Impact of Differential P450c17 Phosphorylation by cAMP Stimulation and by Starvation Conditions on Enzyme Activities and Androgen Production in NCI-H295R Cells

Kempná, Petra; Hirsch, Andrea; Hofer, Gaby; Mullis, Primus Ε.; Flück, Christa E.

doi:10.1210/en.2010-0093

Cited by 33 publications

(52 citation statements)

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“…Thus dephosphorylation did not degrade P450c17, but diminished 17,20 lyase activity without altering the substrate binding (Zhang et al, 1995). Studies in other laboratories have confirmed that P450c17 phosphorylation increases 17,20 lyase activity (Biason-Lauber et al, 2000;Kempna et al, 2010). When a protein is activated by phosphorylation there is generally an equilibrium between phosphorylation by the kinase and dephosphorylation by a phosphatase (reviewed by Virshup, 2000).…”

Section: Phosphorylation Of P450c17 -Initial Studiesmentioning

confidence: 99%

The post-translational regulation of 17,20 lyase activity

Miller

Tee

2015

Molecular and Cellular Endocrinology

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Section: Phosphorylation Of P450c17 -Initial Studiesmentioning

confidence: 99%

The post-translational regulation of 17,20 lyase activity

Miller

Tee

2015

Molecular and Cellular Endocrinology

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“…THE TERM SERUM STARVATION, or simply starvation, as well as serum deprivation, depletion, removal, restriction, withdrawal, and serum limitation have been used to denote various procedures that include growing cells in either reduced serum, serum-free, or serum-and protein-free medium (5,14,16,27,28,34,45,47,50,53,56,57,70). Unfortunately, none of these terms have a well-defined meaning that would unambiguously reflect the actual procedure, and they have all been inconsistently applied to describe different protocols.…”

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confidence: 99%

“…Alternatively, serum starvation may be carried out in medium with low serum concentration (relative to normal growth medium), typically 0.1-0.5% (6,15,36,47,60), but sometimes as high as 2-5% (13,19,59) or as low as 0.05% (32,58). The time frame for serum starvation is equally varied and can include anything from 15 to 30 min up to several weeks (1,3,4,27,28,49,58,63,72). Moreover, while alternative designations such as serum withdrawal or deprivation are sometimes used synonymously and interchangeably with serum starvation (27,34,56,72), others make a clear distinction based on serum concentration (15).…”

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confidence: 99%

“…The time frame for serum starvation is equally varied and can include anything from 15 to 30 min up to several weeks (1,3,4,27,28,49,58,63,72). Moreover, while alternative designations such as serum withdrawal or deprivation are sometimes used synonymously and interchangeably with serum starvation (27,34,56,72), others make a clear distinction based on serum concentration (15). We will not attempt to redefine these terms and incubating cells in reduced serum or serum-free medium in all its different forms will be laxly referred to as serum starvation in this paper.…”

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Serum starvation: caveat emptor

Pirkmajer

Chibalin

2011

American Journal of Physiology-Cell Physiology

235

194

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Serum starvation is one of the most frequently performed procedures in molecular biology and there are literally thousands of research papers reporting its use. In fact, this method has become so ingrained in certain areas of research that reports often simply state that cells were serum starved without providing any factual details as to how the procedure was carried out. Even so, we quite obviously lack unequivocal terminology, standard protocols, and perhaps most surprisingly, a common conceptual basis when performing serum starvation. Such inconsistencies not only hinder interstudy comparability but can lead to opposing and inconsistent experimental results. Although it is frequently assumed that serum starvation reduces basal activity of cells, available experimental data do not entirely support this notion. To address this important issue, we studied primary human myotubes, rat L6 myotubes and human embryonic kidney (HEK)293 cells under different serum starvation conditions and followed time-dependent changes in important signaling pathways such as the extracellular signal-regulated kinase 1/2, the AMP-activated protein kinase, and the mammalian target of rapamycin. Serum starvation induced a swift and dynamic response, which displayed obvious qualitative and quantitative differences across different cell types and experimental conditions despite certain unifying features. There was no uniform reduction in basal signaling activity. Serum starvation clearly represents a major event that triggers a plethora of divergent responses and has therefore great potential to interfere with the experimental results and affect subsequent conclusions.

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“…The weaker interaction between cytochrome b 5 and P450c17 when 17-OH-Preg is the substrate promotes 17,20 lyase activity by enhancing the interaction of P450c17 with POR (36). Third, serine/threonine (Ser/Thr) phosphorylation, but not tyrosine phosphorylation, of P450c17 will increase 17,20 lyase activity (34,(37)(38)(39)(40). Either maximal Ser/Thr phosphorylation of P450c17 or maximal concentrations of b 5 will maximize 17,20 lyase activity so that adding b 5 to phosphorylated P450c17 elicits no further increase in lyase activity (34), indicating that both factors operate via the same mechanism, presumably optimizing interactions with POR.…”

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Phosphorylation of Human Cytochrome P450c17 by p38α Selectively Increases 17,20 Lyase Activity and Androgen Biosynthesis

Tee

Miller

2013

Journal of Biological Chemistry

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Background: Ser/Thr phosphorylation of P450c17 increases 17,20 lyase activity and androgenic capacity. Results: Drug inhibition and siRNA knockdowns in adrenal cells implicate p38␣, which phosphorylated bacterially expressed P450c17, doubling 17,20 lyase activity. Conclusion: Phosphorylation of P450c17 by p38␣ provides a post-translational mechanism distinguishing glucocorticoid from sex steroid synthesis. Significance: p38␣ pathways may participate in hyperandrogenic states and provide targets for glucocorticoid-sparing inhibition of androgen synthesis.Cytochrome P450c17, a steroidogenic enzyme encoded by the CYP17A1 gene, catalyzes the steroid 17␣-hydroxylation needed for glucocorticoid synthesis, which may or may not be followed by 17,20 lyase activity needed for sex steroid synthesis. Whether or not P450c17 catalyzes 17,20 lyase activity is determined by three post-translational mechanisms influencing availability of reducing equivalents donated by P450 oxidoreductase (POR). These are increased amounts of POR, the allosteric action of cytochrome b 5 to promote POR-P450c17 interaction, and Ser/ Thr phosphorylation of P450c17, which also appears to promote POR-P450c17 interaction. The kinase(s) that phosphorylates P450c17 is unknown. In a series of kinase inhibition experiments, the pyridinyl imidazole drugs SB202190 and SB203580 inhibited 17,20 lyase but not 17␣-hydroxylase activity in human adrenocortical HCI-H295A cells, suggesting an action on p38␣ or p38␤. Co-transfection of non-steroidogenic COS-1 cells with P450c17 and p38 expression vectors showed that p38␣, but not p38␤, conferred 17,20 lyase activity on P450c17. Antiserum to P450c17 co-immunoprecipitated P450c17 and both p38 isoforms; however, knockdown of p38␣, but not knockdown of p38␤, inhibited 17,20 lyase activity in NCI-H295A cells. Bacterially expressed human P450c17 was phosphorylated by p38␣ in vitro at a non-canonical site, conferring increased 17,20 lyase activity. This phosphorylation increased the maximum velocity, but not the Michaelis constant, of the 17,20 lyase reaction. p38␣ phosphorylates P450c17 in a fashion that confers increased 17,20 lyase activity, implying that the production of adrenal androgens (adrenarche) is a regulated event.Three classes of steroid hormones are required for mammalian life: mineralocorticoids regulate renal sodium retention, thus regulating intravascular volume and blood pressure; glucocorticoids regulate carbohydrate metabolism and responses to stress; and sex steroids (androgens and estrogens) are required for reproduction of the species. In most mammals, mineralocorticoids are C 21 (21-carbon) 17-deoxy steroids, glucocorticoids are C 21 17-hydroxysteroids, and sex steroids are produced from C 19 steroids. A single steroidogenic enzyme, cytochrome P450c17, encoded by the CYP17A1 gene determines which class of steroid is produced. P450c17 catalyzes both the 17␣-hydroxylase activity needed to convert C 21 17-deoxysteroids to their 17-hydroxy counterparts and the 17,20 lyase activity that cleaves the bond...

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Impact of Differential P450c17 Phosphorylation by cAMP Stimulation and by Starvation Conditions on Enzyme Activities and Androgen Production in NCI-H295R Cells

Cited by 33 publications

References 34 publications

The post-translational regulation of 17,20 lyase activity

The post-translational regulation of 17,20 lyase activity

Serum starvation: caveat emptor

Phosphorylation of Human Cytochrome P450c17 by p38α Selectively Increases 17,20 Lyase Activity and Androgen Biosynthesis

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