The Cardiac Ankyrin Repeat Protein (ANKRD1) Is Associated With Proteins Of The Cytoskeleton

Abstract

The Cardiac Ankyrin Repeat Protein (CARP, ANKRD1) is upregulated in cardiomyocytes following mechanical stress and is therefore related to mechano-transduction in muscle. CARP is also expressed in endothelial cells, where it seems to play a role in angiogenesis. As CARP contains four ankyrin repeats known to constitute protein interaction domains, we studied CARP-dependent signaling via the identification of its binding partners. For this aim we developed a double-tagged CARP-fusion protein (CARP-2T), which enabled tandem affinity purification (TAP) of protein complexes under non-denaturing conditions and also an intracellular localization using immunofluorescence microscopy. Our new TAP tag consisted of a C-terminal FLAG-tag and an internal StrepII-tag separated by two protease cleavage sites. CARP-2T was expressed either transiently using liposomal transfection or stably after lentiviral transduction in HEK293 and bEND2 mouse endothelioma cells. Confocal microscopy showed that CARP-2T was mainly localized in the cytoplasm and to a less extent in the nucleus. Gel filtration analysis of the protein extracts proved CARP-2T (MW: 44kDa) to be present in high molecular protein complexes (MW: 150–700 kDa). The CARP-2T-complexes were purified and isolated by TAP using triple FLAG peptide and biotin for elution of the complexes. Components of the purified complexes were analyzed by tandem mass spectrometry (ESI-MS/MS). Besides CARP-2T, α- and β-tubulin as well as cytoskeletal actin (β- or γ-actin) were identified. Our results show that CARP associates with cytoskeletal proteins in non-muscle cells suggesting that CARP might modulate cell motility and integrity, possibly via β/γ-actin-mediated pathways.