PKD1, PKD2, and Their Substrate Kidins220 Regulate Neurotensin Secretion in the BON Human Endocrine Cell Line

Jing Li,L Andy Chen,Courtney M Townsend,B Mark Evers

doi:10.1074/jbc.m707513200

Jing Li, L Andy Chen + Show 2 more

Open Access

https://doi.org/10.1074/jbc.m707513200

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Abstract

Neurotensin (NT) is a gut peptide that plays an important role in gastrointestinal secretion, motility, and growth as well as the proliferation of NT receptor-positive cancers. Protein kinase D (PKD) family members (PKD1, 2, and 3) have been identified as important regulators of secretory transport at the trans-Golgi network. Previously, we showed that PKD1 contributes to stimulated NT secretion; however, the mechanisms are not entirely clear. Here, we show that Kidins220, which is a substrate of PKD proteins in neuroendocrine cells, is localized in the ends of the processes of BON cells, similar to the expression pattern of NT vesicles, and translocates to the membrane and large vesicle-like structures formed in response to phorbol 12-myristate 13-acetate treatment. The short hairpin RNA targeting Kidins220 inhibits NT secretion in parental BON cells or BON cells stably expressing the gastrin-releasing peptide receptor treated with either phorbol 12-myristate 13-acetate or bombesin, respectively. Furthermore, we demonstrate that endogenous PKD1, PKD2, and Kidins220 co-exist with NT-containing vesicles. Overexpression of the kinase-dead PKD1 abrogates Kidins220 expression and NT vesicle formation. Our data establish a physiological link between the PKD/Kidins220 pathway and NT-containing vesicles and suggest the role of this pathway in the regulation of hormone secretion. Because NT is an important gut hormone that affects secretion, inflammation, and both normal and tumor cell growth, our findings identify a novel signaling pathway that may be amenable to drug targeting for clinical applications.

Highlights

Localized predominantly in the jejunum and ileum
NT Secretion in BON Cells—We have previously demonstrated a role for PKD1 in stimulated NT secretion [10]
Using realtime RT-PCR, we found that PKD1 and PKD2 are abundantly expressed; PKD3 mRNA was detectable but at very low levels in BON cells (Fig. 1A)

Summary

Introduction

Localized predominantly in the jejunum and ileum. The gastrointestinal effects of NT include facilitation of fatty acid translocation from the intestinal lumen, regulation of gut motility, and stimulation of the growth of normal gut mucosa as well as other tissues such as the adrenal gland, hepatocytes, and fibroblasts [2, 3]. We demonstrate that PKD2 and the PKD protein substrate Kidins220 regulate stimulated NT secretion. Discrete significantly decreased in BON/GFP-GRPR cells overexpress- membrane-localized PKD1 (left panel, bottom) and PKD2 ing the kinase-dead PKD1 and PKD2 (Fig. 1D).

Results

Conclusion