YAP tyrosine phosphorylation and nuclear localization in cholangiocarcinoma cells are regulated by LCK and independent of LATS activity

Takaaki Sugihara, Nathan W. Werneburg, Matthew C. Hernandez, Lin Yang, Ayano Kabashima, Petra Hirsova, Lavanya Yohanathan, Carlos Sosa, Mark Truty, George Vasmatzis, Gregory James Gores, Rory Smoot

Research output: Contribution to journalArticle

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Abstract

The Hippo pathway effector, Yes-associated protein (YAP), is a transcriptional coactivator implicated in cholangiocarcinoma (CCA) pathogenesis. YAP is known to be regulated by a serine/threonine kinase relay module (MST1/2-LATS1/2) culminating in phosphorylation of YAP at Serine 127 and cytoplasmic sequestration. However, YAP also undergoes tyrosine phosphorylation, and the role of tyrosine phosphorylation in YAP regulation remains unclear. Herein, YAP regulation by tyrosine phosphorylation was examined in human and mouse CCA cells, as well as patient-derived xenograft (PDX) models. YAP was phosphorylated on tyrosine 357 (Y357) in CCA cell lines and PDX models. SRC family kinase (SFK) inhibition with dasatinib resulted in loss of YAPY357 phosphorylation, promoted its translocation from the nucleus to the cytoplasm, and reduced YAP target gene expression, including cell lines expressing a LATS1/2-resistant YAP mutant in which all serine residues were mutated to alanine. Consistent with these observations, precluding YAPY357 phosphorylation by site-directed mutagenesis (YAPY357F) excluded YAP from the nucleus. Targeted siRNA experiments identified LCK as the SFK that most potently mediated YAPY357 phosphorylation. Likewise, inducible CRISPR/ Cas9-targeted LCK deletion decreased YAPY357 phosphorylation and its nuclear localization. The importance of LCK in CCA biology was demonstrated by clinical observations suggesting LCK expression levels were associated with early tumor recurrence following resection of CCA. Finally, dasatinib displayed therapeutic efficacy in PDX models.

Original languageEnglish (US)
Pages (from-to)1556-1567
Number of pages12
JournalMolecular Cancer Research
Volume16
Issue number10
DOIs
StatePublished - Oct 1 2018

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Long-Acting Thyroid Stimulator
Cholangiocarcinoma
Tyrosine
Phosphorylation
Proteins
Heterografts
Serine
Phosphotransferases
Clustered Regularly Interspaced Short Palindromic Repeats
Cell Line
Protein-Serine-Threonine Kinases
Mutant Proteins
Site-Directed Mutagenesis
Alanine
Small Interfering RNA
Cytoplasm

ASJC Scopus subject areas

  • Molecular Biology
  • Oncology
  • Cancer Research

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YAP tyrosine phosphorylation and nuclear localization in cholangiocarcinoma cells are regulated by LCK and independent of LATS activity. / Sugihara, Takaaki; Werneburg, Nathan W.; Hernandez, Matthew C.; Yang, Lin; Kabashima, Ayano; Hirsova, Petra; Yohanathan, Lavanya; Sosa, Carlos; Truty, Mark; Vasmatzis, George; Gores, Gregory James; Smoot, Rory.

In: Molecular Cancer Research, Vol. 16, No. 10, 01.10.2018, p. 1556-1567.

Research output: Contribution to journalArticle

Sugihara, Takaaki ; Werneburg, Nathan W. ; Hernandez, Matthew C. ; Yang, Lin ; Kabashima, Ayano ; Hirsova, Petra ; Yohanathan, Lavanya ; Sosa, Carlos ; Truty, Mark ; Vasmatzis, George ; Gores, Gregory James ; Smoot, Rory. / YAP tyrosine phosphorylation and nuclear localization in cholangiocarcinoma cells are regulated by LCK and independent of LATS activity. In: Molecular Cancer Research. 2018 ; Vol. 16, No. 10. pp. 1556-1567.
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abstract = "The Hippo pathway effector, Yes-associated protein (YAP), is a transcriptional coactivator implicated in cholangiocarcinoma (CCA) pathogenesis. YAP is known to be regulated by a serine/threonine kinase relay module (MST1/2-LATS1/2) culminating in phosphorylation of YAP at Serine 127 and cytoplasmic sequestration. However, YAP also undergoes tyrosine phosphorylation, and the role of tyrosine phosphorylation in YAP regulation remains unclear. Herein, YAP regulation by tyrosine phosphorylation was examined in human and mouse CCA cells, as well as patient-derived xenograft (PDX) models. YAP was phosphorylated on tyrosine 357 (Y357) in CCA cell lines and PDX models. SRC family kinase (SFK) inhibition with dasatinib resulted in loss of YAPY357 phosphorylation, promoted its translocation from the nucleus to the cytoplasm, and reduced YAP target gene expression, including cell lines expressing a LATS1/2-resistant YAP mutant in which all serine residues were mutated to alanine. Consistent with these observations, precluding YAPY357 phosphorylation by site-directed mutagenesis (YAPY357F) excluded YAP from the nucleus. Targeted siRNA experiments identified LCK as the SFK that most potently mediated YAPY357 phosphorylation. Likewise, inducible CRISPR/ Cas9-targeted LCK deletion decreased YAPY357 phosphorylation and its nuclear localization. The importance of LCK in CCA biology was demonstrated by clinical observations suggesting LCK expression levels were associated with early tumor recurrence following resection of CCA. Finally, dasatinib displayed therapeutic efficacy in PDX models.",
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AU - Kabashima, Ayano

AU - Hirsova, Petra

AU - Yohanathan, Lavanya

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AU - Gores, Gregory James

AU - Smoot, Rory

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