Dataset Information

Differential regulation of PKD isoforms in oxidative stress conditions through phosphorylation of a conserved Tyr in the P+1 loop.

ABSTRACT: Protein kinases are essential molecules in life and their crucial function requires tight regulation. Many kinases are regulated via phosphorylation within their activation loop. This loop is embedded in the activation segment, which additionally contains the Mg²⁺ binding loop and a P?+?1 loop that is important in substrate binding. In this report, we identify Abl-mediated phosphorylation of a highly conserved Tyr residue in the P?+?1 loop of protein kinase D2 (PKD2) during oxidative stress. Remarkably, we observed that the three human PKD isoforms display very different degrees of P?+?1 loop Tyr phosphorylation and we identify one of the molecular determinants for this divergence. This is paralleled by a different activation mechanism of PKD1 and PKD2 during oxidative stress. Tyr phosphorylation in the P?+?1 loop of PKD2 increases turnover for Syntide-2, while substrate specificity and the role of PKD2 in NF-?B signaling remain unaffected. Importantly, Tyr to Phe substitution renders the kinase inactive, jeopardizing its use as a non-phosphorylatable mutant. Since large-scale proteomics studies identified P?+?1 loop Tyr phosphorylation in more than 70 Ser/Thr kinases in multiple conditions, our results do not only demonstrate differential regulation/function of PKD isoforms under oxidative stress, but also have implications for kinase regulation in general.

SUBMITTER: Cobbaut M

PROVIDER: S-EPMC5430542 | biostudies-literature | 2017 Apr

REPOSITORIES: biostudies-literature

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Differential regulation of PKD isoforms in oxidative stress conditions through phosphorylation of a conserved Tyr in the P+1 loop.

Cobbaut Mathias M Derua Rita R Döppler Heike H Lou Hua Jane HJ Vandoninck Sandy S Storz Peter P Turk Benjamin E BE Seufferlein Thomas T Waelkens Etienne E Janssens Veerle V Van Lint Johan J

Scientific reports 20170420 1

Protein kinases are essential molecules in life and their crucial function requires tight regulation. Many kinases are regulated via phosphorylation within their activation loop. This loop is embedded in the activation segment, which additionally contains the Mg<sup>2+</sup> binding loop and a P + 1 loop that is important in substrate binding. In this report, we identify Abl-mediated phosphorylation of a highly conserved Tyr residue in the P + 1 loop of protein kinase D2 (PKD2) during oxidative ...[more]

PMID: 28428613

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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

Differential regulation of PKD isoforms in oxidative stress conditions through phosphorylation of a conserved Tyr in the P+1 loop.

Publications

Differential regulation of PKD isoforms in oxidative stress conditions through phosphorylation of a conserved Tyr in the P+1 loop.

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