In this study, we show that PI3K—Akt signaling participated in the phase-dependent modulation of L-VGCC rhythms. The mechanism underlying the action of PI3K—Akt signaling in the circadian modulation of L-VGCCs was through the regulation of L-VGCCα1D subunit trafficking and insertion into the plasma membrane. It is worth noting that for some circadian regulated genes and their proteins, the mRNA levels are advanced a few hours earlier than their protein expressions. As we reported previously, the mRNA of L-VGCCCα1D peaks at CT 12, while its protein level reaches maximum at CT 16 (Ko et al. 2007
). As inhibition of PI3K—Akt signaling only affected VGCCα1D trafficking without influencing its mRNA rhythmicity, this signaling pathway only served as part of the circadian output pathway to regulate L-VGCCs. If PI3K—Akt also served in the circadian input entrainment, then inhibition of this pathway would change the mRNA levels of L-VGCCs or other core oscillator genes, to which we did not find any effects on cBmal mRNA rhythmicity in cultured photoreceptors after treatment with Akti for 2 h (data not shown).
Activation of Akt requires multiple steps that includes the generation of the second messenger PtdIns(3,4,5)P3
by PI3K, translocation of Akt from cytoplasm to plasma membrane involving its pleckstrin homology domain, and phosphorylation of Thr308 in the kinase domain and Ser473 within the regulatory domain (Fayard et al. 2005
). As a downstream target of PI3K, the overall activity of Akt is the result of an equilibrium between these two phosphorylation sites (Beaulieu et al. 2007
). Even though most reports show that humoral factors can induce Akt phosphorylation on both phosphorylation sites, there is evidence showing differential expression and chemical-induced phosphorylation between pAktThr308 and pAkt-Ser473 in D2
dopaminergic receptor knockout mice (Beaulieu et al. 2007
). We found that only one phosphorylation site of Akt was under circadian control: the phosphorylation at Thr308. Akt phosphorylation at Ser473, as well as total Akt, remained constant throughout the day. To date, no other report deals with the circadian regulation of Akt activity or gene/protein expression of Akt, and little is known about the circadian regulation of PI3K—Akt signaling. In Drosophila
, Susi, a negative regulator of PI3K, is expressed in a circadian fashion (Wittwer et al. 2005
). In rats, intracerebroventricular infusion of melatonin induces Akt phosphorylation in the hypothalamus (Anhe et al. 2004
). Here, we are the first to show that there is a circadian regulation of Akt activity both in ovo
() and in vitro
(). However, we cannot exclude the possibility that the circadian regulation of Akt activity might be because of rhythms in endogenous humoral factors in the intact retina (for example, dopamine released from amacrine cells) that act on photoreceptors. In two transgenic mouse models that lead to overall increases in Akt phosphorylation, the free-running period of these mutant mice is lengthened and closer to 24 h than wild type controls, but their circadian entrainment to LD cycles is normal and similar to the wild type pattern (Harrington et al. 2007
; Ogawa et al. 2007
). Hence, circadian rhythmicity of Akt phosphorylation may be an important process in the regulation of free-running periods, as well as circadian outputs as indicated in this study.
Various ion channel activities are regulated by PI3K—Akt signaling in the presence of extracellular stimulators (Blair et al. 1999
; Kanzaki et al. 1999
; Lhuillier and Dryer 2002
; Le Blanc et al. 2004
; Tajika et al. 2004
). In parasympathetic neurons, PI3K—Akt signaling is necessary for trophic factor-induced protein trafficking and channel insertion of large-conductance calcium activated-potassium channels during development (Lhuillier and Dryer 2002
). Activation of PI3K—Akt signaling promotes translocation of L-VGCCs to the plasma membrane and therefore enhances the L-VGCC currents in cardiomyocytes, neurons, and COS cells (Viard et al
). We have shown here that inhibition of PI3K—Akt signaling significantly dampened the circadian regulation of L-VGCC current amplitude without affecting channel gating (). Using membrane surface biotinylation assays, we found that inhibition of PI3K or Akt caused a significant decrease in membrane-bound VGCCα1D subunit without any increase of cytosolic VGCCα1D (). The mRNA and protein expression of the VGCCα1D subunit are under circadian control, but mRNA levels peak about 4 h ahead of protein expression (Ko et al. 2007
). We postulate that after translation, the channel protein is inserted into the plasma membrane within a short period of time. This notion is supported by our results that show peak activity of pAktThr308 () is concurrent with the peak protein level of L-VGCCα1D (Ko et al. 2007
) and the maximum current amplitudes recorded at CT 16–19. Higher levels of phosphorylated Akt lead to more VGCCα1D subunits inserted into the plasma membrane and greater L-VGCC current amplitudes during the subjective night. Hence, PI3K—Akt signaling is important in the circadian regulation of L-VGCC trafficking and membrane insertion.
It was perplexing that treatment with PI3K/Akti at night decreased the membrane compartment of VGCCα1D without a corresponding increase of cytosolic VGCCα1D (), which would seem to indicate that PI3K/Akti might affect VGCCα1D protein translation and contradict the notion that PI3K—Akt is involved only in channel trafficking and membrane insertion in this case. This view point would be true if VGCCα1D protein synthesis, trafficking to the membrane, membrane retention, as well as protein recycling and degradation were maintained at the same rate during the day and night, with the only difference being in the amount of VGCCα1D mRNA levels. If membrane retention of inserted protein and/or protein degradation of non-inserted/recycled protein are at a higher rate at night than during the day, then we would observe more membrane protein at night without a difference in cytosolic protein between day and night. We recently found that after VGCCα1D is inserted into the plasma membrane, it interacts with an extracellular protein, retinoschisin, that aids in plasma membrane retention of this channel (Shi et al. 2009
). Retinoschisin is an extracellular protein that is secreted mainly from photoreceptors and bipolar cells, and there is a circadian regulation of mRNA and protein expression of retinoschisin in the chick retina (Ko et al. 2008
). The physical interaction between VGCCα1D and retinoschisin in the retina is under circadian control, which is higher at night than during the day, and hence, the membrane retention of VGCCα1D is higher at night (Shi et al. 2009
). In mice, there is a diurnal change in protein expression of Rab3A in whole-brain synaptosome preparations (Darna et al. 2009
). The Rab proteins are small Ras-related GTPases that have emerged as important regulators for endocytic transport, recycling, vesicle sorting, and transportation to lysosomes for degradation (Stein et al. 2003
). Rab3A has been shown to interact with the sodium-selective amiloride-sensitive epithelial channel, and this interaction mediates channel protein recycling and degradation (Saxena et al. 2005
). Recent advances have further shown that ubiquitin-mediate proteolysis and protein degradation serves as one of the post-translational mechanisms regulating circadian rhythms (He and Liu 2005
; Fujiwara et al. 2008
; Yang et al. 2009
). Therefore, there could be a possible circadian regulation of Rab-mediated L-VGCC recycling and protein degradation in the retina yet to be explored.
Even though the Ras—Erk signaling pathway is important as an input pathway in circadian entrainment and phase-shifting (Obrietan et al. 1998
; Butcher et al. 2002
), it also serves as part of the output pathway to regulate cellular responses and behaviors (Ko et al
; Williams et al. 2001
). Both Ras and Erk activities are rhythmic in chick retinas (Ko et al. 2004
), and the L-VGCC current amplitude rhythm (Ko et al. 2007
) and the apparent affinity rhythm of CNGCs (Ko et al
) are under the output control of Ras—Erk. In Drosophila
, mutations of neurofibromatosis-1, an upstream regulator of Ras, do not alter the rhythmicities of oscillator genes, but the locomotor behavior rhythm of 90% of the adult mutants carrying a null mutation in the neurofibromatosis-1 gene by deletion became arrhythmic (Williams et al. 2001
). Hence, Ras—Erk could serve as part of a ‘universal’ output pathway to regulate circadian rhythms.
The PI3K—Akt and Erk signaling pathways are distinct from each other, but these two pathways share very similar functional roles in regulation of protein translation, cell cycles, protein transport and trafficking, cell-survival, and trophic factors-induced differentiation among others (Yoon et al. 2008
). Melatonin induces phosphorylation of both Akt and Erk in the hypothalamus (Anhe et al. 2004
). Both Ras—Erk and PI3K—Akt are necessary for transforming growth factor β1-induced large-conductance calcium activated-potassium channel expression and membrane insertion in chick ciliary ganglion neurons (Lhuillier and Dryer 2002
), as well as insulin growth factor 1-induced chondrogenic differentiation in adult mesenchymal stem cells (McMahon et al. 2008
). As these two pathways could work synergistically for a particular cellular process (Shankar et al. 2008
) or respond to the same stimulation independently (Chavarria et al. 2007
), it is possible that the circadian oscillators can regulate both pathways simultaneously to govern down-stream rhythmic outputs.
Here, we found that Ras is a common upstream regulator for both Erk and PI3K—Akt signaling pathways to regulate L-VGCC rhythms (), where inhibition of Ras abolished the rhythmicity of pErk and pAktThr308 (). In addition, we have shown in this study that PI3K—Akt and Erk signaling are parallel pathways that both regulate the L-VGCC rhythm in photoreceptors. Inhibition of either pathway abolishes the L-VGCC rhythm, but the inhibition of one does not affect the rhythmic activity of the other. Therefore, both pathways are equally important in the circadian regulation of L-VGCC rhythms in retina photoreceptors (). Inhibition of Ras does not cause global perturbations of membrane trafficking in photoreceptors, as we previously showed that Ras—Erk regulates the apparent affinity rhythm of cGMP to CNGCs without affecting channel insertion, and the maximum current amplitude and α subunit expression of CNGCs do not change as a function of time of the day (Ko et al
; Chae et al. 2007
). Treatment with a Ras inhibitor or transfection with a Ras dominant negative gene into photoreceptors does not change the maximum current amplitude of CNGCs (Ko et al. 2004
). The apparent affinity rhythm of CNGCs is because of the circadian regulation of tyrosine phosphorylation of the CNGC auxiliary subunits (Chae et al. 2007
). On the other hand, the circadian regulation of VGCCα1D subunit expression (Ko et al. 2007
) and its insertion into the plasma membrane as shown in this study are required for the circadian rhythm of L-VGCC current amplitudes (Ko et al. 2007
). Inhibition of either Ras—Erk or PI3K—Akt prevented membrane insertion of VGCCα1D as well as dampened L-VGCC maximum current amplitude rhythms. It is possible that these two parallel signaling pathways have different downstream targets that could ultimately lead to the regulation of L-VGCC insertion into the cell membrane, while Ras—Erk signaling participates in additional cellular events related to the circadian regulation of ion channels.
Fig. 8 A model illustrates that both Ras—PI3K—Akt and Ras—Erk signaling pathways play equally important roles in regulating L-VGCC channel trafficking and insertion into the plasma membrane, which leads to the circadian regulation of (more ...)
In conclusion, the circadian oscillators in the retina regulate the activity of Akt, and both Ras—PI3K—Akt and Ras—Erk signaling pathways play equally important roles in regulating L-VGCC channel trafficking that leads to the circadian regulation of L-VGCC current amplitudes. The circadian rhythms of L-VGCCs further leads to the circadian control of retinoschisin secretion from photoreceptors that might contribute to the circadian regulation of synaptic plasticity in the retina (Ko et al. 2008
). Therefore, the circadian regulation of L-VGCCs is essential to the daily rhythms of retina function and physiology.