The cardiac ankyrin repeat protein (CARP) is upregulated in several tissues in stress situations, e.g. in cardiomyocytes during hypertrophy, in smooth muscle cells after vessel injury and in migrating endothelial cells in wound healing models. As CARP contains four ankyrin repeats known to mediate protein interactions, 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. After their purification from HEK293 cell lysates the CARP-2T complex components were analyzed by tandem mass spectrometry revealing cytoplasmic ß-actin to be a new binding partner of CARP. CARP overexpressing HEK293 cells showed a co-localization of CARP with ß-actin.

Four hours after cell seeding during cell adhesion and spreading CARP was increasingly detected in the cytoplasm, whereas eight hours after cell seeding CARP was distributed equally between the nucleus and the cytoplasm. Synchronized HEK293 cells showed an intracellular CARP redistribution with a preferred cytoplasmic localization during mitosis. Confocal microscopy showed a co-localization of CARP with ß-actin at the lamellipodiae of migrating human vein endothelial cells (HUVEC). CARP overexpression did not significantly influence endothelial cell migration or tube formation behaviour. Our results show a new interaction of CARP with cytoplasmic ß-actin at the lamellipodiae of migrating endothelial cells where CARP might be involved in ß-actin filament stability and polymerization. Moreover we hypothesize that the sofar observed upregulation of CARP in several tissues might be directly linked to stress-dependent actin reorganization processes.