A-kinase-anchoring protein AKAP95 is targeted to the nuclear matrix and associates with p68 RNA helicase.
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.
Neurological Sciences Institute, Oregon Health Sciences University, Beaverton, Oregon 97006, USA.
L
L
Akileswaran
J W
JW
Taraska
J A
JA
Sayer
J M
JM
Gettemy
V M
VM
Coghlan
D054758:A Kinase Anchor Proteins; D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D001921:Brain / Q000201:enzymology*; D019556:COS Cells; D002460:Cell Line; D002522:Chlorocebus aethiops; D003001:Cloning, Molecular; D017124:Conserved Sequence; D053487:DEAD-box RNA Helicases; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D017930:Genes, Reporter; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins; D007668:Kidney; D051379:Mice; D008856:Microscopy, Fluorescence; D008969:Molecular Sequence Data; D015530:Nuclear Matrix / Q000201:enzymology*; D009687:Nuclear Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011494:Protein Kinases / Q000378:metabolism*; D020365:RNA Helicases / Q000378:metabolism*; D051381:Rats; D011994:Recombinant Proteins / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics; D016415:Sequence Alignment; D017384:Sequence Deletion; D017386:Sequence Homology, Amino Acid; D014162:Transfection; D016335:Zinc Fingers
D054758:A Kinase Anchor Proteins; C490117:AKAP8L protein, human; C086194:Akap8 protein, rat; D004268:DNA-Binding Proteins; D047908:Intracellular Signaling Peptides and Proteins; D009687:Nuclear Proteins; D011994:Recombinant Proteins; D011494:Protein Kinases; C506749:Ddx5 protein, human; C506751:Ddx5 protein, mouse; D053487:DEAD-box RNA Helicases; C506752:Ddx5 protein, rat; D020365:RNA Helicases