Functional Characterization Of A Novel Oncogene-like Small GTPase, RabJ, Derived From Human Dendritic Cells

In a large-scale sequencing of the cDNA libraries derived from human dendritic cells (DC), we have identified many novel full-length genes, which potentially play crucial roles in the regulation of immune responses and cellular processes. In the present study, we aimed to further investigate the biological functions of the small Rab-like J domain-containing protein RabJ. Full-length RabJ cDNA is derived from the DC cDNA library, potentially containing an open reading frame of 822bp and thus encoding a 173-residue protein with a calculated molecular weight of 30.5 kDa. RabJ mRNA is preferentially expressed in human testis and widely detected in tumor cell lines. Previous studies of ours have preliminarily shown that RabJ is a nuclear protein and can interact with heat shock cognate protein 70 (HSC70). However, the defined biological functions of RabJ have not been assigned. After plentiful experiments, We confirm and suggest that RabJ is possibly involved in the regulation of cell proliferation, control of cell cycle and tumorigenesis.We went first to confirm the expression pattern of RabJ in cell lines and tissues by RT-PCR and immunohistochemistry. RabJ is widely distributed in various human and mouse hematological leukemia cell lines or solid tumor cell lines, including HeLa, Jurkat, Molt-4, NB4, THP-1, U937, MCF-7, PC-3, A20,EL-4, P815, B16, and 3LL, but not in normal cell lines TC-1 and NIH3T3. In murine tissues, expression of RabJ is detected in testis, ovary, kidney, brain, stomach, colon and skeletal muscle, but not in thymus, liver, heart and lung. In adult murine testis, RabJ is distributed in primary spermatocytes and early spermatids, but not in Sertoli, Leydig or mature sperms, which resembles that of cylin D and cylin E. This expression pattern of RabJ indicates that RabJ is possibly involved in regulation of cell proliferation or cell differentiation.Then we examined the subcellular localization of RabJ. A further examination of the N-terminal 18 residues of RabJ protein has facilitated the identification of a segment rich of basic residues, similar to the nuclear localization signal (NLS) Lys-Arg/Lys-X-Arg/Lys. Therefore, RabJ can be divided into three subdomains, that is, the N-terminal NLS (1-18), the internal Rab domain (19-208) and the C-terminal J domain (209-273). We constructed eukaryotic vectors for expression of full-length RabJ ( 1-273) and mutated RabJ, including the 1-208, 19-208, 209-273, and 1 9-273 segments. After transient transfection of these vectors in NIH3T3 cells, and under the assistance of immunofluorescence confocal microscope, we show that RabJ is a nuclear protein, which is abolished when the NLS and J domain are simultaneously deleted, indicating that the nuclear localization of RabJ is dependent on both the NLS and J domain. In wild type HeLa and MCF-7 cells, both of which are RabJ-positive, RabJ is also found in the nucleus after subcellular fractionation or under the assistance of immunofluorescence confocal microscope and immunoelectron microscope. Therefore, RabJ is a nuclear protein, and the N-terminal segment of RabJ presumably serves as NLS.To investigate the possible roles of RabJ in regulation of cell proliferation, we established NIH3T3 cells stably expressing full-length RabJ and RabJ mutants, including hRabJ (1-208), hRabJ (209-273), hRabJ (19-208), and hRabJ (19-273), after selection in 600-800 /jg/ml neomycin and subsequent limited dilution. Based on the [3H]-TdR incorporation assay, we examined the proliferation of NIH3T3 cell lines under low serum conditions (0.5% fetal calf serum, PCS). Full-length RabJ overexpression increases proliferation of NIH3T3 cells 20 to 30 folds to that of the wild type and mock-control cells. RabJ can also promote the cell cycle transition into G2/M and S phases, but Rab domain alone can not. When plated on soft agar, NIH3T3 cells stably expressing full-length RabJ as well as the RabJ (1 -208) and RabJ (209-273) can form focus and colonies, indicating that NIH3T3 cells are transformed, which is dependent on the NLS+R...