Expression cloning of cxr-1: a sos homologue potentially regulated by diacylglycerol and calcium

We have been interested in screening cDNA libraries constructed from different cell lines using a retroviral based transformation assay in order to identify genes that are involved in Ras activation. Upon screening a T cell hybridoma cell line (T28) cDNA library we initially identified a cDNA who's overexpression in these cells caused transformation phenotypically similar to that of Ras overexpression. Sequence analysis indicated that this cDNA contained several unique structural features such as regions homologous to the CDC25/Sos family of guanine nucleotide exchange factors (GEFs), a Cl domain highly similar to the diacylglycerol-binding C1 domains of protein kinase Cs, and a pair of calcium binding motifs known as EF hands. Deletion constructs were created and it was determined that although the EF hands are not required for transformation, an active GEF domain is essential. The C1 domain is also necessary for transformation and can be mimicked by a prenylation signal or replaced by the Cl domain of PKCO to completely restore transforming activity. GFP-tagged Cl domain containing constructs localized to the plasma membrane upon PMA stimulation and to lipid droplets within the cytoplasm upon PLC stimulation. Taken together, these results suggest that the Cl domain may play a role in localizing CXR to plasma membranes where activation of Ras-type effector molecules might take place via the GEF domain. CXR is expressed extensively in brain, thymus, spleen, and bone marrow, which points to a possible role of CXR in neuronal and lymphoid signaling. Both human and C. elegans homologues of CXR-1 have been identified and show a high degree of sequence conservation. An additional family member, designated CXR-2, has been identified in both the human and murine systems and its pattern of expression is similar to that of CXR-1.