Epidermal Growth Factor-Induced Selective Phosphorylation of Cultured Rat Hepatocyte 55-kD Cytokeratin before Filament Reorganization and DNA Synthesis

• Published in: The Journal of cell biology Vol. 109; no. 4; pp. 1665 - 1676
• Main Authors: Baribault, Helene, Blouin, Richard, Bourgon, Lise, Marceau, Normand
• Format: Journal Article
• Language: English
• Published: New York, NY Rockefeller University Press 01.10.1989; The Rockefeller University Press
• Subjects: Actin Cytoskeleton - drug effects; Actin Cytoskeleton - ultrastructure; Animals; Biological and medical sciences; Cell structures and functions; Cells; Cells, Cultured; Cultured cells; Cytoskeleton - ultrastructure; Cytoskeleton, cytoplasm. Intracellular movements; Dexamethasone - pharmacology; DNA Replication - drug effects; Embryonic cells; Epidermal Growth Factor - pharmacology; Epithelial cells; Fluorescent Antibody Technique; Fundamental and applied biological sciences. Psychology; Hepatocytes; Insulin; Keratins; Keratins - biosynthesis; Keratins - metabolism; Kinetics; Liver - drug effects; Liver - metabolism; Liver - ultrastructure; Liver cells; Molecular and cellular biology; Molecular Weight; Phosphoproteins - biosynthesis; Phosphoproteins - isolation & purification; Phosphorylation; Rats; Rats, Inbred F344; Cell culture; Phosphorylation; Intermediary filament; Radiolabelling; Rat; Mitosis; Rodentia; Microfilament; DNA synthesis; Indirect immunofluorescence; Cell differentiation; Proteins; Keratin; Vertebrata; Mammalia; Epidermal growth factor; Hepatocyte; Aminoacid; Immunoblotting assay; Molecular rearrangement; Cytoskeleton; Microtubule; Fluorescence microscopy
• ISSN: 0021-9525; 1540-8140
• DOI: 10.1083/jcb.109.4.1665

We have reported previously that the addition of dexamethasone to cultured quiescent suckling rat hepatocytes in the presence of insulin, a culture condition which does not cause growth activation, induces a selective increase in the synthesis of the 49-kD/55-kD cytokeratin (CK49/CK55) pair over a 24-h period. This increased synthesis coincides with the formation of dense filament networks reminiscent of those observed in situ at the cell periphery (Marceau, N., H. Baribault, and I. Leroux-Nicollet. 1985. Can. J. Biochem. Cell Biol. 63:448-457). We show here for the first time that when EGF is added 48 h after insulin and dexamethasone, there is an early preferential phosphorylation of the CK55 of the CK49/CK55 pair, an induced filament rearrangement from the cell periphery to the cytoplasm, and a subsequent entry into S phase and mitosis after a lag period of 8 h. Indirect immunofluorescence microscopy with monoclonal antibodies to CK49 and CK55 indicate that, while before EGF treatment the cytokeratin filaments were mainly distributed near the cell periphery, the addition of EGF resulted in their reorganization to a predominantly cytoplasmic localization within <3 h. Anti-tubulin and anti-actin antibodies showed no detectable alteration in the distribution of microtubules and microfilaments. Pulse-chase measurements with [35 S]methionine showed no apparent change in the turnover of either CK49 or CK55 during the period that precedes the initiation of DNA synthesis. 32 P-labeling in vivo followed by SDS-PAGE demonstrated that CK55 was phosphorylated at a much higher level than CK49 in nonstimulated hepatocytes, and that the addition of EGF resulted in a selective stimulation of 32 P- CK55 labeling within <30 min. Comparative analyses by two-dimensional PAGE of [35 S]methionine and 32 P-labeled cytokeratins at various times after EGF stimulation demonstrated a rapid increase in a first phosphorylated form of CK55 and the appearance of a second phosphorylated form at 30 min postistimulation. The changes in the relative proportion of non-phosphorylated and phosphorylated forms were confirmed by immunoblotting with the anti-CK55 monoclonal antibody. Determinations of the 32 P-labeled phosphoamino acids of CK55 extracted from the gels demonstrated that the radioactivity was mostly in serine residues. Labeling of Triton-permeabilized hepatocytes with γ 32 P-ATP after treatment with EGF for 30 min to 3 h at 37°C, also demonstrated a phosphorylation of CK55 and CK49 as well, implying that the EGF-responsive serine protein kinase is detergent insoluble and probably part of the surface membrane skeleton. We propose that early selective CK55 phosphorylation at serine residues and subsequent rearrangements of CK49/CK55 filaments from cell periphery to cytoplasm are EGF receptor driven cell surface reactions that are part of the cascade of events that culminate in the initiation of DNA synthesis and subsequent cell division of hepatocytes.