Submitted on March 31, 2004
Accepted on August 25, 2004
Differential Subcellular Targeting of PKC
in Response to Pharmacological or Ischemic Stimuli in Intestinal Epithelia
Joshua M. V. Mammen1, J. Cecilia Song1, James Yoo1, Peter S. Kim1, Harold W. Davis1, M. Isabel Calvo1, Roger T. Worrell1, Karl S. Matlin1, and Jeffrey B. Matthews1*
1 Epithelial Pathobiology Research Group, Department of Surgery, University of Cincinnati College of Medicine, Cincinnati, OH, USA
* To whom correspondence should be addressed. E-mail: Jeffrey.Matthews{at}uc.edu.
Ischemia is the central pathogenic factor underlying a spectrum of intestinal
disorders. The study of the cellular signaling responses to ischemic stress in non-epithelial
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 Protein
Kinase C (PKC) family of proteins has been implicated in ischemic stress in non-epithelial
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 MARCKS (myristoylated alanine-rich C kinase substrate) is also studied.
Chemical ischemia resulted in the transient activation of only the isoform PKC
as
detected by translocation employing the subcellular fractionation technique. The
pharmacologic agonists PMA and Carbachol also led to the translocation of PKC. 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 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 is not to the lateral membrane, but rather basally. The data suggests the differential
translocation of PKC in response to pharmacologic agonists versus ischemic stress may
lead to different effects on downstream targets.
