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Expression of Cdk5, p35, and Cdk5-associated kinase activity in the developing rat lens
1Laboratory of Molecular and Developmental Biology, National Eye Institute, National Institutes of Health, Bethesda, Maryland 20892-2730, USA.
Insights
Cyclin-dependent kinase 5 (Cdk5) and its activator p35 are expressed in the developing rat lens. This suggests Cdk5/p35 kinase activity may be involved in lens fiber cell differentiation.
Area of Science:
- Ophthalmology
- Developmental Biology
- Cell Biology
Background:
- Cyclin-dependent kinase 5 (Cdk5) is a key regulator of neuronal development.
- Its role in non-neuronal tissues, such as the lens, is less understood.
Purpose of the Study:
- To investigate the expression and activity of Cdk5 and its regulatory subunit p35 in the developing rat lens.
- To determine the potential role of Cdk5/p35 in lens fiber cell differentiation.
Main Methods:
- Reverse transcription and polymerase chain reaction (RT/PCR) to detect mRNA expression.
- In situ hybridization to localize mRNA expression.
- Immunocytochemistry to visualize protein localization.
- Immunoprecipitation and in vitro kinase assays to assess protein complex formation and activity.
Main Results:
- Cdk5 and p35 mRNA and proteins were detected in rat lens epithelial and fiber cells during development (E16-P8).
- High expression was observed in differentiating lens fibers in the bow region.
- Cdk5 was found in the cytoplasm and nucleus of maturing fiber cells.
- Cdk5/p35 complexes exhibited kinase activity in vitro.
Conclusions:
- Cdk5/p35 kinase is expressed and active in the developing lens.
- These findings indicate that Cdk5/p35 is not exclusive to neurons and may play a role in lens fiber cell differentiation.
Abstract:
We have investigated the expression of Cdk5 and its regulatory subunit, p35, in the developing rat lens from embryonic day 16 (E16) to postnatal day 8 (P8). Reverse transcription and polymerase chain reaction (RT/PCR) detected Cdk5 and p35 mRNA expression in lens epithelial cells and in differentiating lens fibers throughout this developmental period. Subsequent sequencing of the RT/PCR products confirmed their identifies. In sity hybridization with Cdk5 and p35 riboprobes showed especially high expression of both mRNAs in the newly formed lens fiber cells in the bow region of the lens. Immunocytochemistry at E18 showed that Cdk5 was present in the cytoplasm of lens epithelial cells and fiber cells, with especially strong immunostaining at the anterior ends of the fibers. Fiber cells in the final stages of maturation, immediately prior to nuclear degeneration, showed positive staining for Cdk5 in the nucleus. Immunoprecipitation of proteins with Cdk5 antibody followed by immunoblotting with either N-terminal specific or C-terminal specific p35 antibodies demonstrated that p35 is complexed with Cdk5 in lens epithelial cells and lens fibers. Immunoprecipitates of Cdk5 from epithelia and fibers showed kinase activity in vitro using histone H1 as a substrate. These findings demonstrate that p35/Cdk5 activity is not restricted to neurons and raise the possibility that this kinase may play a role in lens fiber cell differentiation.