Abstract
Purpose: :
Visual arrestins quench light–induced phototransduction by binding to light–activated, phosphorylated opsins, which was widely believed to be their only function. Non–visual arrestins directly interact with a variety of non–receptor partners, such as clathrin, adaptin2, and the ubiquitin ligase Mdm2. They also serve as scaffolds for MAP kinase cascades leading to the activation of ERK1/2 and JNK3. Here we studied the interactions of rod and cone arrestins with two proteins involved in anti– and pro–apoptotic pathways: the kinase JNK3 and the ubiquitin ligase Mdm2.
Methods: :
Rod and cone arrestins are virtually excluded from the nuclei of photoreceptors and HEK cells. JNK3 and Mdm2 localize primarily to the nucleus. We used the ability of arrestins to redistribute their partners to the cytoplasm to study the interaction of rod and cone arrestins with these two proteins. Tagged and untagged rod and cone arrestins were expressed in HEK–293A cells with or without GFP–JNK3 and GFP–Mdm2. Cells expressing GFP–JNK3 and GFP–Mdm2 alone served as controls.
Results: :
Coexpression of both arrestins with JNK3 and Mdm2 results in the relocalization of these two proteins from the nucleus to the cytoplasm (with JNK3 showing more dramatic changes). Leptomycin B (LMB) is a potent, specific inhibitor of nuclear export signal (NES)–dependent protein export from the nucleus. The effect of LMB on the transport of these two proteins by arrestins was tested to determine their dependence on this pathway. The ability of rod arrestin to exclude JNK3 and Mdm2 is LMB–sensitive. In contrast, the ability of cone arrestin to exclude JNK3 and Mdm2 from the nucleus is less LMB–sensitive. The removal of two putative NES sequences from rod arrestin by mutagenesis redirects its export to an LMB–insensitive pathway, but does not prevent arrestin–dependent exclusion of Mdm2 and JNK3 from the nucleus.
Conclusions: :
The size of arrestin is just small enough to allow its free diffusion to and from the nucleus through the nuclear pore. Therefore, exclusive localization of arrestins in the cytoplasm suggests they are actively exported from the nucleus. The ability of arrestins to redistribute nuclear JNK3 and Mdm2 suggests that free visual arrestins (both rod and cone) interact with these partners. The effect of LMB indicates that arrestins and their complexes with JNK3 and Mdm2 can use alternative export pathways to exit the nucleus.
Keywords: photoreceptors • apoptosis/cell death • signal transduction