Motif analysis reveals diurnal utilization of phosphorylation
sites
We examined our phosphoproteome data for enrichment of phosphorylation
motifs that are connected to known protein kinases using Motif-X
(motif-x.med.harvard.edu; Chou & Schwartz, 2011; Schwartz & Gygi,
2005). The significantly changing phosphorylated peptides at each
transition were analyzed against all quantified phosphopeptides
(P value ≤ 0.05). Motifs corresponding to serine (pS) phosphorylation
sites were enriched at each transition, while enrichment of
phosphorylated threonine (pT) or tyrosine (pY) motifs was absent
(Supplemental Table 7). The lack of pY motif enrichment has also been
reported in other studies examining phosphoproteome changes under either
ED vs EN (Reiland et al., 2009; Uhrig et al., 2019) or free-running
circadian cycles (Choudhary et al., 2015; Krahmer et al., 2019). Only
one pT motif (pTP) has been previously associated with ED vs EN
phosphoproteome changes (Uhrig et al., 2019). The lack of an enriched
pTP motif here is likely due to our stringent multi-time point threshold
requirement for each phosphorylation site versus the comparison
previously performed between ED and EN only (Uhrig et al., 2019).
Furthermore, pS accounts for 84-86% of all phosphorylation events in
plants, compared to only 10-12% pT and 1-4% pY (Nakagami et al., 2010;
Sugiyama et al., 2008), which makes it less likely to find an enrichment
of pT and/or pY motifs in the phosphoproteome. Of the phosphorylation
sites (site probability score ≥ 0.8 we quantified, 82.8%, 16.5% and
0.7% were pS, pT and pY respectively, which is similar to previously
reported distributions of phosphorylation events (Nakagami et al., 2010;
Sugiyama et al., 2008).
At the L-D transition, we found 16 motifs of which 10 correspond to
phosphorylation sites previously identified as targets of protein
kinases CaMKII, PAK1, extracellular signal-regulated kinase (ERK 1/2),
proto-oncogene c-RAF (RAF1), and cell division cycle 2 (CDC2) protein
kinase A and B (Supplemental Table 7). Six phosphorylation sites did not
correspond to known kinase motifs, therefore likely representing
currently uncharacterized and possibly plant-specific motifs considering
the large expansion of protein kinases in plants relative to humans
(Lehti-Shiu & Shiu, 2012). At the D-L transition, four of five
identified motifs are known phosphorylation sites for checkpoint
kinase 1 (CHK1), PAK2, calmodulin kinase IV (CaMKIV) and casein kinase
(CKII) (Supplemental Table 7). CKII phosphorylates the core circadian
clock transcription factors LHY and CCA1 (Lu et al., 2011), which also
peak at the D-L transition (Kusakina & Dodd, 2012). Consistent with
this, we find enrichment of phosphorylation events corresponding to the
CKII phosphorylation motif for these proteins at the D-L transition.
Calcium (Ca2+) has been implicated in circadian
regulation (Marti Ruiz et al., 2018), suggesting that calcium-dependent
calmodulin (CaM) protein kinase orthologs are interesting candidates for
mediating circadian clock signaling. Unlike the enrichment of CKII
motifs at only the D-L transition, we find enrichment of
Ca2+ related kinases CaMKII (D-L and L-D) and CaMKIV
(D-L) phosphorylation motifs at both transitions (Supplemental Table 7).
Previous analyses identified other Ca2+ kinase motifs
enriched at both ED and EN (CDPK-like motifs; Uhrig et al., 2019).
SnRK1-related motifs were identified in the phosphoproteome data from
Arabidopsis CCA1-Ox plants growing in a free-running cycle (Krahmer et
al., 2019). SnRK1 is a central mediator of energy signaling between
different organelles and also phosphorylates CDPKs (Wurzinger,
Nukarinen, Nagele, Weckwerth, & Teige, 2018). Together, these studies
and the results presented here suggest a broader role for
Ca2+ in diurnal plant cell regulation during the L-D
and D-L transitions.
Compared to humans, plants have more protein kinases (Lehti-Shiu &
Shiu, 2012), but most of their targets remain unknown. Our
phosphoproteome results, together with previously reported
phosphoproteome datasets (Choudhary et al., 2015; Krahmer et al., 2019;
Reiland et al., 2009; Uhrig et al., 2019) provide a compilation of
specific phosphorylation motifs that are rapidly modified at light-dark
diurnal transitions. When combined with transcriptional expression data
for lower abundant proteins kinases (Uhrig et al., 2019), the
phosphoproteome changes uncovered here in other studies (Choudhary et
al., 2015; Krahmer et al., 2019; Reiland et al., 2009; Uhrig et al.,
2019) narrow the protein kinase sub-families to those most likely
catalyzing diurnal phosphorylation events.