Protein kinase C-mediated phosphorylation of RKIP regulates inhibition of
Na-alanine cotransport by leukotriene D(4) in intestinal epithelial cells
#MMPMID25231108
Arthur S
; Sundaram U
Am J Physiol Cell Physiol
2014[Dec]; 307
(11
): C1010-6
PMID25231108
show ga
Leukotriene D4 (LTD4) is an important immune inflammatory mediator that is known
to be elevated in the mucosa of chronically inflamed intestine and alter nutrient
absorption. LTD4 inhibits Na-alanine cotransport in intestinal epithelial cells
by decreasing the affinity of the cotransporter ASCT1. LTD4 is known to increase
intracellular Ca(++) and cAMP concentrations. However, the intracellular
signaling mechanism of LTD4-mediated ASCT1 inhibition is unknown. In the present
study, pretreatment with calcium chelator BAPTA-AM or inhibition of
Ca(++)-dependent protein kinase C (PKC), specifically PKC?, resulted in the
reversal of LTD4-mediated inhibition of ASCT1, revealing the involvement of the
Ca(++)-activated PKC pathway. PKC? is known to phosphorylate Raf kinase inhibitor
protein (RKIP), thus activating its downstream signaling pathway. Immunoblotting
with anti-RKIP-Ser(153) antibody showed an increase in phosphorylation levels of
RKIP in LTD4-treated cells. Downregulation of endogenous RKIP showed no decrease
in ASCT1 activity by LTD4, thus confirming its involvement in ASCT1 regulation.
Phosphorylation of RKIP by PKC is known to activate different signaling pathways,
and in this study it was found to activate cAMP-activated protein kinase A (PKA)
pathway. Although protein abundance of ASCT1 was not altered in any of the
experimental conditions, there was an increase in the levels of phosphothreonine
in ASCT1 protein, thus showing that phosphorylation changes were responsible for
the altered affinity of ASCT1 by LTD4. In conclusion, LTD4 inhibits ASCT1 through
PKC-mediated phosphorylation of RKIP, leading to the subsequent activation of PKA
pathway, possibly through ?2-andrenergic receptor activation.
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