Akileswaran L, Taraska J W, Sayer J A, Gettemy J M, Coghlan V M
Neurological Sciences Institute, Oregon Health Sciences University, Beaverton, Oregon 97006, USA.
J Biol Chem. 2001 May 18;276(20):17448-54. doi: 10.1074/jbc.M101171200. Epub 2001 Feb 22.
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.
细胞核在结构和功能上由核基质组织。我们研究了细胞核中依赖cAMP的蛋白激酶锚定蛋白AKAP95是否包含定位于亚核区室以及与其他蛋白相互作用的特定信号。AKAP95在哺乳动物细胞中表达，并且发现它仅定位于核基质。通过突变分析来确定AKAP95的核定位和核基质靶向的决定因素。发现这些位点与先前鉴定的DNA和蛋白激酶A结合结构域不同。核基质靶向位点是独特的，但在AKAP95家族成员中保守。在原位证实了AKAP95与分离的核基质的直接结合，并且发现其依赖于核基质靶向位点。此外，Far Western印迹分析在从大鼠脑分离的核基质中鉴定出至少三种AKAP95结合蛋白。酵母双杂交克隆鉴定出一个结合伴侣为p68 RNA解旋酶。该解旋酶和AKAP95在哺乳动物细胞的核基质中共定位，在体外相互作用，并从溶解的细胞提取物中作为复合物沉淀出来。这些结果定义了核基质蛋白之间新的蛋白质-蛋白质相互作用，并提示AKAP95作为协调激素反应性转录复合物组装的支架的潜在作用。
