Cyclin B, p34cdc2, and H1-kinase activity in terminally differentiating lens fiber cells.
Terminal differentiation of lens fiber cells is marked by chromatin condensation, abrupt dissolution of the nuclear lamina, vesicularization of the nuclear membrane, and complete degradation of the nucleus and other organelles. Since these events resemble the chromosomal condensation and nuclear envelope breakdown associated with mitosis, we investigated whether a similar biochemical mechanism might be involved by testing for the presence of cyclin B/p34cdc2 complexes and p34cdc2-associated histone kinase activity in differentiating lens fiber cells. A coupled reverse transcription/polymerase chain reaction using RNA from E7, E15, or E20 embryonic chicken lens fibers amplified a cyclin B product of the expected size, whose identity was confirmed by sequencing. In situ hybridization showed that cyclin B mRNA was present in nucleated lens fiber cells at E19. Immunoblotting of proteins isolated from E6 or E15 lens fibers by p13-agarose affinity chromatography with anti-cyclin B antibody detected the 45-kDa cyclin B protein, while immunoblotting with anti-PSTAIRE antibody detected a single, 34-kDa band, identified as p34cdc2. The p13-affinity purified fraction from E6 or E15 lens fibers showed histone H1 kinase activity in vitro. Immunocytochemistry of E6 lenses with anti-chicken cyclin B antiserum showed positive staining in the nuclei of postmitotic annular pad and fiber cells. These results demonstrate that cyclinB/p34cdc2 complexes and p34cdc2-associated histone kinase activity are present in postmitotic, differentiating lens fiber cells and support the possibility that phosphorylation of specific nuclear substrates by p34cdc2 may play a role in the denucleation of lens fiber cells.
Gao
C Y
CY
Laboratory of Molecular and Developmental Biology, National Eye Institute, National Institutes of Health, Bethesda, Maryland 20892, USA.
Bassnett
S
S
Zelenka
P S
PS
D000818:Animals; D001483:Base Sequence; D016203:CDC2 Protein Kinase / Q000096:biosynthesis* / Q000235:genetics; D002454:Cell Differentiation; D002642:Chick Embryo; D016213:Cyclins / Q000096:biosynthesis* / Q000235:genetics; D007150:Immunohistochemistry; D017403:In Situ Hybridization; D007908:Lens, Crystalline / Q000166:cytology / Q000254:growth & development*; D016200:Maturation-Promoting Factor / Q000096:biosynthesis* / Q000235:genetics; D008969:Molecular Sequence Data; D012333:RNA, Messenger / Q000032:analysis*
D016213:Cyclins; D012333:RNA, Messenger; D016203:CDC2 Protein Kinase; D016200:Maturation-Promoting Factor