AKAP95 transports Cx43 into nucleus, regulating G1/S transition of A549 cells.

Abstract Purpose: Cx43 (connexin 43) has been found to inhibit cell cycle of tumor cells. No nuclear localization sequence of Cx43 was found but the protein has been found in nucleus. The process remains unclear. We are aiming at providing the model that AKAP95 (A-kinase anchoring protein) binds to and transports Cx43 into nucleus of lung cancer cells.Methods: Lung cancer cells were arrested at preliminary stage (P), middle and late stage (M) of G1 phase and restriction point (R). Immunocoprecipitation (Co-IP) and Western blot (WB) were used to analyze expression and binding of AKAP95, Cx43, cyclin D1 and cyclin E1 at different stages of lung cancer cells during G1 phase. Confocal laser scanning microscopy (CLSM) was used to detect the location of proteins in arrested cells. Transmission electron microscope (TEM) with higher resolution was used to detect binding/aggregating and location of the above four proteins at subcellular level.Results: 1) AKAP95 transports Cx43 into the nucleus through the nuclear pore mainly at R stage. Some AKAP95 and Cx43 form protein aggregates during the process. 2) Cyclin E1 was found to be bind/aggregate with AKAP95-Cx43 complex and involved in the nuclear transport of Cx43 mainly at R stage. 3)Cyclin D1 was found to bind with AKAP95 and Cx43 respectively but not to be involved in their aggregating and nuclear transport of Cx43.Conclusions: 1) AKAP95 and Cx43 bind/aggregate to each other during G1 phase, forming AKAP95-Cx43 complexes/aggregates. After their binding/aggregating, AKAP95 targets nuclear pore and matrix, transporting Cx43 into nucleus. Formed AKAP95-Cx43 complexes/ aggregates do not dissociate in nucleus. The process can be summarized as ‘forming AKAP95-Cx43 complexes/aggregates; transporting Cx43 into nucleus; functioning remaining binding/aggregating’. 2) Cyclin D1 was only found to bind to AKAP95 and Cx43 respectively and function in cytoplasm while cyclin E1 was involved in the binding/aggregation of AKAP95 and Cx43, as well as the nuclear transport of Cx43.