17alpha-hydroxylase/17,20-lyase deficiency as a model to study enzymatic activity regulation: role of phosphorylation.
Cytochrome P450 17alpha-hydroxylase (CYP17) is a single gene-encoded protein with two activities: 17alpha-hydroxylase and 17,20-lyase. The two catalytic activities are differentially regulated in health and disease. We took advantage of naturally occurring human mutations to understand the molecular bases of this differential regulation. We identified eight novel mutations in the CYP17 gene, different in nature and spread throughout the gene. As posttranslational modifications appear to be important for activity control, we investigated the phosphorylation state of wild-type and mutant CYP17 proteins. Although phospholabeled protein was seen when the wild-type and most mutant proteins were expressed, no phosphorylation was detected for the F417C mutant. F417C is the only 17,20-lyase deficiency case confirmed at the molecular level and represents the first phosphorylation CYP17-deficient mutant. In search of the physiological agents involved in this process, the effect of cAMP was tested on activity and phosphorylation state of our mutant CYP17 proteins. cAMP stimulates activity and phosphorylation in all cases, except in the F417C and R35L mutants. The lack of response to the physiological second messenger might explain the different phenotypes. The F417C mutant protein, which is already shown to be associated with the lack of electron transfer, provides for the first time a link between the electron transfer system and the phosphorylation state of the CYP17 enzyme in the control of 17,20-lyase activity.
Biason-Lauber
A
A
Department of Pediatrics, University of Zurich, Switzerland. alauber@kispi.unizh.ch
Kempken
B
B
Werder
E
E
Forest
M G
MG
Einaudi
S
S
Ranke
M B
MB
Matsuo
N
N
Brunelli
V
V
Schönle
E J
EJ
Zachmann
M
M
D015124:8-Bromo Cyclic Adenosine Monophosphate / Q000378:metabolism; D000312:Adrenal Hyperplasia, Congenital; D000818:Animals; D019556:COS Cells; D002522:Chlorocebus aethiops; D004252:DNA Mutational Analysis; D004579:Electron Transport / Q000235:genetics; D006579:Heterozygote; D006720:Homozygote; D006801:Humans; D010766:Phosphorylation; D011233:Precipitin Tests; D011499:Protein Processing, Post-Translational / Q000235:genetics; D012333:RNA, Messenger / Q000096:biosynthesis / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013254:Steroid 17-alpha-Hydroxylase / Q000235:genetics
D012333:RNA, Messenger; D015124:8-Bromo Cyclic Adenosine Monophosphate; D013254:Steroid 17-alpha-Hydroxylase