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Mutations that disrupt PHOXB interaction with the neuronal calcium sensor HPCAL1 impede cellular differentiation in

W Wang1, Q Zhong2, L Teng3

  • 1Department of Pediatric Oncology, Dana-Farber Cancer Institute, Harvard Medical School, Boston, MA, USA.

Oncogene
|July 23, 2013
PubMed

Insights

PHOX2B mutations linked to neuroblastoma impair its interaction with HPCAL1, hindering sympathetic neuron differentiation. This protein-protein interaction defect may predispose to neuroblastoma by blocking normal neuronal development.

Area of Science:

  • Genetics and Molecular Biology
  • Developmental Biology
  • Oncology

Background:

  • Heterozygous germline mutations in PHOX2B are associated with sympathetic nervous system disorders, including neuroblastoma and congenital central hypoventilation syndrome (CCHS).
  • PHOX2B variants in CCHS typically involve polyalanine expansions, while neuroblastoma-associated mutations are often frameshift or truncation mutations.
  • The distinct mutation types suggest different pathomechanisms, potentially involving altered protein-protein interactions.

Purpose of the Study:

  • To investigate whether neuroblastoma-associated PHOX2B variants exert their effects through altered protein-protein interactions.
  • To identify proteins that interact with wild-type (WT) and mutant PHOX2B.
  • To elucidate the functional consequences of PHOX2B-protein interactions on sympathetic neuronal differentiation.

Main Methods:

  • A large-scale yeast two-hybrid screen was conducted using WT and six mutant PHOX2B proteins against over 10,000 human genes.
  • Interactions were validated, and the subcellular localization of HPCAL1 was assessed in the presence of different PHOX2B variants.
  • The impact of PHOX2B-HPCAL1 interaction on DBH promoter transactivation and neurite outgrowth in neuroblastoma cells was evaluated.

Main Results:

  • The neuronal calcium sensor protein HPCAL1 strongly bound to WT PHOX2B and a CCHS-associated mutant, but weakly or not at all to neuroblastoma-associated variants.
  • WT PHOX2B and some mutants induced nuclear translocation of HPCAL1, while neuroblastoma-associated mutants caused cytoplasmic retention.
  • Knockdown of HPCAL1 in neuroblastoma cells expressing PHOX2B impaired neurite outgrowth and inhibited sympathetic neuronal differentiation.

Conclusions:

  • Neuroblastoma-associated PHOX2B variants may predispose to malignancy by failing to bind HPCAL1, thereby impeding sympathetic neuron differentiation.
  • The PHOX2B-HPCAL1 interaction is crucial for normal sympathetic neuronal development.
  • Altered protein-protein interactions of PHOX2B represent a key mechanism in neuroblastoma pathogenesis.

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