Myristoylated alanine-rich C kinase substrate-mediated neurotensin release via protein kinase C-delta downstream of the Rho/ROK pathway.
Myristoylated alanine-rich protein kinase C substrate (MARCKS) is a cellular substrate for protein kinase C (PKC). Recently, we have shown that PKC isoforms-alpha and -delta, 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-delta siRNA, ROKalpha siRNA, and C3 toxin (a Rho protein inhibitor), suggesting that the PKC-delta and the Rho/ROK pathways are necessary for MARCKS activation. The phosphorylation of PKC-delta was inhibited by C3 toxin, demonstrating that the role of MARCKS in NT secretion was regulated by PKC-delta 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-delta proteins, in stimulated gut peptide secretion.
Li
Jing
J
Department of Surgery and Sealy Center for Cancer Cell Biology, The University of Texas Medical Branch, Galveston, Texas 77555, USA.
O'Connor
Kathleen L
KL
Greeley
George H
GH
Blackshear
Perry J
PJ
Townsend
Courtney M
CM
Evers
B Mark
BM
D015153:Blotting, Western; D001839:Bombesin / Q000494:pharmacology; D001905:Botulinum Toxins / Q000737:chemistry; D002460:Cell Line; D045744:Cell Line, Tumor; D002462:Cell Membrane / Q000378:metabolism; D003600:Cytosol / Q000378:metabolism; D019886:Gastrin-Releasing Peptide / Q000737:chemistry; D005786:Gene Expression Regulation; D020868:Gene Silencing; D049452:Green Fluorescent Proteins / Q000378:metabolism; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins / Q000378:metabolism / Q000502:physiology*; D008565:Membrane Proteins / Q000378:metabolism / Q000502:physiology*; D018613:Microscopy, Confocal; D008856:Microscopy, Fluorescence; D009227:Myristic Acids / Q000737:chemistry*; D000076250:Myristoylated Alanine-Rich C Kinase Substrate; D009496:Neurotensin / Q000378:metabolism*; D010455:Peptides / Q000737:chemistry; D010766:Phosphorylation; D020033:Protein Isoforms; D011493:Protein Kinase C / Q000378:metabolism*; D051745:Protein Kinase C-delta; D021381:Protein Transport; D034622:RNA Interference; D034741:RNA, Small Interfering / Q000378:metabolism; D011863:Radioimmunoassay; D015398:Signal Transduction; D013755:Tetradecanoylphorbol Acetate / Q000737:chemistry; D013997:Time Factors; D014162:Transfection; D020741:rho GTP-Binding Proteins / Q000378:metabolism*
D047908:Intracellular Signaling Peptides and Proteins; C000619710:MARCKS protein, human; D008565:Membrane Proteins; D009227:Myristic Acids; D010455:Peptides; D020033:Protein Isoforms; D034741:RNA, Small Interfering; D000076250:Myristoylated Alanine-Rich C Kinase Substrate; D049452:Green Fluorescent Proteins; D009496:Neurotensin; D019886:Gastrin-Releasing Peptide; C497258:PRKCD protein, human; D011493:Protein Kinase C; D051745:Protein Kinase C-delta; D001905:Botulinum Toxins; D020741:rho GTP-Binding Proteins; C096324:botulinum toxin type C; D013755:Tetradecanoylphorbol Acetate; D001839:Bombesin