Phosphorylation of the enzyme's catalytic subunit at a classical phosphorylation consensus motif has been observed with purified enzyme. Demonstration of phosphorylation at the same site in normal living cells or tissues has been more difficult, however, making it uncertain that the Na-K-ATPase is a direct physiological substrate of the kinase. Recently, the structure of the homologous sarco(endo)plasmic reticulum Ca-ATPase (SERCA1a) has been determined at 2.6 Å resolution (Toyoshima C, Nakasako M, Nomura H, and Ogawa H. Nature 405: 647–655, 2000.), and the Na-K- ATPase should have the same fold. Here, the Na-K-ATPase sequence has been aligned with the Ca-ATPase structure to examine the predicted disposition of the phosphorylation site. The location is close to the membrane and partially buried by adjacent loops, and the site is unlikely to be accessible to the kinase in this conformation. Conditions that may expose the site or further bury it are discussed to highlight the issues facing future research on regulation of Na-K-ATPase by cAMP-dependent pathways.

Regulation of spontaneous and induced resumption of meiosis in mouse oocytes by different intracellular pathways

Reproduction ◽  
2000 ◽  
pp. 377-383 ◽  
Author(s):  
L Leonardsen ◽  
A Wiersma ◽  
M Baltsen ◽  
AG Byskov ◽  
CY Andersen
Keyword(s):  
Signal Transduction ◽  
Protein Kinase ◽  
Protein Kinase A ◽  
Mitogen Activated Protein Kinase ◽  
Meiotic Maturation ◽  
Mouse Oocytes ◽  
Mitogen Activated Protein ◽  
Dependent Signal ◽  
Kinase Pathway ◽  
Kinase A

The mitogen-activated protein kinase-dependent and the cAMP-protein kinase A-dependent signal transduction pathways were studied in cultured mouse oocytes during induced and spontaneous meiotic maturation. The role of the mitogen-activated protein kinase pathway was assessed using PD98059, which specifically inhibits mitogen-activated protein kinase 1 and 2 (that is, MEK1 and MEK2), which activates mitogen-activated protein kinase. The cAMP-dependent protein kinase was studied by treating oocytes with the protein kinase A inhibitor rp-cAMP. Inhibition of the mitogen-activated protein kinase pathway by PD98059 (25 micromol l(-1)) selectively inhibited the stimulatory effect on meiotic maturation by FSH and meiosis-activating sterol (that is, 4,4-dimethyl-5alpha-cholest-8,14, 24-triene-3beta-ol) in the presence of 4 mmol hypoxanthine l(-1), whereas spontaneous maturation in the absence of hypoxanthine was unaffected. This finding indicates that different signal transduction mechanisms are involved in induced and spontaneous maturation. The protein kinase A inhibitor rp-cAMP induced meiotic maturation in the presence of 4 mmol hypoxanthine l(-1), an effect that was additive to the maturation-promoting effect of FSH and meiosis-activating sterol, indicating that induced maturation also uses the cAMP-protein kinase A-dependent signal transduction pathway. In conclusion, induced and spontaneous maturation of mouse oocytes appear to use different signal transduction pathways.

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