Kinase Domain Autophosphorylation Rewires the Activity and Substrate Specificity of CK1 Enzymes

Abstract

CK1s are acidophilic serine/threonine kinases with multiple critical cellular functions; their misregulation contributes to cancer, neurodegenerative diseases, and sleep phase disorders. Here, we describe an evolutionarily conserved mechanism of CK1 activity: autophosphorylation of a threonine (T220 in human CK1δ) located at the N‐terminus of helix αG, proximal to the substrate binding cleft. Crystal structures and molecular dynamics simulations uncovered inherent plasticity in αG that increased upon T220 autophosphorylation. The phosphorylation‐induced structural changes significantly altered the conformation of the substrate binding cleft, affecting substrate specificity. In T220 phosphorylated yeast and human CK1s, activity toward many substrates was decreased, but we also identified a high‐affinity substrate that was phosphorylated more rapidly, and quantitative phosphoproteomics revealed that disrupting T220 autophosphorylation rewired CK1 signaling in Schizosaccharomyces pombe. T220 is present exclusively in the CK1 family, thus its autophosphorylation may have evolved as a unique regulatory mechanism for this important family.

Dual-specific autophosphorylation of kinase IKK2 enables phosphorylation of substrate IκBα through a phosphoenzyme intermediate

“…Phosphorylation of substrates by protein kinases is regulated in various ways (Cullati et al, 2022;Sang et al, 2022). IKK2, a kinase central to inflammation, phosphorylates serine residues of its primary substrate IB with -phosphate of ATP.…”

“…Kinase domain autophosphorylation is known to regulate function of other kinases as well, e.g., autophosphorylation at T220 greatly influenced both the activity and substrate specificity of CK1 (Cullati et al, 2022).…”

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

“…In addition, the presence of an Asparagine (N) at position 220 in fly DBT, as opposed to a Threonine (T) at this position in CKIδ, might contribute to the opposite temperature compensation phenotypes of K224D mutants in flies and mice. Replacing the fly Asparagine (N220) with a Threonine would be interesting, not only in the light of temperature compensation differences, but also in the context of recently published autophosphorylation of Threonine, which is the preferred amino acid in corresponding positions in the majority of homologous kinases (Cullati et al, 2022). Secondly, the possible interaction with BDBT in the fruitfly might be an interesting and experimentally testable explanation for the altered temperature compensation in K224D.…”

“…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.…”

Circadian rhythms in the Drosophila eye may regulate adaptation of vision to light intensity

“…Moreover, the nuclear localization signal to which BDBT binds (Venkatesan et al, 2015(Venkatesan et al, , 2019 is preceded by an Asparagine (N) in Drosophila and by a phosphorylatable Threonine (T) in mammals and yeast (Cullati et al, 2022;Thakkar et al, 2022). This T can be phosphorylated in mammals and yeast to change the substrate preference for CKI.…”