Journal of cyclic nucleotide and protein phosphorylation research最新文献

Forskolin is a unique diterpene activator of adenylate cyclase which has been extensively used in the study of cAMP generating systems. This report describes the production of antibodies to forskolin and the optimization of two sensitive assay methods for such antibodies. 7-0-Hemisuccinyl 7-deacetyl forskolin, coupled to either human serum albumin or goat IgG, was injected into goats to elicit antibodies to the forskolin hapten. Two assay methods, a radioimmunoassay with [12-3H]forskolin as a tracer and a colorimetric enzyme-linked immunosorbent assay (ELISA) with horse radish peroxidase-labelled rabbit anti-goat IgG as an indicator, were optimized to test for the presence of forskolin antibodies in antisera and isolated IgG fractions. The titers for forskolin antisera were 4000-10000. Both assay methods can be adapted to quantify forskolin and its protein conjugates. The availability of antibodies to this diterpene will be useful in accelerating the understanding of the mechanism of adenylate cyclase activation by forskolin.

The adenylate cyclase activity of membranes prepared from rat lung, measured under standard assay conditions, was markedly increased by the presence of a crude supernatant fraction prepared from rat lung, liver, or brain. This was not due to an increase in the initial rate of cyclic AMP (cAMP) synthesis, but to the maintenance of a constant rate of cAMP synthesis for periods of at least 10 min. After incubating lung membranes in the cyclase reaction mixture until cAMP synthesis had virtually ceased (10 min), the addition of alpha-(32P)-ATP caused a marked increase in the activity of the enzyme. This was the only component of the original reaction mixture that supported re-initiation of cAMP synthesis. Re-initiation also occurred when supernatant was added. This implies that substrate depletion occurs in the presence of membranes and that lung supernatant can catalyze rapid regeneration of substrate. Chromatographic analysis confirmed that ATP was rapidly hydrolyzed to AMP in the presence of the membranes, that this rapid destruction of ATP did not occur when supernatant was present, and that ATP was resynthesized from AMP when supernatant was added to a reaction mixture in which most of the ATP initially present had been destroyed. The effects of supernatant were mimicked by commercially available adenylate kinase. Addition of adenylate kinase did not affect adenylate cyclase activity measured in membranes prepared from brain, heart, or kidney, suggesting that lung membranes may contain more nucleotide pyrophosphatase and/or less endogenous adenylate kinase activity. Studies of soluble factors that affect adenylate cyclase must carefully control for differential substrate depletion in the presence and absence of tissue extracts.

There is increasing evidence that the recently discovered Atrial Natriuretic Factor, a potent vasorelaxing, natriuretic and diuretic hormone, may achieve some of its diverse physiological effects by regulating the cellular level of cGMP. Since activation of a cGMP-dependent protein kinase is an established effect of cGMP, we have studied the cellular localization of cGMP-dependent protein kinase within the rat kidney cortex using both immunocytochemical and immunochemical procedures. cGMP-dependent protein kinase is selectively concentrated in contractile cells of the kidney vasculature, including both intra- and extraglomerular mesangial cells, vascular smooth muscle cells and microvascular pericytes, as well as in interstitial myofibroblasts. cGMP-dependent protein kinase is not detectable in significant amounts in tubular epithelial cells, podocytes, or endothelial cells. These results support the hypothesis that Atrial Natriuretic Factor may achieve some, and maybe all, of its renal effects by regulating hemodynamic parameters via activation of cGMP-dependent protein kinase within vascular contractile cells.

A monoclonal antibody (CGI-5) directed against the cGMP-inhibited phosphodiesterase isolated from bovine heart was used to examine the phosphorylation of this isozyme in human platelets. PGE1 promoted the phosphorylation of this isozyme, identified as a 110 kDa peptide following SDS-gel electrophoresis. Phosphorylation resulted in approximately a 40% increase in the cGMP-inhibited phosphodiesterase activity. Cell-free experiments demonstrated that cAMP-dependent protein kinase phosphorylated the cGMP-inhibited phosphodiesterase, and that this could be blocked by the heat stable inhibitor peptide (PKI). Phosphorylation of the cGMP-inhibited phosphodiesterase increases the Vmax for cAMP hydrolysis approximately 50%, but does not affect the Km for cAMP (0.12 microM).

Undegraded tyrosine aminotransferase was purified to near homogeneity from rat liver and was confirmed to be a substrate for the beef heart cyclic AMP dependent protein kinase catalytic subunit. Specific antibody was used to quantitate the amount of phosphate incorporated into the enzyme. Phosphate incorporation was maximal at a catalytic subunit to tyrosine aminotransferase molar ratio of 7:1 using 200 microM ATP for 30 to 60 min at 30 degrees C. Phospho-peptide maps of tyrosine aminotransferase phosphorylated in vitro by the catalytic subunit were compared with those of amino-transferase immunoprecipitated from 32P labeled cells treated with and without 8-Br cAMP. Whereas the phospho-peptide maps of tyrosine aminotransferase isolated from cells treated with and without 8-Br cAMP were identical, differences were observed in the peptide map of tyrosine aminotransferase phosphorylated in vitro and in vivo. These results were taken to indicate that the catalytic subunit is not responsible for tyrosine aminotransferase phosphorylation in vivo.

Thio-substituted ATP is a sensitive probe for detecting protein kinase C activity as demonstrated in bovine adrenocortical cell membrane preparations. A single endogenous protein substrate with a molecular weight of approximately 47 Kd was rapidly phosphorylated with [3 5S] gamma-thio-ATP as phosphate donor. Phosphorylation was significantly increased in 30 seconds and reached a plateau by 3 minutes. The activity of the endogenous membrane kinase was unaffected by ACTH, cAMP, calmodulin or trifluoperazine but was responsive to combinations of calcium (Ca), diolein and phosphatidyl serine (PS). In addition, the kinase was activated by the tumor promoting phorbol ester, 12-0-tetradecanoylphorbol-13-acetate, indicating that the membrane contains a protein kinase C and a single 47 Kd phosphorylatable protein substrate. The same substrate is phosphorylated by Ca/diolein/PS activated kinase in membrane preparations from a broad range of rat tissues. Attempts to identify the substrate indicate that it is neither the type I regulatory subunit of cAMP dependent protein kinase nor mitochondrial cytochrome P450.

Photolysis of solutions containing 4-azido-7-phenylpyrazolo-[1,5a]-1,3,5-triazine (APPT) and calmodulin-sensitive cyclic nucleotide phosphodiesterase resulted in reduction of both cyclic GMP and cyclic AMP hydrolytic activity. The inactivation was dependent upon both time of exposure to ultraviolet irradiation and the initial concentration of APPT. The photo-induced inactivation could be attenuated by the presence of cyclic GMP, 1-methyl-3-isobutylxanthine, and papaverine. alpha-Chymotrypsin treatment caused the enzyme to be fully active in the absence of calmodulin but this treatment did not alter the ability of APPT to inactivate the enzyme. Thus, inhibition of calmodulin-binding did not contribute to the photo-induced inactivation. These data indicate that APPT acts as a photoaffinity agent to covalently modify the APPT-binding site of calmodulin-sensitive phosphodiesterase.

In conclusion, multiple protein kinases and phosphatases are involved in the mediation of the physiological response of a specific cell to extracellular stimuli. This can be achieved by changes in the intracellular levels of "mediators" or "second messengers", which alter the activity of selective protein kinases and/or phosphatases. The involvement of reversible protein phosphorylation in the modulation of the function of cell surface receptors, in the synthesis and degradation of the second messengers, altered cellular metabolism, protein synthesis and gene expression suggests a vital role for this regulatory mechanism in the control of cellular function. Protein phosphorylation may be implicated in the interplay between various second messenger systems resulting in a complex regulation of target proteins by multi-site phosphorylation. The presence of unique as well as common targets for the multiple protein kinases and phosphatases suggests a coordinated mechanism for the regulation of cellular function, which can also account for the diversity of cellular response. Future studies will establish the temporal and spatial organization of the regulatory pathways utilized by specific hormones to alter the function of subcellular compartments featured in the overall physiological response.

This report presents the results of detailed examinations of cAMP metabolism in B1r, an S49 lymphoma protein kinase variant with very low phosphodiesterase activity. Among the conclusions reached are: As expected from the low phosphodiesterase activity previously reported, the cAMP turnover rate was relatively slow (t1/2 was 18-23 min at 37 degrees C). Basal cAMP levels ranged from about 1% to 5% of cellular ATP (i.e., 20-100 microM or 100-500 pmol/mg protein). These were many times higher than in most S49 wild type cells. The high basal cAMP levels made measurements of turnover in the absence of a hormone possible. The turnover constant for cAMP in unstimulated cells was 0.030 +/- 0.003 min-1. This was not significantly different than the value measured during epinephrine stimulation, which was 0.035 +/- 0.004 min-1. These turnover values were used to determine precise levels of adenylate cyclase activity throughout the time course of epinephrine stimulation. Desensitization was both rapid and profound, with the level of adenylate cyclase activity falling by 70% within the first 4 minutes of stimulation. This suggested that desensitization may be a very major factor in the attenuation of catecholamine action, at least in some cell types.

In order to identify common mechanisms of action by which both the platelet-derived growth factor (PDGF) and the tumor promoter tetradecanoyl phorbol acetate (TPA) initiate cell growth, the effects of PDGF and TPA on phosphorylation of cellular proteins were examined in density-inhibited Balb/c-3T3 cells. Cultures were incubated with 32Pi and growth factor, and 32P-labeled cellular proteins were examined after separation by SDS-polyacrylamide gel electrophoresis and autoradiography. TPA and PDGF each induced phosphorylation of a major cytosol protein of approximately 75,000 molecular weight (pp75). Phosphorylation of this protein was not induced by either epidermal growth factor or insulin, neither of which initiate 3T3 cell growth but enhance growth later in the 3T3 cell cycle. pp75 was a single band under reduced and non-reduced conditions, and a single spot was seen on two-dimensional gels. Phosphorylation did not occur at 4 degrees C. Phosphorylation of the protein was observed within 3 min and reached a maximum in 10-30 min. Submitogenic doses of TPA and PDGF induced submaximal phosphorylation. The phosphoprotein was labeled only on serine. Cell free phosphorylation of pp75 occurred at 4 degrees C in the presence of Mg++ and Ca2+. Homogenates from cultures pretreated with TPA phosphorylated pp75 in the presence or absence of Ca2+. Phosphorylation of this protein may possibly be related to activation of the Ca2+-dependent, phospholipid sensitive protein kinase C.