The goal of this line of inquiry was to begin to identify inducible translation control pathways in the SCN. To this end, we focused on the mTOR signaling cassette. We report that light evokes phase-dependent activation of the mTOR pathway and that mTOR is a downstream target of the MAPK cascade. Together, these data reveal a new light-actuated signaling cassette in the SCN.
mTOR signaling is a key regulator of inducible gene transcription, ribosome biogenesis, and mRNA translation (Sarbassov et al., 2005
; Wullschleger et al., 2006
; Proud, 2007
). In one of the best-characterized routes to its activation, growth factors, including insulin, signal to mTOR through a phosphatidylinositol-3-kinase (PI3K)/AKT -dependent process. AKT activation leads to phosphorylation of tuberous sclerosis protein2 (TSC2), which, in turn, blocks its GTPase-activating protein (GAP) activity for the small G protein Rheb, thus allowing the GTP-loaded form of Rheb to activate mTOR (as part of the mTORC1 complex) (Gao et al., 2002
; Inoki et al., 2002
; Long et al., 2005
; Li et al., 2004
). In this study, we examined two principal downstream effectors of mTORC1 which have been implicated in inducible translation control: p70 S6K and 4E-BP1.
The activation of p70 S6K is coordinately regulated by a complex series of phosphorylation steps. A key step in this process is mTOR-mediated phosphorylation of Thr-389 (Weng et al., 1998
). In turn, this event creates a docking site for phosphoinositide-dependent kinase 1 (PDK1), which then phosphorylates p70 S6K within the catalytic domain at Thr-229 (Pullen et al., 1998
; Frödin et al., 2002
), thus leading to enzymatic activation of p70 S6K (Pullen and Thomas, 1997
). In our study, we found that a brief photic stimulus triggered p70 S6K phosphorylation at Thr-389. Interestingly, in the absence of photic stimulation, immunohistochemical labeling was not able to detect the Thr-389 phosphorylated form of p70 S6K in the SCN. This lack of a signal raised the possibility that the SCN exhibits low basal levels of mTOR activity. However, Western analysis was able to detect phosphorylated p70 S6K under control conditions, thus suggesting that our immunohistochemical staining approach lacked the necessary sensitivity to detect a baseline level of phosphorylation. Furthermore, data from S6 and 4E-BP1 (discussed below) supports the idea that the SCN exhibits a relatively high level of mTOR activity.
Although most SCN neurons express mTOR1, light-induced p-p70 S6K expression was only detected in a subset of cells. There are a number of potential explanations for this. Along these lines, limited p-p70 S6K expression may be due to the low sensitivity of the antibody (noted above). Another possibility is that we may be identifying only a subset of the “activated cells’; hence activation may be rapid and transient in some cells whereas others cells may show delayed activation and rapid inactivation. Thus, data collected from a single time point may only capture a subset of the responsive cells. Finally, at a cellular level, the degree of RHT innervation may also likely contribute to the efficiency of activation.
To test whether light-induced phosphorylation of p70 S6K and 4E-BP1 were dependent on mTOR, we employed an intraventricular infusion method to deliver the mTORC1 inhibitor rapamycin to the ventricular system. Similar rapamycin infusion approaches have been used by other groups to study the effects of mTOR in the brain (Tischmeyer et al., 2003
; Narita et al., 2005
). The infusion of rapamycin led to a complete inhibition of p70 S6K activity, its downstream target, S6, as well as 4E-BP1. Together, these data strongly support the idea that light triggers activation of mTOR-dependent signaling. It should be noted that rapamycin does not affect mTOR as part of the mTORC2 complex (Wullschleger et al., 2006
). Interestingly, mTORC2 is regulated by the same uspstream TSC2/Rheb2 signaling cassette, but actuates a distinct set of signaling events from mTORC1, such as actin polymerization (Jacinto et al., 2004
). Additional work will be required to assess inducible activation and function of mTORC2 in the SCN.
Interestingly, in contrast to p70 S6K, we detected relatively high levels of 4E-BP1 phosphorylation under control conditions and light treatment led to a relatively modest increase in 4E-BP1 phosphorylation. The activation of 4E-BP1 is mediated by a sequential set of phosphorylation events. Activation appears to be initiated by the phosphorylation of Thr-37 and Thr-46 (the two sites examined in this study). This dual phosphorylation is a priming event that allows for phosphorylation at Ser-65 and Thr-70 to occur (Gingras et al., 1999
; Gingras et al., 2001
). Ser-65 and Thr-70 phosphorylation are thought to be the trigger that initiates dissociation of 4E-BP1 from eIF4E, thereby allowing cap-dependent mRNA translation to occur (Lin et al., 1994
; Pause et al., 1994
). A number of studies have shown that the phosphorylation of Thr-37 and Thr-46 is mediated by mTOR, whereas Ser-65 and Thr-70 phosphorylation is regulated by both mTOR-dependent and -independent signaling (Gingras et al., 1999
; Nojima et al., 2003
). Of note, the finding that rapamycin infusion triggered a decrease in 4E-BP1 phosphorylation supports the idea that the SCN exhibits a relatively high tonic level of mTOR activity, thus raising the possibility that the mTOR pathway plays a broader role in the SCN than simply conveying photic information.
Next, our attention turned to potential effectors of p70 S6K in the SCN. To this end, we focused on S6. The rationale for examining S6 was two-fold: first, its phosphorylation can be used as a functional read-out for p70 S6K activity, and second, several studies have shown that it regulates the translation of a subset of mRNAs which contain a 5' tract of oligopyrimidine (TOP) (Jefferies et al.,1994
; Terada et al.,1994
; Jefferies et al.,1997
; Kawasome et al., 1998
; Schwab et al., 1999
). Interestingly, a number of 5'TOP mRNAs are components of the translation machinery and thus may influence the overall translational potential of the cell. However, the precise role of S6 in this process is not clear, and several studies have reported that S6 does not directly regulate translation of 5'TOP mRNAs (Tang et al., 2001
; Ruvinsky et al., 2005
). Our results show that light induced a marked increase in S6 phosphorylation at Ser-240 and Ser-244 and that rapamycin potently blocked this process. Interestingly, pS6 was detected in the absence of photic input, further supporting the idea that the SCN exhibits tonic mTOR activity.
The phase-restricted capacity of light to activate mTOR-dependent signaling is consistent with a large body of work showing that photic stimulation exerts a dominant effect during the night time domain. Along these lines, phase-restricted light responses have been characterized at both a behavioral and molecular level. For example, light exposure during the early night causes a phase delay in clock timing, whereas light exposure during the late night causes a phase advance in clock timing (Daan and Pittendrigh, 1976
). At the molecular level, the capacity of light to trigger the expression of the immediate early genes such as Fos, JunB and early growth response factor 1 (EGR-1) and core clock timing genes period 1
is restricted to the night (Aronin et al., 1990
; Kornhauser et al., 1990
; Rusak et al., 1990
, Albrecht et al., 1997
; Zylka et al., 1998
; Mendoza et al., 2007
). Likewise, light activation of the CREB/CRE transcription pathaway and kinases such as protein kinase C and the MAPK cascade is phase-restricted to the night time domain (Ginty et al., 1993
; Obrietan et al., 1998
; Lee et al., 2007
). The data reported here add the mTOR signaling pathway to this phase-restricted photic response network.
One key question for this study relates to the upstream signaling cascade that couples light to rapid activation of mTOR. To this end, we examined the MAPK cascade, a key signaling intermediate in light-mediated SCN entrainment (Obrietan et al., 1998
; Butcher et al., 2002
; Coogan and Piggins, 2003
). As an initial assessment of the potential role of MAPK signaling in light-induced mTOR activity, SCN sections were double labeled for activated ERK and p70 S6K. These assays detected a light-induced temporal and cellular correlation between MAPK pathway activity and p70 S6K phosphorylation. Furthermore, infusion of the MEK1/2 inhibitor U0126 potently repressed p70 S6K activity, thus indicating that light-induced MAPK signaling is an upstream activator of mTOR in the SCN.
There are several potential mechanisms by which the MAPK pathway could stimulate mTOR activity. First, recent work has revealed that ERK triggers phosphorylation-dependent inactivation of TSC2 GAP activity (Ma et al., 2005
). Second, ERK-regulated 90 kDa ribosomal kinase 1 (RSK1) has been shown to block TSC2 activity (Roux et al., 2004
), and thereby stimulate mTOR1 signaling. Interestingly, we recently reported that RSK1 is activated by light in the SCN (Butcher et al., 2004
). Additional work will be required to delineate the precise mechanism by which MAPK signaling regulates mTOR activity in the SCN.
In the SCN, MAPK signaling is thought to couple light to the core clock timing mechanism via a process that elicits transcription factor activation and, in turn, clock gene expression. Along these lines, the MAPK cascade has been shown to facilitate CRE-dependent transcription and period1
transcription (Obrietan et al., 1999
; Dziema et al., 2003
; Travnickova-Bendova et al., 2002
; Butcher et al., 2005
). Interestingly, the data presented here reveal that light triggers coordinate activation of CREB and mTOR-mediated signaling in the SCN. Given that MAPK signaling regulates both of these processes, these data raise the possiblity that the MAPK cascade serves coordinate roles as a regulator of gene transcription and mRNA translation (). Further studies aimed at addressing inducible translation regulation will shed new light on the key cellular signaling events that shape circadian clock timing and entrainment.
Schematic of proposed mTOR signaling pathway in the SCN