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System-wide investigation of ErbB4 reveals 19 sites of Tyr phosphorylation that are unusually selective in their recruitment properties.

ABSTRACT: The first three members of the ErbB family of receptor tyrosine kinases activate a wide variety of signaling pathways and are frequently misregulated in cancer. Much less is known about ErbB4. Here we use tandem mass spectrometry to identify 19 sites of tyrosine phosphorylation on ErbB4, and protein microarrays to quantify biophysical interactions between these sites and virtually every SH2 and PTB domain encoded in the human genome. Our unbiased approach highlighted several previously unrecognized interactions and led to the finding that ErbB4 can recruit and activate STAT1. At a systems level, we found that ErbB4 is much more selective than the other ErbB receptors. This suggests that ErbB4 may enable ErbB2 and ErbB3 to signal independently of EGFR under normal conditions, and provides a possible explanation for the protective properties of ErbB4 in cancer.

SUBMITTER: Kaushansky A

PROVIDER: S-EPMC2606095 | biostudies-literature | 2008 Aug

REPOSITORIES: biostudies-literature

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System-wide investigation of ErbB4 reveals 19 sites of Tyr phosphorylation that are unusually selective in their recruitment properties.

Kaushansky Alexis A Gordus Andrew A Budnik Bogdan A BA Lane William S WS Rush John J MacBeath Gavin G

Chemistry & biology 20080801 8

The first three members of the ErbB family of receptor tyrosine kinases activate a wide variety of signaling pathways and are frequently misregulated in cancer. Much less is known about ErbB4. Here we use tandem mass spectrometry to identify 19 sites of tyrosine phosphorylation on ErbB4, and protein microarrays to quantify biophysical interactions between these sites and virtually every SH2 and PTB domain encoded in the human genome. Our unbiased approach highlighted several previously unrecogni ...[more]

PMID: 18721752

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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.

Project description:ObjectiveAlternative splicing of ErbB4 transcripts is dysregulated in the dorsolateral prefrontal cortex in schizophrenia. ErbB4 regulates the activity of parvalbumin interneurons, and therefore dysregulated ErbB4 splicing could contribute to lower parvalbumin interneuron activity and consequently lower parvalbumin levels in schizophrenia. However, ErbB4 is also present in calretinin interneurons, which are not affected in schizophrenia. Therefore, the authors hypothesized that dysregulated ErbB4 splicing occurs selectively in parvalbumin interneurons and is associated with lower parvalbumin levels in schizophrenia.MethodTissue samples enriched in calretinin and parvalbumin interneurons were laser microdissected from dorsolateral prefrontal cortex layers 2 and 4, respectively, from matched pairs of schizophrenia and comparison subjects. Transcript levels for pan-ErbB4, four ErbB4 splicing variants (JM-a, JM-b, CYT-1, CYT-2), parvalbumin, and calretinin were quantified by quantitative polymerase chain reaction (qPCR) in each layer. Transcript levels for myocardial infarction associated transcript (MIAT), which regulates ErbB4 splicing, were quantified in gray matter by qPCR and in parvalbumin interneurons by microarray.ResultsCalretinin and parvalbumin mRNAs were preferentially expressed in layers 2 and 4, respectively. In schizophrenia subjects, lower parvalbumin levels, higher CYT-1 and JM-a levels, and lower CYT-2 and JM-b levels were detected selectively in layer 4. In layer 4, the JM-a/JM-b ratio was inversely correlated with parvalbumin levels in schizophrenia subjects. MIAT levels were preferentially higher in parvalbumin interneurons in schizophrenia subjects.ConclusionsThese findings suggest that elevated MIAT expression alters ErbB4 splicing selectively in parvalbumin interneurons in schizophrenia. Dysregulated ErbB4 splicing in schizophrenia may contribute to lower activity of parvalbumin interneurons and an activity-dependent down-regulation of parvalbumin expression.

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System-wide investigation of ErbB4 reveals 19 sites of Tyr phosphorylation that are unusually selective in their recruitment properties.

Publications

System-wide investigation of ErbB4 reveals 19 sites of Tyr phosphorylation that are unusually selective in their recruitment properties.

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