Abstract

Interleukin-4 (IL4) induces proliferation, differentiation and survival of B lymphocytes. IL4 protects CLL B cells from death by apoptosis. Gene expression analysis suggest that IL4 pathways are activated in CLL cells. We have identified DOCK10/Zizimin3 as an IL4-induced gene in CLL cells, and have obtained its full length sequence after cloning 1960 bp at its 5′ terminus by RACE-PCR. The human DOCK10/ZIZ3 sequence coded for a protein with 2180 amino acids and a predicted Mr of 250K. DOCK10/ZIZ3 shared homology with the other two members of the Zizimin family, and is the largest among them: DOCK9/ZIZ1 (2069 amino acids) and DOCK11/ZIZ2 (2073 amino acids) are 52% and 50% identical, respectively, to DOCK10/ZIZ3, and 58% identical between them. DOCK10 was predominantly expressed in hematopoietic tissues, particularly in peripheral blood (PB), but also in lymph nodes, thymus and spleen. Among the PB subpopulations, DOCK10 was expressed in B and T lymphocytes and, at lower levels, in monocytes. DOCK10 was also expressed in several non-hematopoietic tissues, most significantly in brain and kidney. Its homologue DOCK9, compared to DOCK10, was predominantly expressed in placenta, and less significantly in hematopoietic tissues, particularly in B lymphocytes and monocytes. DOCK11, like DOCK10, was predominantly expressed in PB. Compared to DOCK10, DOCK11 was expressed more prominently in placenta, thyroid and PB monocytes, and less significantly in brain and lymph nodes. Therefore, each of the Zizimin family members had a specific tissue distribution. Among the three genes, only DOCK10 was induced by IL4 in CLL cells in vitro. Induction of DOCK10 by IL4 was a common event in CLL, since it was observed in 10 out of 10 cases. IL4 also induced DOCK10 expression in normal PB B lymphocytes, suggesting that DOCK10 induction by IL4 in CLL cells may be normal, rather than pathological. Western blot analysis using a polyclonal antibody raised against a peptide which mapped at the N terminus of DOCK10, detected a band of the expected size of 250K. Interestingly, IL4 did not induce DOCK10 expression in CD4 or CD8 T lymphocytes in vitro. Expression of DOCK10 was also studied in 4 B-ALL, 2 T-ALL, and 1 T-CLL. DOCK10 neither was expressed at significant levels nor induced by IL4 in vitro in these patients, except for a weak induction in a common B-ALL case, suggesting that expression of DOCK10, and its induction with IL4, may be restricted to certain stages of B cell differentiation, and/or certain B cell malignancies. DOCK10 was distributed both in cytosolic and nuclear extracts of CLL cells, and IL4 increased its expression in both compartments. K562 clones stably transfected with DOCK10 using the inducible tet-off expression system showed significantly higher levels of DOCK10 in cytoplasm than in nucleus. Immunofluoresce analysis of HA-tagged DOCK10 K562 clones showed preferent staining of the cytoplasm, and dotted structures were frequently observed. GST-pulldown assays showed that DOCK10 bound to nucleotide-free (nf) Cdc42, but not to GTP- or GDP-loaded Cdc42. In addition, DOCK10 bound to nf Rac1, albeit with less affinity than to Cdc42. DOCK10 did not bind to RhoA. These results suggest that, like DOCK9 and DOCK11, DOCK10 may act as a novel Cdc42 guanine-nucleotide exchange factor (GEF) and, in addition, as a Rac1 GEF.

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