The KH-Tudor Domain of A-Kinase Anchoring Protein 149 Mediates RNA-Dependent Self-Association † Marie Rogne, ‡ Helga B. Landsverk, ‡ Aleyde Van Eynde, § Monique Beullens, § Mathieu Bollen, § Philippe Collas,* ,‡ and Thomas Ku ¨ntziger ‡ Institute of Basic Medical Sciences, Department of Biochemistry, Faculty of Medicine, UniVersity of Oslo, P.O. Box 1112 Blindern, 0317 Oslo, Norway, and DiVision of Biochemistry, Catholic UniVersity of LeuVen, Campus Gasthuisberg, Herestraat 49, 3000 LeuVen, Belgium ReceiVed July 14, 2006; ReVised Manuscript ReceiVed October 4, 2006 ABSTRACT: A-Kinase anchoring proteins (AKAPs) control the subcellular localization and temporal specificity of protein phosphorylation mediated by cAMP-dependent protein kinase. AKAP149 (AKAP1) is found in mitochondria and in the endoplasmic reticulum-nuclear envelope network where it anchors protein kinases, phosphatases, and a phosphodiesterase. AKAP149 harbors in its COOH-terminal part one KH and one Tudor domain, both known to be involved in RNA binding. We investigated the properties of the COOH-terminal domain of AKAP149. We show here that AKAP149 is a self-associating protein with RNA binding features. The KH domain of AKAP149 is sufficient for self-association in a RNA- dependent manner. The Tudor domain is not necessary for self-association, but it is required together with the KH domain for targeting to well-defined nuclear foci. These foci are spatially closely related to nucleolar subcompartments. We also show that the KH-Tudor-containing domain of AKAP149 binds RNA in vitro and in RNA coprecipitation experiments. AKAP149 emerges as a scaffolding protein involved in the integration of intracellular signals and possibly in RNA metabolism. In the context of the wide array of intracellular effects mediated by cAMP signaling through cAMP-dependent protein kinase (PKA), 1 subcellular localization and temporal specificity of protein phosphorylation by PKA are mediated by proteins collectively termed A-kinase anchoring proteins (AKAPs) (1, 2). AKAPs bind a PKA regulatory subunit dimer through a consensus sequence, whereas a targeting domain specifies subcellular localization. AKAPs also interact with other signaling molecules such as protein kinase C (PKC), phosphodiesterases, and protein phosphatases in a space- and time-regulated fashion (2, 3). AKAP149 (4), also designated AKAP1, is a human 149 kDa anchoring protein homologous to mouse AKAP121 identified in mitochondria (5-7) and in the endoplasmic reticulum-nuclear envelope (ER-NE) network (7, 8). AKAP149 harbors the hallmarks of a protein phosphatase 1 (PP1) regulatory subunit and targets a fraction of nuclear PP1 to the vicinity of the NE upon nuclear reassembly in vitro and at the end of mitosis (8, 9). AKAP149 has been shown to enhance PP1 phosphatase activity toward B-type lamins upon re-formation of NE at mitosis exit, thus promoting lamin dephosphorylation and polymerization (8, 9). AKAP149 has also been shown to bind PKCR in immunoprecipitation experiments from NE fractions (8, 10) and phosphodiesterase PDE4A (11). In addition, AKAP121 binds protein tyrosine phosphatase D1, a classical nonre- ceptor protein tyrosine phosphatase known to bind and activate the tyrosine kinase Src (12), and binds Src itself (13). These findings suggest that AKAP121 focuses PKA and Src signaling to mitochondria. AKAP121/AKAP149 has been shown in two different instances to bind RNA. AKAP121 binds in vitro to the 3′ untranslated region (UTR) of two transcripts for mitochon- drial proteins (14). AKAP121/AKAP149 also binds to the mRNA 3′ UTR of lipoprotein lipase, an extracellular enzyme hydrolyzing the triglyceride core of circulating chylomicrons (15, 16). This interaction is critical for the decrease in the extent of LPL mRNA translation following PKA stimulation in white adipose tissue (15, 16). In both cases, binding of AKAP121 to RNA is mediated by a single COOH-terminally situated K homology (KH) domain, which is a known RNA- binding motif (17, 18). KH domains have also been implicated in the oligomerization of proteins such as fragile X mental retardation protein (FMRP), the expression of which is altered in fragile X syndrome (19). In addition to a KH domain, AKAP121/AKAP149 contains a single COOH-terminal Tudor domain, which is also found in many RNA-interacting proteins (20). Tudor domains may also be involved in DNA binding (21) and protein-protein interac- tion through binding to arginine-glycine (RG) motifs (22, 23) and to methylated lysine (K4 or K79) in core histone † This work was supported by the Fund for Scientific Research- Flanders (Grant G.0290.05) (M. Beullens), the Research Council of Norway, and the Norwegian Cancer Society (P.C.). * To whom correspondence should be addressed. Telephone: 472- 2851066. Fax: 4722851058. E-mail: philc@medisin.uio.no. ‡ University of Oslo. § Catholic University of Leuven. 1 Abbreviations: AKAP, A-kinase anchoring protein; BSA, bovine serum albumin; EGFP, enhanced green fluorescent protein; ER, endoplasmic reticulum; NE, nuclear envelope; PKA, protein kinase A; PKC, protein kinase C; PP1, protein phosphatase 1; RFP, red fluorescent protein; UTR, untranslated region; KH, K homology; UBF, upstream binding factor. 14980 Biochemistry 2006, 45, 14980-14989 10.1021/bi061418y CCC: $33.50 © 2006 American Chemical Society Published on Web 11/22/2006