Autophosphorylation of the CK1 kinase domain regulates enzyme activity and function

Abstract

CK1 enzymes are conserved, acidophilic serine/threonine kinases with a variety of critical cellular functions; misregulation of CK1 contributes to cancer, neurodegenerative diseases, and sleep phase disorders. Despite this, little is known about how CK1 activity is controlled. CK1 kinases have highly similar catalytic domains, plus a conserved extension to that kinase domain that is important for enzyme stability and activity. In contrast to the catalytic domains, the C‐terminal tails of CK1 family members diverge in sequence and length; however, they all appear to serve as substrates of autophosphorylation. The autophosphorylated tails are proposed to inhibit kinase activity by acting as pseudosubstrates. In addition to C‐terminal autophosphorylation, autophosphorylation of the CK1□ kinase domain has been detected, but its effect on CK1 activity and cellular function has never been explored. Here, we addressed the role of kinase domain autophosphorylation in human CK1□ and CK1□, as well as Hhp1 and Hhp2, their homologues in Schizosaccharomyces pombe. In each case, we found that autophosphorylation of a conserved threonine residue in the kinase domain inhibited enzyme activity. This site resides in the mobile L‐EF loop proximal to the active site, distinct from the well‐characterized T‐loop autophosphorylation that occurs in other kinase families. We found that yeast and human enzymes with phosphoablating mutations at this site are hyperactive in vitro. In vivo, mutation of this site protects yeast cells from heat shock, indicating a change in substrate profile, a prediction we are currently testing using quantitative phosphoproteomics. We have also found that autophosphorylation of the kinase domain may affect autophosphorylation of the C‐terminus, so there is likely interplay between the two different modalities of CK1 autoregulation that ultimately determines the extent to which substrates are phosphorylated. We propose that phosphorylation on the L‐EF loop prevents substrate docking with the kinase domain by shielding the positively charged substrate binding pocket and/or sterically hindering the active site. Due to the strong sequence conservation of this autophosphorylation site and the functional importance of the L‐EF loop, which is unique to the CK1 family of kinases, this mechanism is likely to regulate the majority of CK1 enzymes in vivo. Support or Funding Information This work was supported by R35‐GM131799 (to KLG) and T32‐CA119925 (to SNC).

Assessing the Inhibitory Potential of Kinase Inhibitors In Vitro: Major Pitfalls and Suggestions for Improving Comparability of Data Using CK1 Inhibitors as an Example

“…Although depletion of ATP catalyzed by all tested enzymes was less than 10% of total ATP after 60 min, the reaction velocity already decreased after 10 min (as shown for 7 nM GST-CK1δ and 70 nM 6×His-CK1δ in Figure 3 and Figure 5 ) or 5 min (as shown for 70 nM 6×His-CK1ε in Figure 9 ). Since substrate depletion cannot explain the decline in reaction velocity, CK1δ might be inhibited due to time-dependent increase in autophosphorylation [ 18 , 34 , 35 ]. Unfortunately, autophosphorylation events within the enzyme reaction cannot be prevented in an easy way, e.g., by the addition of phosphatases.…”

“…Lastly, the subsequent separation of the kinase reactions via SDS-PAGE also allows for discrimination between substrate phosphorylation and autophosphorylation of the kinase protein, which is essential in order to correctly and reliably determine kinase activity and thus inhibitor potency. Autophosphorylation modulates kinase activity and is a feature shared by almost all eukaryotic protein kinases [ 17 , 18 ], most of them becoming catalytically active by phosphorylation of their activation loop or autophosphorylation domains [ 19 ]. The levels of kinase autophosphorylation can be highly variable between kinases originating from different expression systems [ 20 ].…”

“…By decreasing the enzyme concentration (red curve) the initial velocity region can be extended while simultaneously lowering the limit of detectable IC 50 values. The shape of the initial velocity region can furthermore be affected by the loss of enzymatic activity due to inhibition (e.g., mediated by autophosphorylation [ 18 ], product inhibition [ 25 ]) or degradation (green curve). Determining the initial velocity region by testing different enzyme concentrations is a mandatory requirement for the correct subsequent determination of the kinetic parameters and should be considered as the first step of the inhibitor screening procedure.…”

“…Due to their regulatory function in DNA damage-related signal transduction including the direct phosphorylation of central components like p53 and MDM2 as well as their involvement in Wnt, Hedgehog and Hippo signal transduction pathways CK1 isoforms have evolved as potential targets in cancer therapy [ 28 , 29 ]. Dysregulation and mutations of CK1 isoforms were shown to be involved in cancer progression but also play a critical role in the development of neurodegenerative diseases like Alzheimer’s disease [ 18 , 30 ]. According to this, the development of SMIs that exclusively inhibit disease-associated CK1 isoforms has been promoted in recent years.…”