Adenosine 3':5'-Monophosphate-Dependent Protein Kinase
from Human Placenta:
Characterization of the Catalytic Subunit

Tamanini, Anna; Berton, Giorgio; Cabrini, Giulio

doi:10.1159/000468874

Cited by 5 publications

(1 citation statement)

References 8 publications

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“…K I values were derived from the IC 50 plots using eq 1: where [S] is the substrate concentration and K M is the Michaelis constant for the enzyme and the ATP substrate. Using a K M of 50 mM for ATP, the IC 50 values translated to K I values of 22 ± 3 mM and 28 ± 4 nM, respectively, which compare well to literature K I values of 8.3 mM and 48 nM. , The slight variability in the numbers may be due to the choice of K M for ATP when calculating K I . Reported K M values for ATP binding to PKA range from 10 to 100 mM; thus, we used the mean of these values as the K M for ATP in the calculation.…”

Section: Resultssupporting

confidence: 71%

Nanovolume Kinase Inhibition Assay Using a Sol−Gel-Derived Multicomponent Microarray

2005

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We report on the development of a new class of kinase microarrays based on the coimmobilization of both kinase and substrate components within a single pin-printed sol-gel microarray element and the use of such arrays for nanovolume inhibition assays. We successfully immobilized the alpha-catalytic subunit of cAMP-dependent protein kinase (PKA) and the peptide substrate kemptide within sol-gel-derived microarrays for the purpose of monitoring phosphorylation and inhibition. Using Pro-Q Diamond stain as an end-point indicator of phosphorylation, we demonstrate the selective detection of phosphoproteins over nonphosphorylated controls and the ability to detect phosphorylated proteins over a 500-fold concentration range. Limits of detection for the phosphoprotein beta-casein were 7.5 pg, and the detectable signal remained linear up to 3.75 ng of protein per array spot. PKA is demonstrated to be active when coentrapped with two different substrates, and inhibition assays for PKA with the inhibitors H7 and H89 demonstrate the ability to detect kinase inhibition as well as derive IC50 plots from a single array using an overprinting method to deliver approximately 0.6 nL of reagent per array element, or a total of 72 nL of reagents to generate a full, 12-point IC50 curve in pentuplicate.

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Section: Resultssupporting

confidence: 71%

Nanovolume Kinase Inhibition Assay Using a Sol−Gel-Derived Multicomponent Microarray

2005

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Activation of protein kinase A is a pivotal step involved in both BMP-2- and cyclic AMP-induced chondrogenesis

Lee

Chuong

1997

J. Cell. Physiol.

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We studied the roles of protein kinase A (PKA) activation and cyclic AMP response element binding protein (CREB) phosphorylation in chondrogenesis using serum-free chicken limb bud micromass cultures as a model system. We showed the following points: 1) in micromass cultures, activation of PKA enhances chondrogenesis and increases the phosphorylation of CREB; 2) BMP-2, a chondrogenic stimulator, increases PKA activity and the level of phosphorylated CREB (P-CREB); 3) H8, a PKA inhibitor, inhibits chondrogenesis; 4) the chondrogenic activities of BMP-2 and cAMP are suppressed by H8; and 5) long-term TPA treatment (a protein kinase C (PKC) modulator) inhibits chondrogenesis and decreases the levels of CREB and P-CREB. These results suggest that activation of PKA is a physiological event during chondrogenesis that is involved in the chondrogenic effects of both BMP-2 and cyclic AMP (cAMP)-dependent pathways.

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Parathyroid hormone-related peptide inhibits the expression of bone morphogenetic protein-4 mRNA through a cyclic AMP/protein kinase A pathway in mouse clonal chondrogenic EC cells, ATDC5

Ito

Akiyama

Shigeno

et al. 2000

Biochimica et Biophysica Acta (BBA) - Molecular Cell Research

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The bone morphogenetic proteins (BMPs) play crucial roles in chondrogenic differentiation. Little is known, however, regarding the regulation of BMP gene expression. Here we examined the effect of parathyroid hormone-related peptide (PTHrP) (1-141), a full-length form of PTHrP molecules, on the expression of BMP-4 mRNA in clonal mouse chondrogenic EC cells, ATDC5. In differentiated ATDC5 cells, the expression of BMP-4 mRNA was inhibited by PTHrP (1-141), which stimulated cAMP accumulation and protein kinase A (PKA) activity in these cells. Dibutyryl cAMP, a permeable analog of cAMP, mimicked and H-89, a selective PKA inhibitor, blocked this effect of PTHrP (1-141). Moreover, actinomycin D attenuated the inhibition of BMP-4 mRNA expression by PTHrP (1-141). These results indicate that PTHrP (1-141) transcriptionally inhibits the expression of BMP-4 mRNA through a cAMP/PKA pathway in ATDC5 cells.

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Adenosine 3':5'-Monophosphate-Dependent Protein Kinase from Human Placenta: Characterization of the Catalytic Subunit

Cited by 5 publications

References 8 publications

Nanovolume Kinase Inhibition Assay Using a Sol−Gel-Derived Multicomponent Microarray

Nanovolume Kinase Inhibition Assay Using a Sol−Gel-Derived Multicomponent Microarray

Activation of protein kinase A is a pivotal step involved in both BMP-2- and cyclic AMP-induced chondrogenesis

Parathyroid hormone-related peptide inhibits the expression of bone morphogenetic protein-4 mRNA through a cyclic AMP/protein kinase A pathway in mouse clonal chondrogenic EC cells, ATDC5

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