To determine which Akt isoforms are expressed in bovine adrenal chromaffin cells, cell lysates were run on SDS-PAGE alongside recombinant Akt protein standards and western blotted with isoform-specific Akt antibodies (). Anti-Akt1 gave a readily detectable band in chromaffin cells, equivalent to between 5 and 10 ng of recombinant Akt1. This antibody was not entirely specific, however, as bands of lesser intensity were also detected using 10 ng of Akt2 and Akt3 (, upper panel). In contrast, the Akt2 and Akt3 antibodies were absolutely specific for their corresponding recombinant protein. The Akt2 and Akt3 signals in chromaffin cells were much lower than those seen with 5 ng of recombinant Akt2/3. Therefore, the partial cross-reactivity of anti-Akt1 with Akt2/3 can at most account for only a small proportion of the staining intensity seen with anti-Akt1. We therefore concluded that Akt1 is the predominant isoform expressed by bovine chromaffin cells, although lower levels of Akt 2 and 3 are also present.
Akt is activated by Ca2+ in chromaffin cells
Exocytosis is triggered by Ca2+ in chromaffin cells, so we investigated whether secretory stimuli resulted in activation of cellular Akt. To do this we treated cells with nicotine, which activates acetylcholine receptors and triggers Ca2+ entry through channels to elicit exocytosis. Akt activity was assayed by western blotting of cell lysates with a phospho-serine473-specific antibody. Nicotine treatment resulted in a clear increase in phospho-Akt staining, despite no change in total Akt levels (, left panel). To determine if Ca2+ directly drives Akt activation, we used digitonin-permeabilized cells. Application of 10 μM Ca2+, the optimal concentration for triggering exocytosis, again resulted in increased Akt phosphorylation (, right panel). To determine the time course of Akt activation relative to secretion, we simultaneously monitored catecholamine release and Akt phosphorylation (). Both processes followed broadly similar time courses, with Ca2+-induced Akt phosphorylation being detectable within 2 minutes of stimulation.
To determine if Akt regulates exocytosis in adrenal chromaffin cells, we transfected cell cultures with HA-tagged Akt1 constructs and analysed individual exocytotic release events using carbon fibre amperometry. Transfected cells were visualised by the endogenous fluorescence of an EGFP reporter plasmid that was co-transfected as a marker. After stimulation of regulated exocytosis with digitonin and calcium, transient spikes of catecholamine oxidation were recorded, indicative of individual chromaffin granule fusion events (). Transfection of wild type Akt had no significant effect on the frequency of exocytotic fusion events elicited (). In contrast, analysis of individual exocytotic events revealed a 43% increase in the amount of catecholamine released per fusion event (). This increased quantal size was due to a slowing of catecholamine release kinetics, as evidenced by an increase in both the rise- and fall-time of amperometric spikes (). Transfection of a myristoylated, constitutively active form of Akt produced a similar increase in quantal size of individual fusion events and likewise had no effect on overall frequency of release events (data not shown). To confirm that the Akt constructs were active upon transfection into chromaffin cells, double label immunofluorescence was used. Total transfected Akt was visualised using a HA-tag antibody (red) while active Akt was visualised using a phospho-serine473-specific antibody (green). A clear increase in cellular activated Akt (as defined by phospho-serine473 immunoreactivity) was seen in all HA-positive cells transfected with wt-Akt () or constitutively active Akt (data not shown).
Akt alters exocytotic release kinetics and increases quantal size
In order to establish whether these effects of Akt on exocytosis were due to Akt phosphorylation of target proteins, or alternatively reflected a kinase-independent function, we transfected chromaffin cells with a mutant Akt construct, AAA-Akt. This construct encodes Akt1 containing three point mutations: K179A, T308A and S473A. These mutations result in a kinase-dead, phosphorylation-deficient form of Akt that has been shown to act as a dominant negative mutant in some cell types (26
). Stimulation of AAA-Akt transfected cells by digitonin and calcium again resulted in transient spikes of catecholamine release due to exocytosis (). As with wild type Akt, there was no statistically significant effect on the overall frequency of exocytotic fusion events (). In pointed contrast to wt-Akt, however, no significant changes in charge, rise-time or fall-time were observed upon transfection of AAA-Akt (). These data strongly suggest that the effect of Akt on quantal size and the kinetics of exocytotic release requires Akt kinase activity. In order to rule out the possibility that the AAA mutant was not stably expressed in the transfected cells, transfected Akt was visualised using an HA-tag antibody (, red). Similar transfection efficiency and fluorescence intensity was seen for both AAA-Akt and wt-Akt, thus ruling out this trivial explanation. In contrast to wt-Akt, however, no increase in phospho-serine473 immunoreactivity (, green) was apparent in AAA-Akt transfected cells, as predicted for this mutant protein.
The effect of Akt on exocytotic release requires kinase activity
Transfection of CSP in chromaffin cells has two effects on exocytosis as measured by amperometry: a reduction in the frequency of exocytotic fusion events, and an increased quantal size as a result of a slowing of release kinetics (36
). This latter effect on quantal size (but not the effect on frequency) is abolished in a mutant CSP(S10A) construct that cannot be phosphorylated on serine-10 (17
) (). This raised the possibility that the similar effect of Akt and CSP transfection in amperometry was due to direct Akt phosphorylation of CSP on this residue. A comparison of the coding sequence of CSP with the minimal Akt consensus phosphorylation motif revealed a potential Akt phosphorylation site at serine-10 (), the same residue that is phosphorylated in vitro by PKA (17
). An in vitro kinase assay using activated Akt and recombinant purified CSP in the presence of 32
P-labelled ATP was then set up to test this experimentally. As can be seen in , 32
P was readily incorporated into wild type CSP, indicating phosphorylation by Akt. However, when the assay was performed using S10A mutant recombinant CSP, which has a non-phosphorylatable alanine in place of serine-10, 32
P incorporation was barely detectable despite equal levels of recombinant protein being present in both cases (). To determine the efficiency of Akt phosphorylation, we analysed the kinetics of 32
P incorporation under initial rate conditions using synthetic peptides. The peptides used corresponded to amino acids 4-14 of CSP, the same peptide but with an S10A substitution, or the optimal Akt substrate peptide, Crosstide. This revealed the N-terminal CSP peptide to be a comparable Akt substrate to Crosstide, whereas the S10A peptide exhibited no detectable 32
P incorporation under the same conditions ().
Overexpression of active Akt or phosphorylatable CSP has similar effects on single granule release properties
CSP is efficiently 32P-phosphorylated by Akt in vitro
Although these data clearly showed that CSP was an efficient in vitro Akt substrate, a different approach was required to assess whether Akt was a cellular CSP kinase. To address this issue we raised an antibody (P-CSP) against a synthetic CSP4-14 peptide containing a phosphorylated serine 10 residue (33
). As can be seen in , this P-CSP antibody specifically detected CSP that had been phosphorylated in vitro by PKA, but displayed no observable binding to mock-phosphorylated CSP or to serine-phosphorylated recombinant Munc18-1. The phospho-specificity of the antibody was further confirmed by the abolition of binding by the phospho-CSP4-14 peptide used for immunisation and the lack of effect of a non-phosphorylated version of the same peptide (). When in vitro phosphorylation reactions were blotted and probed with the P-CSP antibody, a strong signal was observed with both Akt- and PKA-phosphorylated wild type CSP, but not with S10A mutant CSP (), confirming that CSP is efficiently phosphorylated by Akt on serine-10 in vitro. To determine if this also occurred within living cells, we co-transfected HEK293 cells with plasmids encoding CSP and Akt constructs and monitored CSP phosphorylation using the P-CSP antibody (). CSP-transfected cells co-transfected with constitutively active Akt (myr-Akt) gave a much stronger P-CSP signal than those co-transfected with AAA-Akt, despite similar levels of total expressed CSP. In contrast, CSP phosphorylation was barely detectable in S10A mutant CSP-transfected cells co-transfected with either Akt construct.
Phospho-CSP antibody demonstrates Akt phosphorylation of CSP on serine-10 in vitro and in HEK cells
Finally, we sought to determine if Akt phosphorylates CSP in the adrenal chromaffin cells used for our functional exocytosis experiments. Unlike HEK cells, chromaffin cells express endogenous CSP and have a very low transfection efficiency, thus precluding the use of the western blotting approach. We therefore used a triple-labelling immunofluorescence approach on chromaffin cells transfected with CSP and Akt plasmids. For this purpose, we raised a new sheep polyclonal antibody to recombinant CSP protein to enable simultaneous detection of total CSP and phospho-CSP in the same cells. Characterisation of this sheep antiserum on recombinant protein, transfected cells and native tissue revealed a very similar specificity to the previously described rabbit antiserum (30
) (data not shown). Double CSP/Akt-co-transfected cells were identified via the HA-tag antibody (red) marker for recombinant Akt and in all cases a large increase in total CSP expression (blue) was observed. Despite these similar levels of total CSP, however, there was a striking increase in the phospho-CSP (green) signal in wt-Akt transfected cells compared to AAA-Akt transfected cells (). Quantification of the fluorescence intensity of these double transfected cells using confocal microscopy revealed an approximately 3-fold higher CSP phosphorylation in wt-Akt-transfected cells compared to AAA-Akt transfected cells (). Transfection of CSP alone resulted in a similar level of phosphorylation to that seen with AAA-Akt, further demonstrating the specificity of recombinant CSP phosphorylation by Akt in chromaffin cells ( and ). In cells transfected with Akt constructs alone, a significant increase in phosphorylation of endogenous CSP was also observed with wt-Akt in comparison to AAA-Akt and non-transfected controls (). Therefore, Akt is a chromaffin cell CSP kinase capable of phosphorylating both transfected and native CSP on serine-10. To determine if Ca2+
-induced activation of Akt would further increase CSP phosphorylation, we permeabilized untransfected chromaffin cells and stimulated them with Ca2+
(). Tonic Akt activity was evident in the absence of Ca2+
, and this was increased by Ca2+
application. With CSP, a high level of basal phosphorylation on serine-10 was observed, but no further increase in this signal occurred in response to Ca2+
. It is likely that this high level of tonic CSP phosphorylation would prevent detection of small Ca2+
- induced acute changes in phosphorylation using our phospho-specific antibody, however. Indeed, using a 32
P-metabolic labelling approach, in which the high ‘noise’ of basal phosphorylation is reduced, it has previously been shown that nicotine increases CSP phosphorylation (18
Akt is a CSP serine-10 kinase in chromaffin cells
Overexpression of active Akt induces CSP Ser10 phosphorylation in chromaffin cells