Dataset Information

Fuzzy interactions between the auto-phosphorylated C-terminus and the kinase domain of CK1δ inhibits activation of TAp63α

ABSTRACT: The p53 family member TAp63α plays an important role in maintaining the genetic integrity in oocytes. DNA damage, in particular DNA double strand breaks, lead to the transformation of the inhibited, only dimeric conformation into the active tetrameric one that results in the initiation of an apoptotic program. Activation requires phosphorylation by the kinase CK1 which phosphorylates TAp63α at four positions. The third phosphorylation event is the decisive step that transforms TAp63α into the active state. This third phosphorylation, however, is ~20 times slower than the first two phosphorylation events. This difference in the phosphorylation kinetics constitutes a safety mechanism that allows oocytes with a low degree of DNA damage to survive. So far these kinetic investigations of the phosphorylation steps have been performed with the isolated CK1 kinase domain. However, all CK1 enzymes contain C-terminal extensions that become auto-phosphorylated and inhibit the activity of the kinase. Here we have investigated the effect of auto-phosphorylation of the C-terminus in the kinase CK1δ and show that it slows down phosphorylation of the first two sites in TAp63α but basically inhibits the phosphorylation of the third site. We have identified up to ten auto-phosphorylation sites in the CK1δ C-terminal domain and show that all of them interact with the kinase domain in a “fuzzy” way in which not a single site is particularly important. Through mutation analysis we further show that hydrophobic amino acids following the phosphorylation site are important for a substrate to be able to successfully compete with the auto-inhibitory effect of the C-terminal domain. This auto-phosphorylation adds a new layer to the regulation of apoptosis in oocytes.

INSTRUMENT(S): Q Exactive HF

ORGANISM(S): Homo Sapiens (human)

SUBMITTER: Christian Münch

LAB HEAD: Christian Münch

PROVIDER: PXD043233 | Pride | 2023-10-06

REPOSITORIES: Pride

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Publications

Fuzzy interactions between the auto-phosphorylated C-terminus and the kinase domain of CK1δ inhibits activation of TAp63α.

Lambert Mahil M Gebel Jakob J Trejtnar Charlotte C Wesch Nicole N Bozkurt Süleyman S Adrian-Allgood Martin M Löhr Frank F Münch Christian C Dötsch Volker V

Scientific reports 20230930 1

The p53 family member TAp63α plays an important role in maintaining the genetic integrity in oocytes. DNA damage, in particular DNA double strand breaks, lead to the transformation of the inhibited, only dimeric conformation into the active tetrameric one that results in the initiation of an apoptotic program. Activation requires phosphorylation by the kinase CK1 which phosphorylates TAp63α at four positions. The third phosphorylation event is the decisive step that transforms TAp63α into the ac ...[more]

PMID: 37777570

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Project description:Reactivation of androgen receptor (AR) may drive recurrent prostate cancer in castrate patients. Ack1 tyrosine kinase is overexpressed in prostate cancer and promotes castrate resistant xenograft tumor growth and enhances androgen target gene expression and AR recruitment to enhancers. Ack1 phosphorylates AR at Tyr-267 in the N-terminal transactivation domain. In this study, the role of this phosphorylation site was investigated by characterizing the phosphorylation site mutant in the context of full length and truncated AR lacking the ligand-binding domain. The Y267F mutant showed decreased transactivation of reporters. Expression of wild type full length and truncated AR in LNCaP cells increased cell proliferation in androgen-depleted conditions and increased colony formation. However, the Y267F mutant of full length and truncated AR was defective in stimulating cell proliferation. The full length AR Y267F mutant was defective in nuclear translocation induced by androgen or Ack1 kinase. The truncated AR was constitutively localized to the nucleus. Chromatin immunoprecipitation analysis showed that it was recruited to the target enhancers without androgen. The truncated Y267F AR mutant did not exhibit constitutive nuclear localization and androgen enhancer binding activity. Expression of AR responsive genes in cells expressing truncated AR wt and AR-Y267F mutant under androgen deprived conditions was assessed by microarray gene expression analysis. The AR pathway score was increased in cells expressing truncated AR wt and decreased in cells expressing truncated AR-Y267F. These results support the concept that phosphorylation of Tyr-267 is required for AR nuclear translocation and recruitment and DNA binding and transcription of AR responsive genes. Gene expression profiling was performed using RNA samples from LNCaP cells expressing truncated AR-wt and truncated AR-Y267F growing in androgen deprived conditions. The RNA sample from vector control cells were used as reference sample. Two biological replicates were used per each cell line.

Dataset Information

Fuzzy interactions between the auto-phosphorylated C-terminus and the kinase domain of CK1δ inhibits activation of TAp63α

Publications

Fuzzy interactions between the auto-phosphorylated C-terminus and the kinase domain of CK1δ inhibits activation of TAp63α.

Similar Datasets

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