Attenuation of cell cycle regulator p27(Kip1) expression in vertebrate epithelial cells mediated by extracellular signals in vivo and in vitro.
Cell cycle arrest in potentially dividing cells is often mediated by inhibitors of G1/S-phase cyclin-dependent kinases. The cyclin E/CDK2-inhibitor p27(Kip1) has been implicated in this context in epithelial cells. We cloned and sequenced p27(Kip1) of ducklings (Anas platyrhynchos) and used an in vitro assay system to study the mechanism of p27(Kip1) downregulation in the nasal gland which precedes an increase in proliferation rate upon initial exposure of the animals to osmotic stress. Western blot studies revealed that p27(Kip1) is downregulated during 24 h of osmotic stress in ducklings with the steepest decline in protein levels between 5 and 8 h. As indicated by the results of Northern blot and semi-quantitative PCR studies, protein downregulation is not accompanied by similar changes in mRNA levels indicating that Kip1 is regulated mainly at the translational (synthesis) or posttranslational level (degradation). Using recombinant duck Kip1 protein expressed in E. coli, we showed that Kip1 is subject to polyubiquitinylation by cytosolic enzymes from nasal gland cells indicating that loss of Kip1 may be regulated, at least in part, by acceleration of protein degradation. In cultured nasal gland tissue, attenuation of Kip1 expression could be induced by activation of the muscarinic acetylcholine receptor indicating that mAChR-receptor signalling may play a role in the re-entry of quiescent gland cells into the cell cycle.
Rohlfing
Anne-Katrin
AK
Animal Physiology and Biochemistry, Ernst Moritz Arndt-University Greifswald, Biotechnikum, Walther Rathenau-Strasse 49 a, 17489 Greifswald, Germany.
Schill
Tillmann
T
Müller
Christian
C
Hildebrandt
Petra
P
Prowald
Alexandra
A
Hildebrandt
Jan-Peter
JP
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002453:Cell Cycle; D003001:Cloning, Molecular; D050760:Cyclin-Dependent Kinase Inhibitor p27 / Q000235:genetics* / Q000378:metabolism*; D018076:DNA, Complementary / Q000235:genetics; D004372:Ducks / Q000033:anatomy & histology / Q000235:genetics / Q000378:metabolism; D004847:Epithelial Cells / Q000378:metabolism; D007150:Immunohistochemistry; D066298:In Vitro Techniques; D008969:Molecular Sequence Data; D009997:Osmotic Pressure; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D011976:Receptors, Muscarinic / Q000378:metabolism; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D012491:Salt Gland / Q000166:cytology / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D015398:Signal Transduction
D018076:DNA, Complementary; D012333:RNA, Messenger; D011976:Receptors, Muscarinic; D011994:Recombinant Proteins; D050760:Cyclin-Dependent Kinase Inhibitor p27