Involvement of protein kinase C in the mechanism of action of Escherichia coli heat-stable enterotoxin (STa) in a human colonic carcinoma cell line, COLO-205

The characteristics of the transport of the dipeptide glycylsarcosine were studied in the human colon carcinoma cell line Caco-2 grown as a monolayer on impermeable plastic support. Transport of glycylsarcosine in these cells was found to be Na(+)-independent, but was stimulated by an inwardly directed H+ gradient. This H(+)-dependent transport of glycylsarcosine was inhibited by di- and tri-peptides and also by the beta-lactam antibiotic cephalexin, but was unaffected by the amino acids glycine and leucine. The transport system exhibited a Michaelis-Menten constant (Kt) of 1.1 +/- 0.1 mM for glycylsarcosine. The specific activity of the transport system in this cell line was found to be maximal when the cultures were confluent. Treatment of the cells with phorbol esters which activate protein kinase C resulted in a significant inhibition of the transport system. This inhibition was specific and could be blocked if treatment was done in the presence of staurosporine, an inhibitor of protein kinase C. Kinetic analysis revealed that the inhibition was associated with a decrease in the maximal velocity, the Kt remaining unaffected. The phorbol-ester-induced inhibition of the peptide-transport system was not prevented by co-treatment with cycloheximide, an inhibitor of cellular protein synthesis. In addition, there was no change in the intracellular pH following treatment with the phorbol ester, suggesting that the effect was not due to alterations in the transmembrane pH gradient. It is concluded that the peptide/H+ co-transport system, which is known to exist in the normal intestine, is expressed in Caco-2 cells and that the function of the transport system is under the regulatory control of protein kinase C.

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Interleukin-3, GM-CSF, and TPA induce distinct phosphorylation events in an interleukin 3-dependent multipotential cell line

Blood ◽

10.1182/blood.v73.2.406.bloodjournal732406 ◽

1989 ◽

Vol 73 (2) ◽

pp. 406-418 ◽

Cited By ~ 32

Author(s):

PH Sorensen ◽

AL Mui ◽

SC Murthy ◽

G Krystal

Keyword(s):

Protein Kinase C ◽

Protein Kinase ◽

Membrane Protein ◽

Cell Line ◽

Mechanism Of Action ◽

Interleukin 3 ◽

Gm Csf ◽

Kinase C ◽

Macrophage Colony ◽

Hemopoietic Growth Factor

The mechanism of action of the hemopoietic growth factor, murine interleukin-3 (mIL-3), was investigated using an mIL-3-dependent multipotential hematopoietic cell line, B6SUtA1. Murine granulocyte- macrophage colony-stimulating factor (mGM-CSF) was as potent as mIL-3 in stimulating these cells. In addition, sodium orthovanadate, an inhibitor of phosphotyrosine phosphatase, and 12-O-tetradecanoyl- phorbol-13-acetate (TPA), a known activator of protein kinase C, also stimulated DNA synthesis in these cells, suggesting that protein phosphorylation might be involved in the mechanism of action of mIL-3 and mGM-CSF. To assess this possibility, intact B6SUtA1 cells exposed for brief periods to mIL-3, mGM-CSF, and TPA were analyzed for changes in phosphorylation patterns using metabolic 32P-labeling and antibodies to phosphotyrosine. Both mIL-3 and mGM-CSF induced the serine-specific phosphorylation of a 68-Kd cytosolic protein, whereas all three agents stimulated the serine-specific phosphorylation of a 68-Kd membrane protein. Furthermore, mIL-3 stimulated tyrosine phosphorylation of the 68-Kd membrane protein, as well as of 140-, 90-, 55, and 40-Kd proteins. The 90-Kd protein was also tyrosine phosphorylated in response to mGM-CSF. These phosphotyrosine containing proteins were not detected in TPA-treated cells. These results indicate that protein phosphorylations on tyrosine and serine residues occur in B6SUtA1 cells following short-term incubation with mIL-3 or mGM-CSF and that most of these phosphorylation events are mediated by kinases other than protein kinase C (PkC).

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