Cloning and characterization of a cDNA encoding an A-kinase anchoring protein located in the centrosome, AKAP450

A combination of protein kinase A type II (RII) overlay screening, database searches and PCR was used to identify a centrosomal A-kinase anchoring pro tein. A cDNA with an 11.7 kb open reading frame was characterized and found to correspond to 50 exons of genomic sequence on human chromosome 7q21-22, This cDNA clone encoded a 3908 amino acid protein of 453 kDa, that was des ignated AKAP350 (DDBJ/ EMBL/GenBank accession No. J131693), Sequence compar ison demonstrated that the open reading frame contained a previously charac terized cDNA encoding Yotiao, as well as the human homologue of AKAP120, Nu merous coiled-coil structures were predicted from AKAP450, and weak homolog y to pericentrin, giantin and other structural proteins was observed. A put ative RII-binding site was identified involving amino acid 2556 of AKAP450 by mutation analysis combined with RII overlay and an amphipatic helix was predicted in this region. Immunoprecipitation of RII from RIPA-buffer extra cts of HeLa cells demonstrated coprecipitation of AKAP450. By immunofluorec ent labeling with specific antibodies it was demonstrated that AKAP450 loca lized to centrosomes. Furthermore, AKAP450 was shown to co-purify in centro somal preparations. The observation of two mRNAs and several splice product s suggests additional functions for the AKAP350 gene.