Kinase domain autophosphorylation rewires the activity and substrate specificity of CK1 enzymes

Cullati, Sierra; Chaikuad, A.; Chen, Jun‐Song; Gebel, Jakob; Tesmer, Laura; Zhubi, Rezart; Navarrete‐Perea, Jose; Guillen, Rodrigo X.; Gygi, Steven P.; Hummer, Gerhard; Dötsch, Volker; Knapp, Stefan; Gould, Kathleen L.

doi:10.1016/j.molcel.2022.03.005

Cited by 21 publications

(46 citation statements)

References 120 publications

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“…Site 1 harbors the location of the tau mutation (R178C) that shortens circadian period (Lowrey et al, 2000). We did not observe strong density for K224, the other basic residue that could coordinate an anion in Site 1, suggesting flexibility in the Fɑ helix consistent with previous studies (Cullati et al, 2022; Philpott et al, 2020; Shinohara et al, 2017). Moving down the pFASP peptide, we observed additional backbone-backbone interactions and docking of the pFASP side chains of V663 & A664 into small hydrophobic pockets within the substrate binding cleft ( Figure 4b , d ).…”

Section: Resultssupporting

confidence: 91%

“…Moving down the pFASP peptide, we observed additional backbone-backbone interactions and docking of the pFASP side chains of V663 & A664 into small hydrophobic pockets within the substrate binding cleft ( Figure 4b , d ). We did not observe strong density for K224, the other basic residue that could coordinate an anion in Site 1, suggesting flexibility in the Fɑ helix consistent with previous studies (Cullati et al, 2022; Philpott et al, 2020; Shinohara et al, 2017). Moving down the pFASP peptide, we observed additional backbone-backbone interactions and docking of the pFASP side chains of V663 & A664 into small hydrophobic pockets within the substrate binding cleft ( Figure 4b , d ).…”

Section: Resultssupporting

confidence: 91%

“…As expected, pS662 forms stable electrostatic interactions in Site 1 ( Figure 5c ). In addition to being close to R178 and K224, pS662 also engages in stable electrostatic interactions with Q214, located in the highly flexible loop-EF (Cullati et al, 2022; Shinohara et al, 2017). In the active site, pS665 is locked in place by a cluster of electrostatic interactions involving D128, K130, D148 and a Na + ion ( Figure 5d ).…”

Section: Resultsmentioning

confidence: 99%

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PERIOD phosphorylation leads to feedback inhibition of CK1 activity to control circadian period

Philpott

Freeberg

Park

et al. 2022

Preprint

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PERIOD (PER) and Casein Kinase 1δ regulate circadian rhythms through a phosphoswitch that controls PER stability and repressive activity in the molecular clock. CK1δ phosphorylation of the Familial Advanced Sleep Phase (FASP) serine cluster embedded within the Casein Kinase 1 binding domain (CK1BD) of mammalian PER1/2 inhibits its activity on phosphodegrons to stabilize PER and extend circadian period. Here, we show that the phosphorylated FASP region (pFASP) of PER2 directly interacts with and inhibits CK1δ. Co-crystal structures in conjunction with accelerated molecular dynamics simulations reveal how pFASP phosphoserines dock into conserved anion binding sites near the active site of CK1δ. Limiting phosphorylation of the FASP serine cluster reduces product inhibition, decreasing PER2 stability and shortens circadian period in human cells. We found that Drosophila PER also regulates CK1δ via feedback inhibition through the phosphorylated PER-Short domain, revealing a conserved mechanism by which PER phosphorylation near the CK1BD regulates CK1 kinase activity.

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Section: Resultssupporting

confidence: 91%

Section: Resultssupporting

confidence: 91%

Section: Resultsmentioning

confidence: 99%

See 1 more Smart Citation

PERIOD phosphorylation leads to feedback inhibition of CK1 activity to control circadian period

Philpott

Freeberg

Park

et al. 2022

Preprint

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“…Notably, D. melanogaster DBT contains Asparagine (N) instead of Threonine (T) in position 220, which contrasts with DBT homologs from Thermobia , mouse, ( Figure 1A ), and more distant kinases. A recent study indicates that autophosphorylation of T220 influences substrate specificity ( Cullati et al, 2022 ), thus the N at 220 prevents this posttranslational regulatory modification. Remarkable sequence divergence is observed in the C-terminal tail.…”

Section: Resultsmentioning

confidence: 99%

Evolution of casein kinase 1 and functional analysis of new doubletime mutants in Drosophila

et al. 2022

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Circadian clocks are timing devices that rhythmically adjust organism’s behavior, physiology, and metabolism to the 24-h day-night cycle. Eukaryotic circadian clocks rely on several interlocked transcription-translation feedback loops, where protein stability is the key part of the delay between transcription and the appearance of the mature proteins within the feedback loops. In bilaterian animals, including mammals and insects, the circadian clock depends on a homologous set of proteins. Despite mostly conserved clock components among the fruit fly Drosophila and mammals, several lineage-specific differences exist. Here we have systematically explored the evolution and sequence variability of insect DBT proteins and their vertebrate homologs casein kinase 1 delta (CKIδ) and epsilon (CKIε), dated the origin and separation of CKIδ from CKIε, and identified at least three additional independent duplications of the CKIδ/ε gene in Petromyzon, Danio, and Xenopus. We determined conserved regions in DBT specific to Diptera, and functionally tested a subset of those in D. melanogaster. Replacement of Lysine K224 with acidic residues strongly impacts the free-running period even in heterozygous flies, whereas homozygous mutants are not viable. K224D mutants have a temperature compensation defect with longer free-running periods at higher temperatures, which is exactly the opposite trend of what was reported for corresponding mammalian mutants. All DBTs of dipteran insects contain the NKRQK motif at positions 220–224. The occurrence of this motif perfectly correlates with the presence of BRIDE OF DOUBLETIME, BDBT, in Diptera. BDBT is a non-canonical FK506-binding protein that physically interacts with Drosophila DBT. The phylogeny of FK506-binding proteins suggests that BDBT is either absent or highly modified in non-dipteran insects. In addition to in silico analysis of DBT/CKIδ/ε evolution and diversity, we have identified four novel casein kinase 1 genes specific to the Drosophila genus.

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“…Further, to investigate whether individually overexpressed PP2A subunits could lead to increased cellular PP2A phosphatase activity, we measured the phosphatase activity of the PP2A holoenzymes isolated from yeast cells by an in vitro dephosphorylation assay. In this assay, autophosphorylated Hhp2, one of the S. pombe CK1s, was used as the artificial substrate for PP2A (Figure S3), because CK1 can autophosphorylate 66 while PP2A can counteract CK1‐dependent phosphorylation 67 . We tagged the PP2A subunit‐encoding genes at their endogenous loci with 3Flag, thus the relative levels of endogenously expressed and overexpressed PP2A subunits were distinguishable due to their size difference (3Flag vs. TetR‐2Flag tags) when they were simultaneously present (Figure 3B).…”

Section: Resultsmentioning

confidence: 99%

Analysis of the potential role of fission yeast PP2A in spindle assembly checkpoint inactivation

et al. 2022

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As a surveillance mechanism, the activated spindle assembly checkpoint (SAC) potently inhibits the E3 ubiquitin ligase APC/C (anaphase-promoting complex/ cyclosome) to ensure accurate chromosome segregation. Although the protein phosphatase 2A (PP2A) has been proposed to be both, directly and indirectly, involved in spindle assembly checkpoint inactivation in mammalian cells, whether it is similarly operating in the fission yeast Schizosaccharomycer pombe has never been demonstrated. Here, we investigated whether fission yeast PP2A is involved in SAC silencing by following the rate of cyclin B (Cdc13) destruction at SPBs during the recovery phase in nda3-KM311 cells released from the inhibition of APC/C by the activated spindle checkpoint. The timing of the SAC inactivation is only slightly delayed when two B56 regulatory subunits (Par1 and Par2) of fission yeast PP2A are absent. Overproduction of individual PP2A subunits either globally in the nda3-KM311 arrest-and-release system or locally in the synthetic spindle checkpoint activation system only slightly suppresses the SAC silencing defects in PP1 deletion (dis2Δ) cells. Our study thus demonstrates that the fission yeast PP2A is not a key regulator actively involved in SAC inactivation.

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Kinase domain autophosphorylation rewires the activity and substrate specificity of CK1 enzymes

Cited by 21 publications

References 120 publications

PERIOD phosphorylation leads to feedback inhibition of CK1 activity to control circadian period

PERIOD phosphorylation leads to feedback inhibition of CK1 activity to control circadian period

Evolution of casein kinase 1 and functional analysis of new doubletime mutants in Drosophila

Analysis of the potential role of fission yeast PP2A in spindle assembly checkpoint inactivation

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