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DR Ledee, PS Zelenka; Interaction Between the CDK5 Activating Proteins, p39 and p35, and an Alternatively Spliced Zinc-Finger Protein in Lens . Invest. Ophthalmol. Vis. Sci. 2002;43(13):2333.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose: Cyclin dependent kinase 5 (CDK5) and its activating proteins, p39 and p35, are highly expressed in lens. CDK5 has been shown to be necessary for lens development in Xenopus, although the underlying mechanisms involved are unknown. Our goal was to identify other proteins that interact with CDK5 and its activators as a means of understanding CDK5 function in the lens. Methods: A cDNA library was constructed using embryonic day 18 rat lens mRNA. Yeast two-hybrid screening using CDK5, p35 or p39 as bait identified clones encoding potential interacting proteins. Clones positive for both the His and LacZ markers were further examined by sequencing, and protein-protein specificity was tested against pLamin C. Interactions were further confirmed via GST-pull down assays. Expression of lens mRNA corresponding to potential interacting proteins was examined by RT-PCR. Results: Analysis of a p39 interacting clone identified the rat homologue of the mouse AK014458 cDNA. Interestingly, AK014458 protein possesses a 5' BTB domain (a protein interaction motif), and a 3' zinc-finger, C2H2 type. This protein was found to interact with the other CDK5 activator, p35, but not with CDK5 or pLamin C, the non-specific control. Sequencing of the clone showed it to be an alternative splice variant, creating a truncated protein that results in the loss of the 3' Zn+ finger motif. RT-PCR of rat lens RNA identified the presence of both the full length mRNA and the splice variant. Conclusion: Although the function of AK014458 is unknown, the BTB and Zn-finger motifs are often characteristic of transcription factors. The alternatively spliced variant suggests a possible dominant-negative role, and the interactions with p35/p39 may be a means of regulation by CDK5.
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