Reorganization of cytoskeletal proteins by Escherichia coli heat-stable enterotoxin (STa)-mediated signaling cascade.

BACKGROUND

IP(3)-mediated calcium mobilization from intracellular stores activates and translocates PKC-alpha from cytosol to membrane fraction in response to STa in COLO-205 cell line. The present study was undertaken to determine the involvement of cytoskeleton proteins in translocation of PKC-alpha to membrane from cytosol in the Escherichiacoli STa-mediated signaling cascade in a human colonic carcinoma cell line COLO-205.

METHODS

Western blots and consequent densitometric analysis were used to assess time-dependent redistribution of cytoskeletal proteins. This redistribution was further confirmed by using confocal microscopy. Pharmacological reagents were applied to colonic carcinoma cells to disrupt the microfilaments (cytochalasin D) and microtubules (nocodazole).

RESULTS

STa treatment in COLO-205 cells showed dynamic redistribution and an increase in actin content in the Triton-insoluble fraction, which corresponds to an increase in polymerization within 1min. Moreover, pharmacological disruption of actin-based cytoskeleton greatly disturbed PKC-alpha translocation to the membrane.

CONCLUSIONS

These results suggested that the organization of actin cytoskeleton is rapidly rearranged following E. coli STa treatment and the integrity of the actin cytoskeleton played a crucial role in PKC-alpha movement in colonic cells. Depolymerization of tubulin had no effect on the ability of the kinase to be translocated to the membrane.

GENERAL SIGNIFICANCE: In the present study, we have shown for the first time that in colonic carcinoma cells, STa-mediated rapid changes of actin cytoskeleton arrangement might be involved in the translocation of PKC-alpha to membrane.