Heterozygous germline mutations in PHOX2B, a transcriptional regulator of sympathetic neuronal differentiation, predispose to diseases of the sympathetic nervous system, including
neuroblastoma and
congenital central hypoventilation syndrome (CCHS). Although the PHOX2B variants in CCHS largely involve expansions of the second
polyalanine repeat within the C-terminus of the
protein, those associated with neuroblastic
tumors are nearly always frameshift and truncation mutations. To test the hypothesis that the
neuroblastoma-associated variants exert their effects through loss or gain of
protein-
protein interactions, we performed a large-scale yeast two-hybrid screen using both wild-type (WT) and six different mutant PHOX2B
proteins against over 10 000 human genes. The neuronal
calcium sensor
protein HPCAL1 (VILIP-3) exhibited strong binding to WT PHOX2B and a CCHS-associated
polyalanine expansion mutant but only weakly or not at all to
neuroblastoma-associated frameshift and truncation variants. We demonstrate that both WT PHOX2B and the
neuroblastoma-associated R100L missense and the CCHS-associated
alanine expansion variants induce nuclear translocation of HPCAL1 in a Ca(2+)-independent manner, while the
neuroblastoma-associated 676delG frameshift and K155X truncation mutants impair subcellular localization of HPCAL1, causing it to remain in the cytoplasm. HPCAL1 did not appreciably influence the ability of WT PHOX2B to transactivate the DBH promoter, nor did it alter the decreased transactivation potential of PHOX2B variants in 293T cells. Abrogation of the PHOX2B-HPCAL1 interaction by
shRNA knockdown of HPCAL1 in
neuroblastoma cells expressing PHOX2B led to impaired neurite outgrowth with transcriptional profiles indicative of inhibited sympathetic neuronal differentiation. Our results suggest that certain PHOX2B variants associated with
neuroblastoma pathogenesis, because of their inability to bind to key interacting
proteins such as HPCAL1, may predispose to this
malignancy by impeding the differentiation of immature sympathetic neurons.