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. A double-tagged CARP-fusion protein (CARP-2T) enabled tandem affinity purification (TAP) of protein complexes under non-denaturing conditions as well as intracellular localization. CARP-2T was expressed either transiently using liposomal transfection or stably after lentiviral transduction in HEK293 and bEND2 mouse endothelioma cells. Gel filtration analysis of the protein extracts proved CARP-2T (MW: 44kDa) to be present in high molecular protein complexes (MW: 150-700 kDa). Components of the purified complexes were analyzed by tandem mass spectrometry (ESI-MS/MS). Besides CARP-2T, a- and b- tubulin as well as cytoskeletal actin (b- or [gamma]-actin) were identified. Confocal microscopy showed that CARP-2T has a dynamic localization depending on the cell cycle.

Our results show that CARP associates with cytoskeletal proteins in non-muscle cells suggesting that it might modulate cell motility and integrity in a cell-cyle dependent manner.