The cell nucleus is structurally and functionally organized by the nuclear matrix. We have examined whether the nuclear cAMP-dependent protein kinase-anchoring protein AKAP95 contains specific signals for targeting to the subnuclear compartment and for interaction with other proteins. AKAP95 was expressed in mammalian cells and found to localize exclusively to the nuclear matrix. Mutational analysis was used to identify determinants for nuclear localization and nuclear matrix targeting of AKAP95. These sites were found to be distinct from previously identified DNA and protein kinase A binding domains. The nuclear matrix-targeting site is unique but conserved among members of the AKAP95 family. Direct binding of AKAP95 to isolated nuclear matrix was demonstrated in situ and found to be dependent on the nuclear matrix-targeting site. Moreover, Far Western blot analysis identified at least three AKAP95-binding proteins in nuclear matrix isolated from rat brain. Yeast two-hybrid cloning identified one binding partner as p68 RNA helicase. The helicase and AKAP95 co-localized in the nuclear matrix of mammalian cells, associated in vitro, and were precipitated as a complex from solubilized cell extracts. The results define novel protein-protein interactions among nuclear matrix proteins and suggest a potential role of AKAP95 as a scaffold for coordinating assembly of hormonally responsive transcription complexes.The nuclear matrix was first described in 1974 by Berezney and Coffey (1, 2) as the insoluble, proteinaceous structural component of the nucleus that remains after digestion and extraction of chromatin and associated nuclear factors. Since then it has become increasingly apparent that the matrix plays an important functional role, coordinating many nuclear activities including DNA replication, RNA processing, and transcription (3-4). Integration of these events is thought to occur within a high level of chromatin structure where the nuclear matrix organizes DNA into functional loops by binding specific sequences or "matrix attachment regions." Not surprisingly, these regions are often found flanking actively transcribed genes and may serve as localization sites for assembly of factors regulating gene expression (5-7).Structurally, the intact nuclear matrix includes nuclear lamina, residual nucleoli, and ribonucleoprotein particles bound via a fibrous network of proteins, RNA, and polysaccharides. Although lamins and heterogeneous nuclear RNA proteins are the predominant protein constituents, the core structure contains more than 100 distinct nuclear matrix proteins, many of which bind DNA (8 -10). To date, relatively few nuclear matrix proteins have been characterized in detail, and little is known about how they are incorporated into the matrix. Complicating the analysis is the fact that the nuclear matrix is a dynamic structure, and its protein composition is affected by environmental stimuli and growth conditions. Indeed, several nuclear matrix proteins have been identified as useful markers for specific typ...