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Pathways leading to phosphorylation of p450c17 and to the post-translational regulation of androgen biosynthesis

ABSTRACT: Cytochrome P450c17 (P450c17) is the single enzyme that catalyzes steroid 17alpha-hydroxylase and 17,20 lyase activities and hence is the crucial decision-making step that determines the class of steroid made in a steroidogenic cell. Although both activities are catalyzed on a single active site, the ratio of these activities is regulated by posttranslational events. Serine phosphorylation of P450c17 increases 17,20 lyase activity by increasing the enzyme's affinity for its redox partner, P450 oxidoreductase. We searched for the relevant kinase(s) that phosphorylates P450c17 by microarray studies and by testing of kinase inhibitors. Microarrays show that 145 of the 278 known serine/threonine kinases are expressed in human adrenal NCI-H295A cells, only six of which were induced more than 2-fold by treatment with 8-Br-cAMP. Key components of the ERK1/2 and MAPK/ERK kinase (MEK)1/2 pathways, which have been implicated in the insulin resistance of PCOS, were not found in NCI-H295A cells, implying that these pathways do not participate in P450c17 phosphorylation. Treatment with various kinase inhibitors that probe the protein kinase A/phosphatidylinositol 3-kinase/Akt pathway and the calcium/calmodulin/MAPK kinase pathway had no effect on the ratio of 17,20 lyase activity to 17alpha-hydroxylase activity, appearing to eliminate these pathways as candidates leading to the phosphorylation of P450c17. Two inhibitors that target the Rho-associated, coiled-coil containing protein kinase (ROCK)/Rho pathway suppressed 17,20 lyase activity and P450c17 phosphorylation, both in NCI-H295A cells and in COS-1 cells transfected with a P450c17 expression vector. ROCK1 phosphorylated P450c17 in vitro, but that phosphorylation did not affect 17,20 lyase activity. We conclude that members of the ROCK/Rho pathway act upstream from the kinase that phosphorylates P450c17 in a fashion that augments 17,20 lyase activity, possibly acting to catalyze a priming phosphorylation.

ORGANISM(S): Homo sapiens

PROVIDER: GSE44311 | GEO | 2013/02/15

SECONDARY ACCESSION(S): PRJNA189440

REPOSITORIES: GEO

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Project description:Cytochrome P450c17 (P450c17) is the single enzyme that catalyzes steroid 17alpha-hydroxylase and 17,20 lyase activities and hence is the crucial decision-making step that determines the class of steroid made in a steroidogenic cell. Although both activities are catalyzed on a single active site, the ratio of these activities is regulated by posttranslational events. Serine phosphorylation of P450c17 increases 17,20 lyase activity by increasing the enzyme's affinity for its redox partner, P450 oxidoreductase. We searched for the relevant kinase(s) that phosphorylates P450c17 by microarray studies and by testing of kinase inhibitors. Microarrays show that 145 of the 278 known serine/threonine kinases are expressed in human adrenal NCI-H295A cells, only six of which were induced more than 2-fold by treatment with 8-Br-cAMP. Key components of the ERK1/2 and MAPK/ERK kinase (MEK)1/2 pathways, which have been implicated in the insulin resistance of PCOS, were not found in NCI-H295A cells, implying that these pathways do not participate in P450c17 phosphorylation. Treatment with various kinase inhibitors that probe the protein kinase A/phosphatidylinositol 3-kinase/Akt pathway and the calcium/calmodulin/MAPK kinase pathway had no effect on the ratio of 17,20 lyase activity to 17alpha-hydroxylase activity, appearing to eliminate these pathways as candidates leading to the phosphorylation of P450c17. Two inhibitors that target the Rho-associated, coiled-coil containing protein kinase (ROCK)/Rho pathway suppressed 17,20 lyase activity and P450c17 phosphorylation, both in NCI-H295A cells and in COS-1 cells transfected with a P450c17 expression vector. ROCK1 phosphorylated P450c17 in vitro, but that phosphorylation did not affect 17,20 lyase activity. We conclude that members of the ROCK/Rho pathway act upstream from the kinase that phosphorylates P450c17 in a fashion that augments 17,20 lyase activity, possibly acting to catalyze a priming phosphorylation. NCI-H295A adrenocortical cells were grown on 10-cm plates in RPMI 1640 with 2% fetal calf serum, gentamicin, insulin, and selenium. Three nearly confluent plates were incubated in the presence and absence of 1 mM 8-Br-cAMP for 18 h; the cells were washed with PBS and lysed with 1 ml Trizol (Invitrogen, Carlsbad, CA). Total RNA was purified further using RNeasy columns (QIAGEN, Valencia, CA), and 4 ?g RNA was used to synthesize double-stranded cDNA using the MessageAmp II-Biotin Enhanced Kit (Ambion, Austin, TX) with oligo-dT primer [5?-GGCCAGTGAATTGTAATACGACTCACTATAGGGAGGCGG-(dT)24] to incorporate a T7 RNA polymerase promoter. Antisense cRNA was labeled with biotin-UTP using T7 RNA polymerase (Ambion), and the cRNA was fragmented to 35–200 nucleotides (nt) at 94 C for 35 min in 40 ?l 40 mm Tris-acetate (pH 8.1), 100 mm KOAc, 30 mm MgOAc. The integrities of total RNAs, cRNAs, and fragmented cRNAs were assessed on a Bio-Rad (Hercules, CA) Experion BioAnalyzer. Each sample of fragmented cRNA was initially hybridized with Affymetrix (Santa Clara, CA) Test3 arrays to determine the quality of the labeled probe before hybridization to Affymetrix U133A plus 2.0 arrays. The chips were stained and washed using an Affymetrix GeneChip Fluidics Station 450, and the arrays were scanned using a GCS 3000 scanner with auto-loader in the Genomics Core laboratory. Hybridization intensity signals from the scanned images were analyzed using Affymetrix GeneChip Operating Software (GCOS, version 1.3) to provide qualitative detection calls and quantitative estimates of gene expression levels (signal values). Raw data were exported to Microsoft Excel for analysis. Transcripts displaying a cAMP-induced increased or decreased signal were selected for further analysis only if they had a mean ± 2.0-fold change and were statistically significant (P < 0.05) in three separate experiments; transcripts displaying no signal change relative to controls in at least three experiments were excluded from further analysis.

Project description:Purpose: Deregulated phosphatidylinositol 3-kinase pathway signaling through AGC kinases including AKT, p70S6 kinase, PKA, SGK and Rho kinase, is a key driver of multiple cancers. The simultaneous inhibition of multiple AGC kinases may increase antitumor activity and minimize clinical resistance compared with a single pathway component. Experimental Design: We investigated the detailed pharmacology and antitumor activity of the novel clinical drug candidate AT13148, an oral ATP-competitive multi-AGC kinase inhibitor. Gene expression microarray studies were undertaken to characterize the molecular mechanisms of action of AT13148. Results: AT13148 caused substantial blockade of AKT, p70S6K, PKA, ROCK and SGK substrate phosphorylation and induced apoptosis in a concentration and time-dependent manner in cancer cells with clinically relevant genetic defects in vitro and in vivo. Antitumor efficacy in HER2-positive, PIK3CA-mutant BT474 breast, PTEN-deficient PC3 human prostate cancer and PTEN-deficient MES-SA uterine tumor xenografts was demonstrated. We show for the first time that induction of AKT phosphorylation at serine 473 by AT13148, as reported for other ATP-competitive inhibitors of AKT, is not a therapeutically relevant reactivation step. Gene expression studies showed that AT13148 has a predominant effect on apoptosis genes, whereas the selective AKT inhibitor CCT128930 modulates cell cycle genes. Induction of upstream regulators including IRS2 and PIK3IP1 due to compensatory feedback loops was observed. Conclusions: The clinical candidate AT13148 is a novel oral multi-AGC kinase inhibitor with potent pharmacodynamic and antitumor activity, which demonstrates a distinct mechanism of action from other AKT inhibitors. AT13148 will now be assessed in a first-in-human Phase I trial. The PTEN-deficient U87MG glioblastoma cell line was treated for 6 hours with vehicle control (DMSO) or to different concentrations of AT13148 and CCT128930 (0.1uM, 1xGI50 and 3XGI50).

Project description:mRNA trafficking and local protein translation are associated with protrusive cellular domains, such as neuronal growth cones, and deregulated control of protein translation is associated with tumor malignancy. We show here that activated RhoA, but not Rac1, is enriched in pseudopodia of MSV-MDCK-INV tumor cells and that Rho, Rho kinase (ROCK) and myosin II regulate the microtubule-independent targeting of RNA to these tumor cell domains. ROCK inhibition does not affect pseudopodial actin turnover but significantly reduces the dynamics of pseudopodial RNA turnover. Gene array analysis shows that 7.3% of the total genes analyzed exhibited a greater than 1.6-fold difference between the pseudopod and cell body fractions. Of these, only 13.2% (261 genes) are enriched in pseudopodia, suggesting that only a limited number of total cellular mRNAs are enriched in tumor cell protrusions. Comparison of the tumor pseudopod mRNA cohort and a cohort of mRNAs enriched in neuronal processes identified tumor pseudopod-specific signaling networks that were defined by expression of M-Ras and the Shp2 protein phosphatase. Pseudopod expression of M-Ras and Shp2 mRNA were diminished by ROCK inhibition linking pseudopodial Rho/ROCK activation to the localized expression of specific mRNAs. Pseudopodial enrichment for mRNAs involved in protein translation and signaling suggests that local mRNA translation regulates pseudopodial expression of less stable signaling molecules as well as the cellular machinery to translate these mRNAs. mRNA trafficking and local protein translation are associated with protrusive cellular domains, such as neuronal growth cones, and deregulated control of protein translation is associated with tumor malignancy. We show here that activated RhoA, but not Rac1, is enriched in pseudopodia of MSV-MDCK-INV tumor cells and that Rho, Rho kinase (ROCK) and myosin II regulate the microtubule-independent targeting of RNA to these tumor cell domains. ROCK inhibition does not affect pseudopodial actin turnover but significantly reduces the dynamics of pseudopodial RNA turnover. Gene array analysis shows that 7.3% of the total genes analyzed exhibited a greater than 1.6-fold difference between the pseudopod and cell body fractions. Of these, only 13.2% (261 genes) are enriched in pseudopodia, suggesting that only a limited number of total cellular mRNAs are enriched in tumor cell protrusions. Comparison of the tumor pseudopod mRNA cohort and a cohort of mRNAs enriched in neuronal processes identified tumor pseudopod-specific signaling networks that were defined by expression of M-Ras and the Shp2 protein phosphatase. Pseudopod expression of M-Ras and Shp2 mRNA were diminished by ROCK inhibition linking pseudopodial Rho/ROCK activation to the localized expression of specific mRNAs. Pseudopodial enrichment for mRNAs involved in protein translation and signaling suggests that local mRNA translation regulates pseudopodial expression of less stable signaling molecules as well as the cellular machinery to translate these mRNAs. Pseudopodial Rho/ROCK activation may impact on tumor cell migration and metastasis by stimulating the pseudopodial translocation of mRNAs and thereby regulating the expression of local signaling cascades. Keywords: tumor cells, protrusive cellular domains, pseudopodia vs cell body, RNA trafficking

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Pathways leading to phosphorylation of p450c17 and to the post-translational regulation of androgen biosynthesis

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