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Phosphochromatome profiling of C-terminally truncated CDK6

ABSTRACT: Pre/pro-B-cell lines were generated from bone marrow cells of Cdk6-/- mice by retroviral overexpression of the BCR-ABL (p185) fusion oncogene. These cell lines were transduced with constructs expressing HA-tagged versions of wildtype Cdk6 or of a C-terminally truncated variant of Cdk6 (HA-Cdk6-ΔC). Given the role of CDK6 in transcriptional regulation, we studied the phosphorylation of proteins associated with the chromatin (the phosphochromatome) using phospho-proteomics in cell lines that expressed C-terminally truncated CDK6, wildtype CDK6, or that had a CDK6 knock-out.

INSTRUMENT(S): Orbitrap Fusion Lumos

ORGANISM(S): Mus Musculus (mouse)

TISSUE(S): B Cell, Cell Culture

DISEASE(S): Acute Leukemia

SUBMITTER: Thorsten Klampfl

LAB HEAD: Karoline Kollmann

PROVIDER: PXD059037 | Pride | 2024-12-19

REPOSITORIES: Pride

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			Action	DRS
	EC_VS_72-M595-D01-F04-PO61-P8653-1_2ml.mzML	Mzml
	EC_VS_72-M595-D01-F04-PO61-P8653-1_2ml.mzid.gz	Mzid
	EC_VS_72-M595-D01-PO61-P8653-1.raw	Raw
	EC_VS_72-M595-D01-PO61-P8653-2.raw	Raw
	EC_VS_72-M595-D02-PO61-P8653-1.raw	Raw

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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.

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Phosphochromatome profiling of C-terminally truncated CDK6

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