Characterization of Cyclase Associated Protein homologue, CAP2

Cyclase associated protein (CAP) is a bifunctional protein having an N-terminal adenylyl cyclase binding domain and a C-terminal actin-binding domain. In-between these two domains there is a proline rich SH3 domain presumably involved in the localization of CAP. CAP is expressed widely and has been found in yeast, Dictyostelium, Drosophila, rat, mouse and humans. Two homologues of CAP have been identified in higher eukaryotes, CAP1 and CAP2. Although CAP proteins have been studied for more than a decade and are present in all organisms, many questions remain about the mechanisms of CAP function. The roles of mammalian CAP2 proteins have not been investigated extensively. In our study we showed that CAP2 has 2 transcripts of 3 and 3.5 kb unlike the ubiquitously expressed CAP1, homologue of CAP2 in mice. In contrast to CAP1, CAP2 is expressed in a tissue specific manner. We found that CAP2 is present in relatively moderate levels in brain, heart and skeletal muscle and lower levels in skin. Furthermore, we investigated the expression pattern in more detail in these tissues and found that CAP2 is present in all parts of the adult brain but is detected only in the cerebellum and brain stem in the newborn mice and expressed only in the cerebellar region of the brain in embryos of E16. In brain CAP2 was expressed uniformly in the cortex and also found in the Purkinje cells and in the myelinated axons of the white matter. In skeletal muscle we observed a striated pattern.CAP2 was strongly expressed in the heart where it was present in cardiomyocytes, endothelial cells and the capillary wall of the blood vessels. We observed a striated pattern, which correlated with an association of CAP2 with the M-bands. In the skin CAP2 was expressed in the basal epidermal layers and in the hair follicle region and was found in the sebaceous glands. CAP2 was also expressed in the keratinocytes in skin. Immunofluorescence studies with PAM212, a mouse keratinocyte cell line, and primary mouse keratinocytes revealed that CAP2 was surprisingly localized in the nucleus. Cell fractionation experiments performed with PAM212 cells confirmed the presence of CAP2 in the nucleus. In the case of primary mouse keratinocytes CAP2 was also localised to the nucleus apart from its presence in the cytosol. In contrast in human keratinocytes CAP2 localised to the cell cortex and present in patches. Immunofluorescence studies and experiments with drugs affecting the cytoskeleton revealed that CAP2 was partially associated with F-actin and the microtubules. The nuclear localization was dependent on the microtubular cytoskeletal network and independent of the actin cytoskeleton. Overexpression studies in HEK293 cells using EGFP and Myc fusion proteins showed that CAP2 is cytosolic and was associated with lamellipodia. Unlike our overexpression studies, the immunofluorescence studies with HL-1, a cardiomyocyte cell line, and with primary cardiomyocytes of embryonic stages E17 and E19 showed that CAP2 was localized to the nucleus as in the PAM212 cells. In cardiomyocytes, CAP2 colocalised partially with Troponin I. In order to investigate the interaction with its homologue CAP1, cotransfection of GFP-CAP1, Myc-CAP2 into HEK293 cells followed by immunoprecipitation experiments was performed. The results revealed that CAP2 interacts with CAP1. While investigating the interaction of CAP2 with other proteins, we found that ACF7, an F-actin crosslinking protein interacts with CAP2 in immunoprecipitation studies carried out on HEK293 cells, coexpressing GFP-C-ACF7 and Myc-CAP2. We further found that the domain of CAP2 interacts with myosin light chain alkali (MLC3nm) in skin lysate. In a wound healing assay CAP2 colocalised with F-actin at certain places suggesting CAP2 plays a role in wound healing. Using a conventional knock out strategy we are currently generating a mice knock out strain of CAP2 in order to learn more about the functions of this protein.