Activation of protein kinase C isozymes is associated with post-mitotic events in intestinal epithelial cells in situ.

M L Saxon,X Zhao,J D Black

doi:10.1083/jcb.126.3.747

Abstract

The mechanisms underlying control of cell growth and differentiation in epithelial tissues are poorly understood. Protein kinase C (PKC) isozymes, members of a large family of serine/threonine kinases of fundamental importance in signal transduction, have been increasingly implicated in the regulation of cell growth, differentiation, and function. Using the rat intestinal epithelium as a model system, we have examined PKC-specific activity as well as individual PKC isozyme expression and distribution (i.e., activation status) in epithelial cells in situ. Increased PKC activity was detected in differentiating and functional cells relative to immature proliferating crypt cells. Immunofluorescence and Western blot analysis using a panel of isozyme-specific antibodies revealed that PKC alpha, beta II, delta, epsilon, and zeta are expressed in rat intestinal epithelial cells and exhibit distinct subcellular distribution patterns along the crypt-villus unit. The combined morphological and biochemical approach used permitted analysis of the activation status of specific PKC isozymes at the individual cell level. These studies showed that marked changes in membrane association and level of expression for PKC alpha, beta II, delta, and zeta occur as cells cease division in the mid-crypt region and begin differentiation. Additional changes in PKC activation status are observed with acquisition of mature function on the villus. These studies clearly demonstrate naturally occurring alterations in PKC isozyme activation status at the individual cell level within the context of a developing tissue. Direct activation of PKC in an immature intestinal crypt cell line was shown to result in growth inhibition and coincident translocation of PKC alpha from the cytosolic to the particulate subcellular fraction, paralleling observations made in situ and providing further support for a role of intestinal PKC isozymes in post-mitotic events. PKC isozymes were also found to be tightly associated with cytoskeletal elements, suggesting participation in control of the structural organization of the enterocyte. Taken together, the results presented strongly suggest an involvement of PKC isoforms in cellular processes related to growth cessation, differentiation, and function of intestinal epithelial cells in situ.

Highlights

Direct activation of Protein kinase C (PKC) in an immature intestinal crypt cell line was shown to result in growth inhibition and coincident translocation of PKC tx from the cytosolic to the particulate subcellular fraction, paralleling observations made in situ and providing further support for a role of intestinal PKC isozymes in post-mitotic events
It is widely accepted that PKC exists in an inactive conformation in the cytosol and that activation of the enzyme results in its translocation from the cytosolic to the particulate subcellular fraction, which consists of membrane and cytoskeletal elements (Kraft and Anderson, 1983; Kiley and Jaken, 1990; Mochly-Rosen et ai., 1990; Gregorio et al, 1992, 1994)
In order to gain insight into the role of PKC in intestinal epithelial cell growth and differentiation in situ, PKC activity was measured in cytosolic, membrane, and cytoskeletal subcellular fractions prepared from crypt, low-to-mid villus and upper villus cells isolated by the method of Weiser (1973)

Summary

Introduction

Direct activation of PKC in an immature intestinal crypt cell line was shown to result in growth inhibition and coincident translocation of PKC tx from the cytosolic to the particulate subcellular fraction, paralleling observations made in situ and providing further support for a role of intestinal PKC isozymes in post-mitotic events. Differences in structure, substrate specificity, cofactor requirements, and localization of individual PKC isozymes have been reported (e.g., Parker et al, 1986; Hidaka et al, 1988; Huang et al, 1988; Hocevar and Fields, 1991; Hocevar et al, 1992; Liyanage et al, 1992). These differences, together with the varied consequences of PKC activation in the same cell, suggest that individual isozymes have distinct and specialized functions in cell signaling. Biochemical and morphological evidence for the redistribution of certain PKC isozymes from the cytosol to the cell periphery in response to specific activators (Ito et al, 1988) or physiological stimuli (Ganesan et al, 1990, 1992) has demonstrated a correlation between redistribution and cellular responses related to growth, differentiation, or functional behavior

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