Tautomycin inhibits phosphatase-dependent transformation of the rat kidney mineralocorticoid receptor.
The binding of aldosterone (ALDO) to the mineralocorticoid receptor (MR) induces a conformational change of the protein referred to as 'transformation'. This feature can be evidenced in vivo by the capacity of the MR to interact with chromatin, and in vitro by the ability of the MR to bind to DNA strands or to shift the sedimentation coefficient (S) to lower values. The transformation process allows MR to work as a transcription factor after interacting with specific sequences of DNA. The signal transduction pathway for the MR transformation remains unknown. As a first step towards elucidating the mechanism of steroid-dependent MR transformation, we asked if the MR-signaling pathway is affected by the phosphorylation status of the MR-heterocomplex, and how that pathway may be regulated. Incubation of preformed [3H]ALDO-MR complex with bovine intestinal alkaline phosphatase led to an increase in the rate of MR-transformation (measured as 9.4-5.4S shift). This alkaline phosphatase-dependent MR transformation was inhibited by the specific alkaline phosphatase-type inhibitor levamisole, and was not evident in incubations performed with acid phosphatases. A direct correlation between the DNA-cellulose binding capacity of the [3H]ALDO-MR complex and the percentage of transformed 5.4S MR form was also observed. When rat kidney cytosol was incubated in the absence of both exogenous phosphatase and stabilizing agents (such as molybdate or vanadate), MR transformation also took place, in a time- and temperature-dependent process. In contrast with the inhibitory effect observed upon alkaline phosphatase-promoted transformation, levamisole was unable to inhibit the endogenous transforming activity of MR, suggesting that an endogenous phosphatase other than those which belong to the alkaline-type may be responsible for that transformation. Tautomycin, a polyketide produced by the soil bacteria Streptomyces which inhibits serine/threonine phosphatases of the PP1/PP2A subgroup, was able to inhibit the endogenous phosphatase activity in a concentration-dependent form (Ki(app)=7.35 nM). These results support the idea that the endogenous renal activity involved in the regulation of rat kidney MR transformation may be a protein phosphatase which belongs to the PP1/PP2A subgroup.
Piwien-Pilipuk
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Departamento de Química Biológica, Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires and PRHOM-CONICET, Argentina.
Galigniana
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MD
D000450:Aldosterone / Q000378:metabolism; D000469:Alkaline Phosphatase / Q000037:antagonists & inhibitors / Q000378:metabolism; D000818:Animals; D000935:Antifungal Agents / Q000494:pharmacology; D002467:Cell Nucleus / Q000378:metabolism; D002482:Cellulose / Q000031:analogs & derivatives / Q000378:metabolism; D002499:Centrifugation, Density Gradient; D002852:Chromatography, Ion Exchange; D003593:Cytoplasm / Q000378:metabolism; D004247:DNA / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D007668:Kidney / Q000201:enzymology / Q000378:metabolism*; D007700:Kinetics; D007978:Levamisole / Q000494:pharmacology; D008297:Male; D010749:Phosphoprotein Phosphatases / Q000037:antagonists & inhibitors / Q000502:physiology*; D011487:Protein Conformation / Q000187:drug effects; D011714:Pyrans; D051381:Rats; D017207:Rats, Sprague-Dawley; D018161:Receptors, Mineralocorticoid / Q000737:chemistry / Q000378:metabolism*; D013141:Spiro Compounds; D013696:Temperature
D000935:Antifungal Agents; C033190:DNA-cellulose; D011714:Pyrans; D018161:Receptors, Mineralocorticoid; D013141:Spiro Compounds; C053079:tautomycin; D007978:Levamisole; D000450:Aldosterone; D002482:Cellulose; D004247:DNA; D000469:Alkaline Phosphatase; D010749:Phosphoprotein Phosphatases