Interactions between kinases and phosphatases in the rapid control of brain aromatase

Abstract

Aromatization of testosterone into oestradiol plays a key role in the activation of male sexual behaviour in many vertebrate species. Rapid changes in brain aromatase activity have recently been identified and the resulting changes in local oestrogen bioavailability could modulate fast behavioural responses to oestrogens. In quail hypothalamic homogenates, aromatase activity is down-regulated within minutes by calcium-dependent phosphorylations in the presence of ATP, MgCl2 and CaCl2 (ATP/Mg/Ca). Three kinases (protein kinases A and C and calmodulin kinase; PKA, PKC and CAMK) are potentially implicated in this process. If kinases decrease aromatase activity in a reversible manner, then it would be expected that the enzymatic activity would increase and/or return to baseline levels in the presence of phosphatases. We showed previously that 0.1 mM vanadate (a general inhibitor of protein phosphatases) significantly decreases aromatase activity but specific protein phosphatases that could up-regulate aromatase activity have not been identified to date. The reversibility of aromatase activity inhibition by phosphorylations was investigated in the present study using alkaline and acid phosphatase (Alk and Ac PPase). Unexpectedly, Alk PPase inhibited aromatase activity in a dose-dependent manner in the presence, as well as in the absence, of ATP/Mg/Ca. By contrast, Ac PPase completely blocked the inhibitory effects of ATP/Mg/Ca on aromatase activity, even if it moderately inhibited aromatase activity in the absence of ATP/Mg/Ca. However, the addition of Ac PPase was unable to restore aromatase activity after it had been inhibited by exposure to ATP/Mg/Ca. Taken together, these data suggest that, amongst the 15 potential consensus phosphorylation sites identified on the quail aromatase sequence, some must be constitutively phosphorylated for the enzyme to be active whereas phosphorylation of the others is involved in the rapid inhibition of aromatase activity by the competitive effects of protein kinases and phosphatases. Two out of these 15 putative phosphorylation sites occur in an environment corresponding to the consensus sites for PKC, PKA (and possibly a CAMK) and, in all probability, represent the sites whose phosphorylation rapidly blocks enzyme activity.

Fig. 1

Effects of alkaline (Alk) or acid (Ac) phosphatase (PPase) on aromatase activity (AA) in preoptic-hypothalamic homogenates from male quail brain exposed or not to phosphorylating conditions (presence of ATP/Mg/Ca). (A) Schematic presentation of the experimental protocol. The sequential treatments that were applied to the homogenate aliquots are indicated by arrows and the duration of incubations and the temperatures at which they took place are listed below the horizontal line representing the time line (see also text for additional details). (B) Effect of increasing concentrations of Alk PPase on AA measured in the absence or presence of ATP/Mg/Ca. The arrow and number 4 point to the dose whose effects were studied in detail in experiments described in the next panel. (C) Effects in 4 different homogenates of a single concentration of Alk PPAse (4 units/assay) on AA measured in the absence or presence of ATP/Mg/Ca. (D) Effect of increasing concentrations of Ac PPase on AA measured in the absence or presence of ATP/Mg/Ca. The arrows and numbers (0.4 and 4) point to the dose whose effects were studied in detail in experiments described in the next panel. (E) Effects in 6 different homogenates of two concentrations of Ac PPAse on AA measured in the absence or presence of ATP/Mg/Ca. Data in C and E were analyzed by two-way ANOVA (see text) followed by Tukey HSD’s post-hoc test adapted for repeated measures whose results are indicated as follows: **: p<0.01 compared to the same concentration of PPase without ATP/Mg/Ca; # (##): p<0.05 (0.01) compared to PPase 0 units in the same condition and ΔΔ p<0.01 compared to compared to PPase 0.4 units in the same condition

Fig. 2

Effects of endogenous phosphatases or of exogenous acid (Ac) phosphatase (PPase; 0.4 units) on aromatase activity (AA) in preoptic-hypothalamic homogenates from male quail brain that had been previously exposed or not to phosphorylating conditions (presence of ATP/Mg/Ca). Panels A through D provide a schematic description of the different protocols that were tested. The sequential treatments that were applied to the homogenate aliquots are indicated by arrows and the duration of incubations and the temperatures at which they took place are listed below the horizontal line representing the time line (see also text for additional details). Panel E illustrates the results of a recovery experiment performed on three independent brain homogenates to further test the best conditions identified in the experiments schematized in A-D (Inhibition for 5 min with ATP/Mg/Ca, recovery in the presence of 0.4 unit Ac PPase for 30 min at 25°C). *= p<0.05 for the main effect in the ANOVA comparing at T30 samples exposed or not to ATP/Mg/Ca. This ANOVA identified no effect of the PPase and no interaction between the two factors. #= p<0.05 for the comparison of samples with or without ATP/Mg/Ca at T0.

Fig. 3

Schematic presentation illustrating three possible mechanisms (indicated by numbers in the figure) illustrating the effect of Ac PPase on AA. Ac PPase could regulate AA by removing specific phosphate groups from the aromatase protein (1), or by inactivating a kinase (2) or by activating an endogenous phosphatase (3). See text for additional explanations.