Protein Kinase C Isozyme-mediated Cell Cycle Arrest Involves Induction of p21waf1/cip1 and p27kip1 and Hypophosphorylation of the Retinoblastoma Protein in Intestinal Epithelial Cells

doi:10.1074/jbc.272.14.9424

Protein Kinase C Isozyme-mediated Cell Cycle Arrest Involves Induction of p21^waf1/cip1 and p27^kip1 and Hypophosphorylation of the Retinoblastoma Protein in Intestinal Epithelial Cells*

From the Departments of Experimental Therapeutics and
^‡ Molecular Medicine, Roswell Park Cancer Institute, Buffalo, New York 14263

^§ To whom correspondence should be addressed:
Dept. of Experimental Therapeutics, Roswell Park Cancer Institute, Elm and Carlton St., Buffalo, NY 14263.
Tel.: 716-845-5766; Fax: 716-845-8857; E-mail: jblack{at}sc3103.med.buffalo.edu

Abstract

The molecular mechanisms underlying protein kinase C (PKC) isozyme-mediated control of cell growth and cell cycle progression are poorly understood. Our previous analysis of PKC isozyme regulation in the intestinal epithelium in situ revealed that multiple members of the PKC family undergo changes in expression and subcellular distribution precisely as the cells cease proliferating in the mid-crypt region, suggesting that activation of one or more of these molecules is involved in negative regulation of cell growth in this system (Saxon, M. L., Zhao, X., and Black, J. D. (1994) J. Cell Biol. 126, 747-763). In the present study, the role of PKC isozyme(s) in control of intestinal epithelial cell growth and cell cycle progression was examined directly using the IEC-18 immature crypt cell line as a model system. Treatment of IEC-18 cells with PKC agonists resulted in translocation of PKC α, δ, and ϵ from the soluble to the particulate subcellular fraction, cell cycle arrest in G₁ phase, and delayed transit through S and/or G₂/M phases. PKC-mediated cell cycle arrest in G₁ was accompanied by accumulation of the hypophosphorylated, growth-suppressive form of the retinoblastoma protein and induction of the cyclin-dependent kinase inhibitors p21^waf1/cip1 and p27^kip1. Reversal of these cell cycle regulatory effects was coincident with activator-induced down-regulation of PKC α, δ, and ϵ. Differential down-regulation of individual PKC isozymes revealed that PKC α in particular is sufficient to mediate cell cycle arrest by PKC agonists in this system. Taken together, the data implicate PKC α in negative regulation of intestinal epithelial cell growth both in vitro and in situ via pathways which involve modulation of Cip/Kip family cyclin-dependent kinase inhibitors and the retinoblastoma growth suppressor protein.

Footnotes

↵* This work was supported by grants from the Crohn's and Colitis Foundation of America and the Buffalo Foundation, by National Science Foundation Grant DCB 8917424, and by National Institutes of Health Grant 16056. The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
↵¹ The abbreviations used are:

PKC

protein kinase C

DAG

diacylglycerol

Rb

retinoblastoma protein

cdk

cyclin-dependent kinase

DMEM

Dulbecco's modified Eagle's medium

FCS

fetal calf serum

PMA

phorbol 12-myristate 13-acetate

PDBu

phorbol 12 13-dibutyrate

DiC₈

12-dioctanoyl-sn-glycerol

PAGE

polyacrylamide gel electrophoresis

PBS

phosphate-buffered saline

TBS

Tris-buffered saline
↵²A. Byrd and J. D. Black, unpublished results.
- Received July 8, 1996.
- Revision received January 22, 1997.