Quantitative analysis of protein phosphorylation status and protein kinase activity on microarrays using a novel fluorescent phosphorylation sensor dye

PROTEOMICS ◽  
2003 ◽  
Vol 3 (7) ◽  
pp. 1244-1255 ◽  
Author(s):  
Karen Martin ◽  
Thomas H. Steinberg ◽  
Laurie A. Cooley ◽  
Kyle R. Gee ◽  
Joseph M. Beechem ◽  
...  
Keyword(s):  
Quantitative Analysis ◽  
Protein Kinase ◽  
Protein Phosphorylation ◽  
Kinase Activity ◽  
Protein Kinase Activity

Fluorescent Polyion Complex Nanoparticle That Incorporates an Internal Standard for Quantitative Analysis of Protein Kinase Activity

2014 ◽  
Vol 25 (5) ◽  
pp. 869-872 ◽  
Author(s):  
Takanobu Nobori ◽  
Shujiro Shiosaki ◽  
Takeshi Mori ◽  
Riki Toita ◽  
Chan Woo Kim ◽  
...  
Keyword(s):  
Quantitative Analysis ◽  
Protein Kinase ◽  
Kinase Activity ◽  
Internal Standard ◽  
Protein Kinase Activity ◽  
Polyion Complex

Phosphorylation-dependent modulation of cardiac calcium current by intracellular free magnesium

2001 ◽  
Vol 281 (4) ◽  
pp. H1532-H1544 ◽  
Author(s):  
Siegried Pelzer ◽  
Chicuong La ◽  
Dieter J. Pelzer
Keyword(s):  
Protein Kinase ◽  
Protein Phosphorylation ◽  
Calcium Current ◽  
Kinase Activity ◽  
Kinase Inhibitor ◽  
Protein Kinase Activity ◽  
Inhibitory Effects ◽  
Maximal Stimulation ◽  
Free Magnesium ◽  
Stimulation Of

We compared the effects of cytosolic free magnesium (Mg[Formula: see text]) on L-type Ca2+ current ( I Ca,L) in patch-clamped guinea pig ventricular cardiomyocytes under basal conditions, after inhibition of protein phosphorylation, and after stimulation of cAMP-mediated phosphorylation. Basal I Ca,L density displayed a bimodal dependence on the concentration of Mg[Formula: see text]([Mg2+]i; 10−6–10−2 M), which changed significantly as cell dialysis progressed due to a pronounced and long-lasting rundown of I Ca,L in low-Mg2+ dialysates. Ten minutes after patch breakthrough, I Ca,L density (at +10 mV) in Mg[Formula: see text]-depleted cells ([Mg2+]i∼1 μM) was elevated, increased to a maximum at ∼20 μM [Mg2+]i, and declined steeply at higher [Mg2+]i. Treatment with the broad-spectrum protein kinase inhibitor K252a (10 μM) reduced I Ca,L density and abolished these effects of Mg[Formula: see text] except for a negative shift of I Ca,L-voltage relations with increasing [Mg2+]i. Maximal stimulation of cAMP-mediated phosphorylation occluded the Mg[Formula: see text]-induced stimulation of I Ca,L and prevented inhibitory effects of the ion at [Mg2+]i <1 mM but not at higher concentrations. These results show that the modulation of I Ca,L by Mg[Formula: see text] requires protein kinase activity and likely originates from interactions of the ion with proteins involved in the regulation of protein phosphorylation/dephosphorylation. Stimulatory effects of Mg[Formula: see text] on I Ca,L seem to increase the cAMP-mediated phosphorylation of Ca2+ channels, whereas inhibitory effects of Mg[Formula: see text] appear to curtail and/or reverse cAMP-mediated phosphorylation.

scholarly journals Inhibition of cerebral protein kinase activity and cyclic AMP-dependent ribosomal-protein phosphorylation in experimental hyperphenylalaninaemia

1982 ◽  
Vol 202 (2) ◽  
pp. 343-351 ◽  
Author(s):  
Sidney Roberts ◽  
Beatrice S. Morelos
Keyword(s):  
Protein Kinase ◽  
Cyclic Amp ◽  
Ribosomal Protein ◽  
Protein Phosphorylation ◽  
Kinase Activity ◽  
Activity Ratio ◽  
Protein Kinase Activity ◽  
Control Infant ◽  
Infant Rats ◽  
Dependent Protein

Studies were carried out to elucidate the mechanisms underlying the diminished phosphorylation of cerebral ribosomal protein in experimental hyperphenylalaninaemia [Roberts & Morelos (1980) Biochem. J.190, 405–419]. Administration of N6,O2′-dibutyryl cyclic AMP or 3-isobutyl-1-methylxanthine, which increased phosphorylation of the S6 protein of cerebral 40S ribosomal subunits in control infant rats, did not counteract the decreased phosphorylation of this ribosomal protein resulting from intraperitoneal administration of a loading dose of l-phenylalanine. N2,O2′-Dibutyryl cyclic GMP had no effect on cerebral ribosomal-protein phosphorylation in either control or hyperphenylalaninaemic animals. The phenylalanine-induced decrease in ribosomal-protein phosphorylation was associated with decreased protein kinase activity in cerebral cytosolic and microsomal preparations. However, the maximal protein kinase response to cyclic AMP added in vitro was unaltered by prior administration of phenylalanine in vivo. The heat-stable protein inhibitor of cyclic AMP-dependent protein kinases decreased the activity of these enzymes by about 90% and eliminated the phenylalanine-induced difference in protein kinase activity in the absence of added cyclic AMP. Intracisternal administration of doses of dibutyryl cyclic AMP or 3-isobutyl-1-methylxanthine which increased the cyclic AMP-dependent protein kinase activity ratio in control infant rats was without effect on this index in phenylalanine-treated animals. Dibutyryl cyclic GMP had no effect on the protein kinase activity ratio in either group of animals. These results suggest that inhibition of cerebral cyclic AMP-dependent protein kinases by abnormally high concentrations of phenylalanine may contribute to the decrease in cerebral ribosomal-protein phosphorylation in experimental hyperphenylalaninaemia.

Comparison of protein kinase activity and protein phosphorylation in the medial vestibular nucleus and prepositus hypoglossi in labyrinthine-intact and labyrinthectomized guinea pigs

2000 ◽  
Vol 10 (2) ◽  
pp. 107-117
Author(s):  
DeWana R. Kerr ◽  
Andrew J. Sansom ◽  
Paul F. Smith ◽  
Cynthia L. Darlington
Keyword(s):  
Protein Kinase ◽  
Protein Phosphorylation ◽  
Guinea Pigs ◽  
Kinase Activity ◽  
Vestibular Nucleus ◽  
Vestibular Compensation ◽  
Medial Vestibular Nucleus ◽  
Protein Kinase Activity ◽  
Sham Operation ◽  
Prepositus Hypoglossi

The aim of the present study was to compare in vitro protein expression, protein kinase activity and protein phosphorylation in the medial vestibular nucleus (MVN) and prepositus hypoglossi (PH) from labyrinthine-intact guinea pigs and from guinea pigs at various stages of vestibular compensation following unilateral labyrinthectomy (UL). The ipsilateral (I-MVN) and contralateral (C-MVN) MVN, and the ipsilateral (I-PH) and contralateral (C-PH) PH, were dissected from 3 naive labyrinthine-intact guinea pigs and 55 guinea pigs at 10 hs or 53 hs following a surgical UL or sham operation. Tissue extracts were incubated with [gamma- 33 P]ATP ± Ca 2 + , phorbal 12, 13 dibutyrate and phosphatidylserine or ± Ca 2 + and calmodulin, to enhance protein kinase C (PKC) or calcium calmodulin kinase (CaMK) activity, respectively. Data were analysed as the ratio of activated to basal 33 P incorporation detected by phosphorimaging. There were similar total protein and phosphoprotein profiles in the MVN and PH, as well as both PKC and CaMKII activity, suggesting that the MVN and PH are similar in the way that proteins undergo rapid modification by phosphorylation. During the development of vestibular compensation, a 46 kDa band in C-PH displayed higher PKC-mediated phosphorylation from 10 hs post-UL compared to sham controls. Significantly greater PKC-mediated phosphorylation of proteins of approximately 18, 46 and 75 kDa was observed in C-PH at 10 hs compared to 53 hs post-UL and in most cases the phosphorylation was greater in C-PH than in the C-MVN. These results suggest that between 10 and 53 hs post-UL, PKC-mediated phosphorylation changes mainly in the C-PH rather than the ipsilateral or contralateral MVN.

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