Autophosphorylation of carboxy‐terminal residues inhibits the activity of protein kinase CK1α

Abstract

CK1 constitutes a protein kinase subfamily that is involved in many important physiological processes. However, there is limited knowledge about mechanisms that regulate their activity. Isoforms CK1delta and CK1epsilon were previously shown to autophosphorylate carboxy-terminal sites, a process which effectively inhibits their catalytic activity. Mass spectrometry of CK1alpha and splice variant CK1alphaL has identified the autophosphorylation of the last four carboxyl-end serines and threonines and also for CK1alphaS, the same four residues plus threonine-327 and serine-332 of the S insert. Autophosphorylation occurs while the recombinant proteins are expressed in Escherichia coli. Mutation of four carboxy-terminal phosphorylation sites of CK1alpha to alanine demonstrates that these residues are the principal but not unique sites of autophosphorylation. Treatment of autophosphorylated CK1alpha and CK1alphaS with lambda phosphatase causes an activation of 80-100% and 300%, respectively. Similar treatment fails to stimulate the CK1alpha mutants lacking autophosphorylation sites. Incubation of dephosphorylated enzymes with ATP to allow renewed autophosphorylation causes significant inhibition of CK1alpha and CK1alphaS. The substrate for these studies was a synthetic canonical peptide for CK1 (RRKDLHDDEEDEAMS*ITA). The stimulation of activity seen upon dephosphorylation of CK1alpha and CK1alphaS was also observed using the known CK1 protein substrates DARPP-32, beta-catenin, and CK2beta, which have different CK1 recognition sequences. Autophosphorylation effects on CK1alpha activity are not due to changes in Km(app) for ATP or for peptide substrate but rather to the catalytic efficiency per pmol of enzyme. This work demonstrates that CK1alpha and its splice variants can be regulated by their autophosphorylation status.

Bacteriophage T7 protein kinase: Site of inhibitory autophosphorylation, and use of dephosphorylated enzyme for efficient modification of protein in vitro

“…Since a previous study indicated that phosphorylation reduces T7PK catalytic activity [7], a method was developed to dephosphorylate H-pT7PK, as well as remove the His-tag, which also could affect activity. H-pT7PK was treated with lambda phage protein phosphatase [20], which has a broad specificity for protein phosphomonoester groups [21,22], then subjected to Ni 2+ -NTA affinity chromatography, providing H-T7PK in a yield of ~10%. Removal of phosphate causes a slight increase in mobility of H-pT7PK upon phosphatase treatment (Fig.…”

The CK1 Family: Contribution to Cellular Stress Response and Its Role in Carcinogenesis

“…For CK1ε amino acid residues Ser-323, Thr-325, Thr-334, Thr-337, Ser-368, Ser-405, Thr-407, and Ser-408 within the C-terminal domain are considered to be potential autophosphorylation sites (203). C-terminal inhibitory autophosphorylation could also be demonstrated for CK1γ1-3 as well as for CK1α and its splice variants CK1αL and CK1αS (16, 206). …”

Pharmacological Assessment Defines Leishmania donovani Casein Kinase 1 as a Drug Target and Reveals Important Functions in Parasite Viability and Intracellular Infection

“…In Leishmania, different protein kinases have already been identified as potential drug targets, such as mitogen-activated protein kinases (MAPKs) or cdc2-related kinase 3 (CRK3) (5)(6)(7). Members of another family of kinases have emerged as potential drug targets, casein kinase 1 (CK1), a family of multifunctional Ser/Thr protein kinases that are characterized by a highly conserved kinase domain and a specific C-terminal domain implicated in its regulation and localization (8). Although CK1 homologs in various protozoa share properties that are similar to those of higher eukaryotes, their functions have not yet been identified (9)(10)(11)(12)(13)(14).…”