Differential subcellular targeting of PKC-epsilon in response to pharmacological or ischemic stimuli in intestinal epithelia.
Ischemia is the central pathogenic factor underlying a spectrum of intestinal disorders. The study of the cellular signaling responses to ischemic stress in nonepithelial cells has progressed substantially in the previous several years, but little is known about the response in epithelial cells. Unique features of the epithelial response to ischemic stress suggest differential regulation with regards to signaling. The PKC family of proteins has been implicated in ischemic stress in nonepithelial systems. The role of PKC isoforms in chemical ischemia in intestinal epithelial cells is evaluated in this study. Additionally, the phosphorylation of the F-actin cross-linking protein myristoylated alanine-rich C kinase substrate (MARCKS) is also studied. Chemical ischemia resulted in the transient activation of only the isoform PKC-epsilon as detected by translocation employing the subcellular fractionation technique. The pharmacological agonists phorbol 12-myristate 13-acetate and carbachol also led to the translocation of PKC-epsilon. By immunofluoresence, MARCKS is noted to be located at the lateral membrane under control conditions. In response to carbachol, MARCKS translocates to the cytosol, indicating its phosphorylation, which is additionally confirmed biochemically. Consistent with this observation, carbachol induces the translocation of PKC-epsilon to proximity with MARCKS at the lateral membrane. In response to chemical ischemia, MARCKS fails to translocate and phosphorylation does not increase. Additionally, the translocation of PKC-epsilon is not to the lateral membrane but rather basally. The data suggest that the differential translocation of PKC-epsilon in response to pharmacological agonists versus ischemic stress may lead to different effects on downstream targets.
Mammen
Joshua M V
JM
Epithelial Pathobiology Research Group, Dept. of Surgery, University of Cincinnati College of Medicine, PO Box 670558, Cincinnati, OH 45267-0558, USA.
Song
J Cecilia
JC
Yoo
James
J
Kim
Peter S
PS
Davis
Harold W
HW
Calvo
M Isabel
MI
Worrell
Roger T
RT
Matlin
Karl S
KS
Matthews
Jeffrey B
JB
D002217:Carbachol / Q000494:pharmacology; D018679:Cholinergic Agonists / Q000494:pharmacology; D004847:Epithelial Cells / Q000502:physiology; D006801:Humans; D000860:Hypoxia; D007422:Intestines / Q000166:cytology* / Q000502:physiology*; D047908:Intracellular Signaling Peptides and Proteins / Q000378:metabolism*; D008565:Membrane Proteins / Q000378:metabolism*; D000076250:Myristoylated Alanine-Rich C Kinase Substrate; D010766:Phosphorylation; D011493:Protein Kinase C / Q000378:metabolism / Q000494:pharmacology*; D051744:Protein Kinase C-epsilon; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology
D018679:Cholinergic Agonists; D047908:Intracellular Signaling Peptides and Proteins; C000619710:MARCKS protein, human; D008565:Membrane Proteins; D000076250:Myristoylated Alanine-Rich C Kinase Substrate; D002217:Carbachol; C497255:PRKCE protein, human; D011493:Protein Kinase C; D051744:Protein Kinase C-epsilon; D013755:Tetradecanoylphorbol Acetate