2. Phosphatases Support the Proper Timing of
Circadian Period and Phase
Phosphatases are a group of widely conserved
post-transcriptional modifiers that remove
phosphate groups and have demonstrated
importance for proper clock function. Three
protein phosphatases, PP1, PP2A, and PP4, all
play a role in supporting the core clock (Cha
et al. 2008; Yang et al. 2004). PP1 and PP4
support FRQ stability and therefore proper
phase timing and period length, and PP2A is
important for maintaining the proper frq
mRNA level (Yang et al. 2004; Cha et al. 2008).
However, the most detailed study of FRQ phosphorylation showed only progressive phosphorylation. Without in vivo evidence for dephosphorylation, the effects of phosphatases on
FRQ may thus be indirect (Baker et al. 2009).
Indeed, some of these phosphatases also act on
the WCC and thereby may affect frq levels and
FRQ protein via the core feedback loop. For
example, PP2A and PP4 de-phosphorylate the
WCC, while it is in the cytosol. This is important as this de-phosphorylation potentially
impacts the ability of the WCC to re-enter the
nucleus and start a new round of transcription,
suggesting that the major impact of phosphatases on FRQ is via the positive arm (Fig. 4.2a)
(Cha et al. 2008; Schafmeier et al. 2005, 2008).
Phosphatases also play an important role in the
circadian clocks of Drosophila and mammals,
modulating the phosphorylation state of clock
proteins such that an approximately 24 h
period is maintained (Reischl and Kramer
2011).
E. Ubiquitination and Degradation Contribute
to a Robust Circadian Clock
While not all phosphorylation leads to degradation, the overall progressive phosphorylation
of FRQ is associated with its regulated turnover
by the ubiquitin-proteasome pathway (Garceau et al. 1997; Liu et al. 2000; Baker et al.
2009; Guo et al. 2010). Following the phosphorylation of FRQ, particularly in the PEST-1
region, FRQ is ubiquitinated by the F-box/
WD40 repeat-containing protein FWD-1,
which promotes FRQ’s degradation via the proteasome (Fig. 4.2a) (He et al. 2003). FWD-1 is
part of an SKP/Cullin/F-box (SCF)-type E3 ubiquitin ligase complex that can bind phosphorylated FRQ motifs, with more FRQ
phosphorylation presenting more potential
FWD-1 binding sites (He et al. 2003; He and
Liu 2005a). When fwd-1 is knocked out, the
resulting strain is overtly arrhythmic; hyperphosphorylated FRQ accumulates to high levels
and conidial banding is lost (He et al. 2003; He
and Liu 2005a). However, recent work using a
luciferase reporter system has shown that FRQ
degradation is not strictly required for the completion of the circadian feedback loop and the
maintenance of period length (Fig. 4.2b) (Larrondo et al. 2015). By measuring in vivo luminescence from a strain expressing luciferase
under the control of a segment from the promoter of frq that contains the c-box, circadian
rhythms in frq transcriptional activation were
found even when fwd-1 was knocked out (Larrondo et al. 2015). This suggests that negative
arm protein degradation is not required for the
core circadian oscillator and is not actually a
determinant of clock period, and has since been
confirmed in a mammalian system (Larrondo
et al. 2015; Ode et al. 2017).
Even though FRQ degradation may not be
required for clock functionality, premature
FRQ degradation still leads to arrhythmic
strains (Guo et al. 2010; Hurley et al. 2013). As
an intrinsically disordered protein (IDP), without its stabilizing FRH “Nanny,” FRQ is rapidly
degraded within 3 h through a passive “degradation by default” pathway that does not
involve ubiquitination (Tsvetkov et al. 2009;
Guo et al. 2010; Hurley et al. 2013). Only when
protected by FRH can FRQ have a chance to
mature and carry out its circadian functions
before undergoing ubiquitination and targeted
degradation. This degradation by default pathway also has implications for mammal clock
systems, since the negative arm PER proteins
are also predicted to be IDPs (Hurley et al.
2013).
90
M. S. Jankowski et al.
Circadian Period and Phase
Phosphatases are a group of widely conserved
post-transcriptional modifiers that remove
phosphate groups and have demonstrated
importance for proper clock function. Three
protein phosphatases, PP1, PP2A, and PP4, all
play a role in supporting the core clock (Cha
et al. 2008; Yang et al. 2004). PP1 and PP4
support FRQ stability and therefore proper
phase timing and period length, and PP2A is
important for maintaining the proper frq
mRNA level (Yang et al. 2004; Cha et al. 2008).
However, the most detailed study of FRQ phosphorylation showed only progressive phosphorylation. Without in vivo evidence for dephosphorylation, the effects of phosphatases on
FRQ may thus be indirect (Baker et al. 2009).
Indeed, some of these phosphatases also act on
the WCC and thereby may affect frq levels and
FRQ protein via the core feedback loop. For
example, PP2A and PP4 de-phosphorylate the
WCC, while it is in the cytosol. This is important as this de-phosphorylation potentially
impacts the ability of the WCC to re-enter the
nucleus and start a new round of transcription,
suggesting that the major impact of phosphatases on FRQ is via the positive arm (Fig. 4.2a)
(Cha et al. 2008; Schafmeier et al. 2005, 2008).
Phosphatases also play an important role in the
circadian clocks of Drosophila and mammals,
modulating the phosphorylation state of clock
proteins such that an approximately 24 h
period is maintained (Reischl and Kramer
2011).
E. Ubiquitination and Degradation Contribute
to a Robust Circadian Clock
While not all phosphorylation leads to degradation, the overall progressive phosphorylation
of FRQ is associated with its regulated turnover
by the ubiquitin-proteasome pathway (Garceau et al. 1997; Liu et al. 2000; Baker et al.
2009; Guo et al. 2010). Following the phosphorylation of FRQ, particularly in the PEST-1
region, FRQ is ubiquitinated by the F-box/
WD40 repeat-containing protein FWD-1,
which promotes FRQ’s degradation via the proteasome (Fig. 4.2a) (He et al. 2003). FWD-1 is
part of an SKP/Cullin/F-box (SCF)-type E3 ubiquitin ligase complex that can bind phosphorylated FRQ motifs, with more FRQ
phosphorylation presenting more potential
FWD-1 binding sites (He et al. 2003; He and
Liu 2005a). When fwd-1 is knocked out, the
resulting strain is overtly arrhythmic; hyperphosphorylated FRQ accumulates to high levels
and conidial banding is lost (He et al. 2003; He
and Liu 2005a). However, recent work using a
luciferase reporter system has shown that FRQ
degradation is not strictly required for the completion of the circadian feedback loop and the
maintenance of period length (Fig. 4.2b) (Larrondo et al. 2015). By measuring in vivo luminescence from a strain expressing luciferase
under the control of a segment from the promoter of frq that contains the c-box, circadian
rhythms in frq transcriptional activation were
found even when fwd-1 was knocked out (Larrondo et al. 2015). This suggests that negative
arm protein degradation is not required for the
core circadian oscillator and is not actually a
determinant of clock period, and has since been
confirmed in a mammalian system (Larrondo
et al. 2015; Ode et al. 2017).
Even though FRQ degradation may not be
required for clock functionality, premature
FRQ degradation still leads to arrhythmic
strains (Guo et al. 2010; Hurley et al. 2013). As
an intrinsically disordered protein (IDP), without its stabilizing FRH “Nanny,” FRQ is rapidly
degraded within 3 h through a passive “degradation by default” pathway that does not
involve ubiquitination (Tsvetkov et al. 2009;
Guo et al. 2010; Hurley et al. 2013). Only when
protected by FRH can FRQ have a chance to
mature and carry out its circadian functions
before undergoing ubiquitination and targeted
degradation. This degradation by default pathway also has implications for mammal clock
systems, since the negative arm PER proteins
are also predicted to be IDPs (Hurley et al.
2013).
90
M. S. Jankowski et al.