Myristoylated alanine-rich C kinase substrate-mediated neurotensin release via protein kinase C-δ downstream of the Rho/ROK pathway

Jing Li, Kathleen L. O'Connort, George H. Greeley, Perry J. Blackshear, Courtney Townsend, B. Mark Evers

Research output: Contribution to journalArticle

30 Citations (Scopus)

Abstract

Myristoylated alanine-rich protein kinase C substrate (MARCKS) is a cellular substrate for protein kinase C (PKC). Recently, we have shown that PKC isoforms-α and -δ, as well as the Rho/Rho kinase (ROK) pathway, play a role in phorbol 12-myristate 13-acetate (PMA)-mediated secretion of the gut peptide neurotensin (NT) in the BON human endocrine cell line. Here, we demonstrate that activation of MARCKS protein is important for PMA- and bombesin (BBS)-mediated NT secretion in BON cells. Small interfering RNA (siRNA) to MARCKS significantly inhibited, whereas overexpression of wild-type MARCKS significantly increased PMA-mediated NT secretion. Endogenous MARCKS and green fluorescent protein-tagged wild-type MARCKS were translocated from membrane to cytosol upon PMA treatment, further confirming MARCKS activation. MARCKS phosphorylation was inhibited by PKC-δ siRNA, ROKα siRNA, and C3 toxin (a Rho protein inhibitor), suggesting that the PKC-δ and the Rho/ROK pathways are necessary for MARCKS activation. The phosphorylation of PKC-δ was inhibited by C3 toxin, demonstrating that the role of MARCKS in NT secretion was regulated by PKC-δ downstream of the Rho/ROK pathway. BON cell clones stably transfected with the receptor for gastrin releasing peptide, a physiologic stimulant of NT, and treated with BBS, the amphibian equivalent of gastrin releasing peptide, demonstrated a similar MARCKS phosphorylation as noted with PMA. BBS-mediated NT secretion was attenuated by MARCKS siRNA. Collectively, these findings provide evidence for novel signaling pathways, including the sequential regulation of MARCKS activity by Rho/ROK and PKC-δ proteins, in stimulated gut peptide secretion.

Original languageEnglish (US)
Pages (from-to)8351-8357
Number of pages7
JournalJournal of Biological Chemistry
Volume280
Issue number9
DOIs
StatePublished - Mar 4 2005

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rho-Associated Kinases
Neurotensin
Protein Kinase C
Alanine
Substrates
Acetates
Bombesin
Small Interfering RNA
Phosphorylation
myristoylated alanine-rich C kinase substrate
Chemical activation
Clone cells
Bombesin Receptors
Gastrin-Releasing Peptide

ASJC Scopus subject areas

  • Biochemistry

Cite this

Myristoylated alanine-rich C kinase substrate-mediated neurotensin release via protein kinase C-δ downstream of the Rho/ROK pathway. / Li, Jing; O'Connort, Kathleen L.; Greeley, George H.; Blackshear, Perry J.; Townsend, Courtney; Evers, B. Mark.

In: Journal of Biological Chemistry, Vol. 280, No. 9, 04.03.2005, p. 8351-8357.

Research output: Contribution to journalArticle

Li, Jing ; O'Connort, Kathleen L. ; Greeley, George H. ; Blackshear, Perry J. ; Townsend, Courtney ; Evers, B. Mark. / Myristoylated alanine-rich C kinase substrate-mediated neurotensin release via protein kinase C-δ downstream of the Rho/ROK pathway. In: Journal of Biological Chemistry. 2005 ; Vol. 280, No. 9. pp. 8351-8357.
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abstract = "Myristoylated alanine-rich protein kinase C substrate (MARCKS) is a cellular substrate for protein kinase C (PKC). Recently, we have shown that PKC isoforms-α and -δ, as well as the Rho/Rho kinase (ROK) pathway, play a role in phorbol 12-myristate 13-acetate (PMA)-mediated secretion of the gut peptide neurotensin (NT) in the BON human endocrine cell line. Here, we demonstrate that activation of MARCKS protein is important for PMA- and bombesin (BBS)-mediated NT secretion in BON cells. Small interfering RNA (siRNA) to MARCKS significantly inhibited, whereas overexpression of wild-type MARCKS significantly increased PMA-mediated NT secretion. Endogenous MARCKS and green fluorescent protein-tagged wild-type MARCKS were translocated from membrane to cytosol upon PMA treatment, further confirming MARCKS activation. MARCKS phosphorylation was inhibited by PKC-δ siRNA, ROKα siRNA, and C3 toxin (a Rho protein inhibitor), suggesting that the PKC-δ and the Rho/ROK pathways are necessary for MARCKS activation. The phosphorylation of PKC-δ was inhibited by C3 toxin, demonstrating that the role of MARCKS in NT secretion was regulated by PKC-δ downstream of the Rho/ROK pathway. BON cell clones stably transfected with the receptor for gastrin releasing peptide, a physiologic stimulant of NT, and treated with BBS, the amphibian equivalent of gastrin releasing peptide, demonstrated a similar MARCKS phosphorylation as noted with PMA. BBS-mediated NT secretion was attenuated by MARCKS siRNA. Collectively, these findings provide evidence for novel signaling pathways, including the sequential regulation of MARCKS activity by Rho/ROK and PKC-δ proteins, in stimulated gut peptide secretion.",
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AU - Townsend, Courtney

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AB - Myristoylated alanine-rich protein kinase C substrate (MARCKS) is a cellular substrate for protein kinase C (PKC). Recently, we have shown that PKC isoforms-α and -δ, as well as the Rho/Rho kinase (ROK) pathway, play a role in phorbol 12-myristate 13-acetate (PMA)-mediated secretion of the gut peptide neurotensin (NT) in the BON human endocrine cell line. Here, we demonstrate that activation of MARCKS protein is important for PMA- and bombesin (BBS)-mediated NT secretion in BON cells. Small interfering RNA (siRNA) to MARCKS significantly inhibited, whereas overexpression of wild-type MARCKS significantly increased PMA-mediated NT secretion. Endogenous MARCKS and green fluorescent protein-tagged wild-type MARCKS were translocated from membrane to cytosol upon PMA treatment, further confirming MARCKS activation. MARCKS phosphorylation was inhibited by PKC-δ siRNA, ROKα siRNA, and C3 toxin (a Rho protein inhibitor), suggesting that the PKC-δ and the Rho/ROK pathways are necessary for MARCKS activation. The phosphorylation of PKC-δ was inhibited by C3 toxin, demonstrating that the role of MARCKS in NT secretion was regulated by PKC-δ downstream of the Rho/ROK pathway. BON cell clones stably transfected with the receptor for gastrin releasing peptide, a physiologic stimulant of NT, and treated with BBS, the amphibian equivalent of gastrin releasing peptide, demonstrated a similar MARCKS phosphorylation as noted with PMA. BBS-mediated NT secretion was attenuated by MARCKS siRNA. Collectively, these findings provide evidence for novel signaling pathways, including the sequential regulation of MARCKS activity by Rho/ROK and PKC-δ proteins, in stimulated gut peptide secretion.

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