Protein Kinase A RII-like (R2D2) Proteins Exhibit Differential Localization and AKAP Interaction Amy E. Hanlon Newell, 1y Sarah E. Fiedler, 1y Jenny M. Ruan, 1 Jieyan Pan, 2 P. Jeremy Wang, 2 Jutta Deininger, 1 Christopher L Corless, 1 and Daniel W. Carr 1 * 1 VA Medical Center and Department of Medicine, Oregon Health & Science University, Portland, Oregon 2 Department of Animal Biology, School of Veterinary Medicine, University of Pennsylvania, Philadelphia, Pennsylvania A-kinase anchoring proteins (AKAPs) bind to protein kinase A (PKA) via an am- phipathic helix domain that interacts with a dimerization/docking domain on the regulatory (R) subunit of PKA. Four other mammalian proteins (ROPN1, ASP, SP17, and CABYR) also contain a highly conserved RII dimerization/docking (R2D2) domain, suggesting all four proteins may interact with all AKAPs in a manner similar to RII. All four of these proteins were originally detected in the flagellum of mammalian sperm. In this report, we demonstrate that all four R2D2 proteins are expressed in a wide variety of tissues and three of the proteins SP17, CABYR, and ASP are located in motile cilia of human bronchus and fallopian tubes. In addition, we detect SP17 in primary cilia. We also provide evidence that ROPN1 and ASP bind to a variety of AKAPs and this interaction can be disrupted with anchoring inhibitor peptides. The interaction of SP17 and CABYR with AKAPs appears to be much more limited. None of the R2D2 proteins appears to bind cAMP, a fundamental characteristic of the regulatory subunits of PKA. These observations suggest that R2D2 proteins utilize docking interactions with AKAPs to accomplish their function of regulating cilia and flagella. Based on location, affinity for AKAPs and lack of affinity for cAMP, it appears that each R2D2 protein has a unique role in this process. Cell Motil. Cytoskeleton 65: 539– 552, 2008. ' 2008 Wiley-Liss, Inc. Key words: PKA; cilia; SP17; CABYR; ROPN1; ROPN1L INTRODUCTION Most proteins that participate in cellular signaling networks contain modular protein-interaction domains. Many kinases and phosphatases utilize docking interac- tions where a short peptide motif in target partners is rec- ognized by a groove on the catalytic domain that is sepa- rate from the active site [Remenyi et al., 2006]. Docking is particularly prevalent in serine/threonine kinases and phosphatases, and is a versatile organization tool for building complex signaling networks; it confers a high degree of specificity and, in some cases, allosteric regu- lation [Remenyi et al., 2006]. The cAMP-dependent protein kinase (PKA) is a prime example of a signaling molecule that uses docking This article contains supplementary material available via the Internet at http://www.interscience.wiley.com/jpages/0886-1544/suppmat y These authors contributed equally to this work. Contract grant sponsor: Lalor Foundation Post-Doctoral Fellowship; Grant sponsor: National Institute of Child Health and Human Devel- opment; Grant number: R01HD036408. The content of this work is solely the responsibility of the authors and does not necessarily represent the official views of the National Insti- tute of Child Health and Human Development or NIH. *Correspondence to: Daniel W. Carr, Veterans Affairs Medical Center, Mail Code R&D8, 3710 SW US Veterans Hospital Road, Portland, Oregon 97239. E-mail: carrd@ohsu.edu Received 28 November 2007; Accepted 12 March 2008 Published online 17 April 2008 in Wiley InterScience (www. interscience.wiley.com). DOI: 10.1002/cm.20279 ' 2008 Wiley-Liss, Inc. Cell Motility and the Cytoskeleton 65: 539–552 (2008)