Presence Of Protein Kinase Activity Research Articles

DNA-hosted copper nanoclusters/graphene oxide based fluorescent biosensor for protein kinase activity detection

A novel fluorescent biosensor for protein kinase activity (PKA) detection was designed by applying double-strands DNA-hosted copper nanoclusters (dsDNA-CuNCs) and graphene oxide (GO). One DNA strand of the dsDNA consisted of two domains, one domain can hybridize with another complementary DNA strand to stabilize the fluorescent CuNCs and another domain was adenosine 5′-triphosphate (ATP) aptamer. ATP aptamer of the dsDNA-CuNCs would be spontaneously absorbed onto the GO surface through π-π stacking interactions. Thus GO can efficiently quench the fluorescence (FL) of dsDNA-CuNCs through fluorescence resonance energy transfer (FRET). In the present of ATP, ATP specifically combined with ATP aptamer to form ATP-ATP aptamer binding complexes, which had much less affinity to GO, resulting in the fluorescence recovery of the system. Nevertheless, in the presence of PKA, ATP could be translated into ADP and ADP could not combine with ATP aptamer resulting in the fluorescence quenching of dsDNA-CuNCs again. According to the change of the fluorescence signal, PKA activity could be successfully monitored in the range of 0.1–5.0 U mL−1 with a detection limit (LOD) of 0.039 U mL−1. Besides, the inhibitory effect of H-89 on PKA activity was studied. The sensor was performed for PKA activity detection in cell lysates with satisfactory results.

Translocation of Activated Heterotrimeric G Protein Gαo to Ganglioside-enriched Detergent-resistant Membrane Rafts in Developing Cerebellum

The association of gangliosides with specific proteins in the central nervous system was examined by co-immunoprecipitation with an anti-ganglioside antibody. The monoclonal antibody to the ganglioside GD3 immunoprecipitated phosphoproteins of 40, 53, 56, and 80 kDa from the rat cerebellum. Of these proteins, the 40-kDa protein was identified as the alpha-subunit of a heterotrimeric G protein, G(o) (Galpha(o)). Using sucrose density gradient analysis of cerebellar membranes, Galpha(o), but not Gbetagamma, was observed in detergent-resistant membrane (DRM) raft fractions in which GD3 was abundant after the addition of guanosine 5'-O-(thiotriphosphate) (GTPgammaS), which stabilizes G(o) in its active form. On the other hand, both Galpha(o) and Gbetagamma were excluded from the DRM raft fractions in the presence of guanyl-5'-yl thiophosphate, which stabilizes G(o) in its inactive form. Only Galpha(o) was observed in the DRM fractions from the cerebellum on postnatal day 7, but not from that in adult. After pertussis toxin treatment, Galpha(o) was not observed in the DRM fractions, even from the cerebellum on postnatal day 7. These results indicate the activation-dependent translocation of Galpha(o) into the DRM rafts. Furthermore, Galpha(o) was concentrated in the neuronal growth cones. Treatment with stromal cell-derived factor-1alpha, a physiological ligand for the G protein-coupled receptor, stimulated [(35)S]GTPgammaS binding to Galpha(o) and caused Galpha(o) translocation to the DRM fractions and RhoA translocation to the membrane fraction, leading to the growth cone collapse of cerebellar granule neurons. The collapse was partly prevented by pretreatment with the cholesterol-sequestering and raft-disrupting agent methyl-beta-cyclodextrin. These results demonstrate the involvement of signal-dependent Galpha(o) translocation to the DRM in the growth cone behavior of cerebellar granule neurons.

Interferon gamma bound to endothelial cells is phosphorylated by ecto-protein kinases.

The presence of protein kinase activity and its phosphorylated products has been demonstrated on the outer surface of the plasma membrane of endothelial cells. Extracellular phosphorylation was detected by incubation of primary endothelial cells (HUVEC's) and endothelial cell line EA.hy 926 with [gamma-32P]ATP. The reaction products were subjected to SDS/PAGE, autoradiography and scanning densitometry. Under the experimental conditions, five proteins with apparent molecular masses of 19, 23, 55, 88, and 110 kDa were prominently phosphorylated in both types of cells. Phosphorylation of the 19 kDa protein was the most rapid reaching maximum after 60 s and then the protein became dephosphorylated. Ecto-protein kinases responsible for the surface labeling of membrane proteins were characterized by using (a) protein kinase C inhibitors: K-252b, chelerythrine chloride, and [Ala113] myelin basic protein (104-118), (b) protein kinase A inhibitor Kemptide 8334, and (c) casein kinase II inhibitor 5,6-dichloro-1-beta-D-ribofuranosyl benzimidazole (DRB). Stimulation of endothelial cells with tumor necrosis factor alpha (TNF alpha) and interferon gamma (IFN gamma) is associated with 20-80% reduction of extracellular phosphorylation of all membrane proteins. IFN gamma bound to membrane receptors becomes rapidly phosphorylated. Only in the case of IFN gamma it was associated with the appearance of a strongly phosphorylated band of 17 kDa corresponding to IFN gamma itself. Phosphorylation of this 17 kDa exogenous substrate was prevented by an ecto-kinase inhibitor K-252b. The existence of ecto-phosphoprotein phosphatase activity in endothelial cells was evidenced by testing the effect of microcystin LR--a membrane impermeable reagent that inhibits both PP-1 and PP-2a phosphoprotein phosphatases. The extent of phosphorylation of 19 kDa and 110 kDa phosphoproteins significantly increased in the presence of microcystin. Our results suggest the presence of at least two ecto-kinase activities on endothelial cells that may play a significant role(s) in the regulation of cytokines function.

Non-histone chromatin proteins in beef thyroid: distinct phosphorylation patterns of several protein kinases

Non-histone chromatin protein (NHCP) fractions were extracted from purified beef thyroid nuclear preparations and tested for the presence of protein kinase activities using several known mediators of thyroid regulation, as well as potential phosphotransferase substrates using purified or partially purified protein kinase activities. The addition of cAMP/3-isobutyl-1-methylxanthine had no effect on NHCP histone kinase activity; the addition of 10 micrograms of the heat-stable cAMP-dependent protein kinase A inhibitor, however, resulted in a 47% reduction in histone H1 kinase activity. Nuclear casein kinase II activity was present in the NHCP fractions as evidenced by the capacity of spermine to stimulate (ED50 = 0.19 mM) and heparin to inhibit (ID50 = 0.09 microgram/ml) the phosphorylation of casein; further, the phosphotransferase activity could be purified by sequential casein-agarose and spermine-agarose affinity chromatography. Neither calcium-calmodulin nor calcium/phosphatidylserine/diolein had an effect on NHCP casein kinase or histone kinase activities, respectively. The addition of cAMP-dependent protein kinase A catalytic subunit, nuclear casein kinase II, calcium-activated calmodulin-dependent protein kinase and diacylglycerol-activated calcium/phospholipid-dependent protein kinase C activities exhibited distinct phosphorylation patterns when NHCP were used as substrates and analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis and autoradiography. We conclude that NHCP fraction from beef thyroid: 1) contains both cAMP-dependent protein kinase A catalytic subunit and nuclear casein kinase II and 2) substrates for cAMP-dependent protein kinase A, calcium-activated calmodulin-dependent protein kinase, protein kinase C, and nuclear casein kinase II.

A purified 124-kDa oat phytochrome does not possess a protein kinase activity.

The presence of protein kinase activity in the purified phytochrome preparations [Wong, et al. (1986) J. Biol. Chem. 261, 12089-12097] has been re-examined. The phytochrome preparations having SAR (specific absorbance ratio, A668/A280 for the Pr form as a measure of phytochrome purity) values of greater than 0.95 were homogeneous on SDS gel, but could be further purified to a SAR value of 1.07 by repeated gel filtrations on a Bio-Gel A-0.5 m column. The protein kinase activity remained in the phytochrome preparations having SAR values less than 1.05, but it became undetectable in the phytochrome preparation with a SAR value of 1.07. Two dimensional gel electrophoresis of the phytochrome preparation (SAR, 0.89) showed that a phytochrome band with pl 5.8 had no kinase activity. Phosphorylating activity of the protein kinase was enhanced to some extent by polycations, polylysine and histone. Phytochrome served as a good substrate for this enzyme. The present data indicate that phytochrome has no intrinsic protein kinase activity, but a protein kinase is present in highly purified phytochrome preparations.

Protein phosphorylation in human placenta stimulation by epidermal growth factor

Membranes prepared from normal human term placenta possess a protein kinase activity which phosphorylates endogenous substrates in the presence of [γ- 32P]ATP. This kinase activity requires either Mg 2+ or Mn 2+, is enhanced by glycerol and appears to be cyclic-nucleotide-independent. Addition of epidermal growth factor to the placental membrane preparation increases the level of total phosphorylation by approx. 35%. When analyzed by sodium dodecyl sulfate gel electrophoresis and autoradiography, the placental proteins whose degree of phosphorylation was enhanced by epidermal growth factor had apparent molecular weights of 170 000, 150 000 and 25 000. This report documents the presence of protein kinase activity in human placenta and demonstrates that epidermal growth factor can enhance protein phosphorylation in normal human tissue.

The presence of protein kinase activity and acceptors of phosphate groups in nonpolysomal cytoplasmic messenger ribonucleoprotein complexes of embryonic chicken muscle.

Nonpolysomal cytoplasmic (free) mRNA.protein (mRNP) complexes of embryonic chicken muscle were purified by a combination of oligo(dT)-cellulose chromatography and sucrose density gradient centrifugation. The protein moieties of the purified mRNP complex were analyzed by two-dimensional gel electrophoresis using separation according to charge in the first dimension and molecular weight in the second. Sixteen polypeptides of Mr = 27,000 to 75,000 were present in the mRNP complex. These mRNP polypeptides displayed different electrophoretic migration properties than those of ribosomal proteins. A protein kinase activity was found associated with the mRNP. This enenzyme was able to transfer phosphate group(s) from ATP to at least three acidic mRNP polypeptides of Mr = 27,000, 38,000, and 73,000 and one basic polypeptide of Mr = 75,000. Among these, the Mr = 38,000 acidic polypeptide was the best acceptor of phosphate groups.

Cyclic 3',5'-AMP-stimulated and non-stimulated phosphorylation of protein fractions from rat-liver cell sap on incubation with (gamma-32P)ATP.

Rat-liver cell sap protein was separated into four fractions by pH 5.5 precipitation and ammonium sulfate fractionation. On incubation with (32P)ATP, protein of two of the fractions was phosphorylated, showing the presence of protein kinase activity and endogenous protein substrates. The phosphorylation of one of the fractions was markedly stimulated by cyclic 3′,5′-AMP. Strong evidence was obtained that this phosphate-incorporating material was neither active phosphorylase, phosphorylase kinase nor glycogen synthetase. The two phosphate-incorporating fractions were chromatographed on DEAE-cellulose with stepwise elution. Several subtractions contained material which was phosphorylated on incubation with (32P)ATP. The phosphorylation of two subfractions was greatly stimulated by cyclic 3′,5′-AMP. By polyacrylamide gel electrophoresis in detergent of (32P)ATP-incubated material from these fractions, three major 32P-labelled components with estimated molecular weights of 62000, 55000 and 47 000 were demonstrated. It is supposed that these components are derived from enzymes or other proteins with functions regulated by phosphorylation-dephosphorylation reactions.