A Tail of Kinase Regulation: How C‐termini Modulate CK1 Substrate Phosphorylation
Authors: Cullati Sierra , Chen Jun‐Song , Gould Kathy 
Published: June 2021

CK1 enzymes signal in a variety of important cellular pathways, including DNA damage repair, mitotic checkpoint signaling, circadian rhythm, Wnt signaling, endocytosis, and neurodegenerative disease progression. Like other multifunctional kinases, CK1 must be regulated in space and time in order to target specific subsets of its substrates in each of the pathways it participates in. However, CK1 is generally regarded as a “rogue” kinase, which is constitutively active, ubiquitous throughout cells and tissues, and unregulated except by autoinhibition.

CK1 enzymes are known to autophosphorylate their C‐terminal non‐catalytic tails, which are proposed to inhibit their activity by acting as pseudosubstrates. This model would require a phosphorylation‐dependent intramolecular interaction between the C‐terminus and the kinase domain, but we are unaware of any evidence demonstrating such an event. Furthermore, this proposed mechanism of autoinhibition has not been tested in vivo in any organism.

We have identified six serine and threonine autophosphorylation sites on the C‐terminus of Schizosaccharomyces pombe Hhp1, one of two soluble CK1 enzymes in this organism, and are testing candidate sites on Hhp2 and the human homologues CK1□/□. When these sites are specifically phosphorylated, the Hhp1 C‐terminus binds the kinase domain via a low‐affinity electrostatic interaction. At concentrations above the Kd of this interaction, the phosphorylated C‐terminus inhibits Hhp1 kinase activity, while mutations that abolish phosphorylation increase the activity of the full‐length kinase.

Structural studies have identified two conserved basic patches on the CK1 kinase domain that are hypothesized to interact with the phosphorylated C‐terminus; however, we show that the tail instead interacts with the substrate binding pocket. The physiological substrate Sid4 has a higher affinity than the tail for the Hhp1 kinase domain, and the model substrate casein can out‐compete the phosphorylated C‐terminus for binding to the kinase domain. Our data support a new model of CK1 activation in which the presence of high‐affinity substrates is sufficient to displace the low‐affinity tail and relieve autoinhibition without the need for prior dephosphorylation of CK1.

Because the C‐termini of CK1 family members are responsible for most of the sequence variation between isoforms, we hypothesize that these different tails may differ in affinity for the conserved kinase domain, leading to displacement by different cohorts of substrates. Thus, this mechanism may also explain variations in substrate specificities between CK1 enzymes. In support of this model, truncating the tail of Hhp1 or Hhp2 eliminates substrate‐specific differences in catalytic efficiency. We are currently using phosphorylation site mutants of hhp1 to confirm that these changes in catalytic efficiency are dependent on autophosphorylation and to investigate the significance of autophosphorylation in vivo.

Support or Funding Information

SNC was supported by the Integrated Biological Systems Training in Oncology Program (2T32CA119925). This work was supported by NIH GM112989 to KLG.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.