similar mechanisms in higher eukaryotic clock
systems (Yoshitane et al. 2009; Mahesh et al.
2014).
D. Post-translational Modifications in Clock
Regulation
1. Kinases Play an Essential Role in Circadian
Timing
Post-translational modifications, such as phosphorylation, are central to the functioning of the
positive and negative arm of the core clock in N.
crassa and other clock systems (for more complete reviews of the role of post-translational
modifications in higher eukaryotic systems, see
Mehra et al. 2009a; Weber et al. 2011; Hirano
et al. 2016). The addition of phosphate groups
by numerous kinases has been shown to affect
circadian timing by altering the stability of the
protein and its ability to form protein-protein
interactions. In N. crassa, these modifications
are carried out by the kinases CAMK1, CHK2
(PRD-4), CK1a, CK1b, CK2, GSK, and PKA,
which act on the core proteins of the negative
and positive arms of the clock (Fig. 4.2a).
As mentioned previously, FRQ is progressively phosphorylated in a circadian manner
with over 85 different residues phosphorylated,
making FRQ one of the most highly phosphorylated proteins ever documented (Baker et al.
2009; Tang et al. 2009). CK1a has been suggested to carry out much of this phosphorylation, as CK1a is found in complex with FRQ
and FRH throughout a large proportion of the
circadian day (Baker et al. 2009). Moreover,
when FRQ is not able to interact with CK1a,
FRQ is in a hypo-phosphorylated state and
shows increased stability (He et al. 2006; Querfurth et al. 2011). In addition, other kinases
such as CK2, PKA, CHK2 (PRD-4), CK1b, and
CAMK-1 interact more transiently with FRQ
and likely play a smaller role in its phosphorylation and circadian regulation (Yang et al.
2001, 2002; Pregueiro et al. 2006; He et al.
2006; Huang et al. 2007).
Throughout the daily cycle, different
regions of FRQ are phosphorylated in a clustered manner by interacting kinases, and the
phosphorylation of these different FRQ segments can variously stabilize or de-stabilize
this protein (Baker et al. 2009). The kinase
PKA increases FRQ stability, while CK1a, CK2,
and CHK2 decrease FRQ stability over a circadian cycle (Pregueiro et al. 2006; Huang et al.
2007). Phosphorylation also regulates FRQ’s
ability to interact with other proteins. The
extensive phosphorylation of FRQ affects its
half-life through increased affinity with ubiquitin ligases, such as FWD-1 (He et al. 2003; also
see Sect. III.D). In addition, FRQ’s phosphorylation by CK2 decreases its ability to interact
with the WCC, necessary for the start of another
round of transcription in the circadian loop
(Cheng et al. 2001b; Yang et al. 2002). Besides
targeting FRQ for eventual degradation via the
ubiquitin-proteasome pathway (Fig. 4.2a),
these phosphorylations may affect FRQ’s IDP
conformation to regulate the time-specific
binding of its different interaction partners to
complete the negative feedback loop that determines clock period (He et al. 2006; Baker et al.
2009; Querfurth et al. 2011; Hurley et al. 2013;
Pelham et al. 2018).
There is also evidence that phosphorylation
regulates the positive arm of the clock, influencing the activity, interactions, and degradation
of the WCC. The WCC is able to bind to the frq
c-box promoter when hypo-phosphorylated,
but its transcriptional activity is reduced when
the WCC is phosphorylated first by PKA, which
acts as a priming kinase, followed by CK1a and
CK2 in a FRQ-dependent manner (He et al.
2006; Huang et al. 2007; Wang et al. 2019).
Beyond the canonical circadian kinases, Glycogen synthase kinase (GSK) phosphorylates both
WC-1 and WC-2, presumably promoting WCC
degradation via phosphorylation, and the resultant decrease in WCC levels lengthens the circadian period (Tatarog ˘lu et al. 2012). Like WC1, WC-2 shows a circadian oscillation in phosphorylation (Schafmeier et al. 2005), with
increasing WC-2 phosphorylation leading to a
decrease in overall WCC activity.
4 From Genetics to Molecular Oscillations: The Circadian Clock in Neurospora crassa
89
systems (Yoshitane et al. 2009; Mahesh et al.
2014).
D. Post-translational Modifications in Clock
Regulation
1. Kinases Play an Essential Role in Circadian
Timing
Post-translational modifications, such as phosphorylation, are central to the functioning of the
positive and negative arm of the core clock in N.
crassa and other clock systems (for more complete reviews of the role of post-translational
modifications in higher eukaryotic systems, see
Mehra et al. 2009a; Weber et al. 2011; Hirano
et al. 2016). The addition of phosphate groups
by numerous kinases has been shown to affect
circadian timing by altering the stability of the
protein and its ability to form protein-protein
interactions. In N. crassa, these modifications
are carried out by the kinases CAMK1, CHK2
(PRD-4), CK1a, CK1b, CK2, GSK, and PKA,
which act on the core proteins of the negative
and positive arms of the clock (Fig. 4.2a).
As mentioned previously, FRQ is progressively phosphorylated in a circadian manner
with over 85 different residues phosphorylated,
making FRQ one of the most highly phosphorylated proteins ever documented (Baker et al.
2009; Tang et al. 2009). CK1a has been suggested to carry out much of this phosphorylation, as CK1a is found in complex with FRQ
and FRH throughout a large proportion of the
circadian day (Baker et al. 2009). Moreover,
when FRQ is not able to interact with CK1a,
FRQ is in a hypo-phosphorylated state and
shows increased stability (He et al. 2006; Querfurth et al. 2011). In addition, other kinases
such as CK2, PKA, CHK2 (PRD-4), CK1b, and
CAMK-1 interact more transiently with FRQ
and likely play a smaller role in its phosphorylation and circadian regulation (Yang et al.
2001, 2002; Pregueiro et al. 2006; He et al.
2006; Huang et al. 2007).
Throughout the daily cycle, different
regions of FRQ are phosphorylated in a clustered manner by interacting kinases, and the
phosphorylation of these different FRQ segments can variously stabilize or de-stabilize
this protein (Baker et al. 2009). The kinase
PKA increases FRQ stability, while CK1a, CK2,
and CHK2 decrease FRQ stability over a circadian cycle (Pregueiro et al. 2006; Huang et al.
2007). Phosphorylation also regulates FRQ’s
ability to interact with other proteins. The
extensive phosphorylation of FRQ affects its
half-life through increased affinity with ubiquitin ligases, such as FWD-1 (He et al. 2003; also
see Sect. III.D). In addition, FRQ’s phosphorylation by CK2 decreases its ability to interact
with the WCC, necessary for the start of another
round of transcription in the circadian loop
(Cheng et al. 2001b; Yang et al. 2002). Besides
targeting FRQ for eventual degradation via the
ubiquitin-proteasome pathway (Fig. 4.2a),
these phosphorylations may affect FRQ’s IDP
conformation to regulate the time-specific
binding of its different interaction partners to
complete the negative feedback loop that determines clock period (He et al. 2006; Baker et al.
2009; Querfurth et al. 2011; Hurley et al. 2013;
Pelham et al. 2018).
There is also evidence that phosphorylation
regulates the positive arm of the clock, influencing the activity, interactions, and degradation
of the WCC. The WCC is able to bind to the frq
c-box promoter when hypo-phosphorylated,
but its transcriptional activity is reduced when
the WCC is phosphorylated first by PKA, which
acts as a priming kinase, followed by CK1a and
CK2 in a FRQ-dependent manner (He et al.
2006; Huang et al. 2007; Wang et al. 2019).
Beyond the canonical circadian kinases, Glycogen synthase kinase (GSK) phosphorylates both
WC-1 and WC-2, presumably promoting WCC
degradation via phosphorylation, and the resultant decrease in WCC levels lengthens the circadian period (Tatarog ˘lu et al. 2012). Like WC1, WC-2 shows a circadian oscillation in phosphorylation (Schafmeier et al. 2005), with
increasing WC-2 phosphorylation leading to a
decrease in overall WCC activity.
4 From Genetics to Molecular Oscillations: The Circadian Clock in Neurospora crassa
89