MgcRacGAP is involved in the control of growth and differentiation of hematopoietic cells by Toshiyuki Kawashima, Koichi Hirose, Takaya Satoh, Azusa Kaneko, Yasuo Ikeda, Yoshito Kaziro, Tetsuya Nosaka, and Toshio Kitamura Blood Volume 96(6):2116-2124 September 15, 2000 ©2000 by American Society of Hematology

An antisense cDNA for mMgcRacGAP inhibits IL-6–induced macrophage differentiation of M1 cells.M1 cells were transduced with the antisense mMgcRacGAP (asMgcRacGAP) using a bicistronic retrovirus vector pMX-asMgcRacGAP-IRES-EGFP (M1/pMX-asMgcRacGAP-IRES-EGFP). An antisense cDNA for mMgcRacGAP inhibits IL-6–induced macrophage differentiation of M1 cells.M1 cells were transduced with the antisense mMgcRacGAP (asMgcRacGAP) using a bicistronic retrovirus vector pMX-asMgcRacGAP-IRES-EGFP (M1/pMX-asMgcRacGAP-IRES-EGFP). As a control, M1 cells were also transduced with the blank vector pMX-IRES-EGFP (M1/pMX-IRES-EGFP). (A) Differentiation of parental M1, M1/pMX-IRES-EGFP, and M1/pMX-asMgcRacGAP-IRES-EGFP cells was evaluated by cell profiles in flow cytometry in the absence (i) or the presence (iii) of murine IL-6. The x-axis indicates forward scatter. The y-axis indicates side scatter. Undifferentiated and differentiated M1 cells were dotted in region R1 and region R2, respectively. GFP expression in parental M1, M1/pMX-IRES-EGFP, and M1/pMX-asMgcRacGAP-IRES-EGFP cells was also evaluated in flow cytometry to confirm the efficiency of expression of EGFP by bicistronic vector pMX-IRES-EGFP (ii). The x-axis indicates fluorescence intensity as a log scale ranging from 100 to 104. The y-axis indicates the number of cells. (B) Morphologic analysis of IL-6–induced differentiation in M1/pMX-IRES-EGFP and M1/pMX-asMgcRacGAP-IRES-EGFP cells. M1/pMX-IRES-EGFP and M1/pMX-asMgcRacGAP-IRES-EGFP cells were cultured in the presence of 5-ng/mL mIL-6 for 4 days, and cells were centrifuged onto glass slides and stained with May-Grunwald-Giemsa stain. Photographs were taken at 400 × magnification. The morphology of parental M1 cells is also shown as a control. Toshiyuki Kawashima et al. Blood 2000;96:2116-2124 ©2000 by American Society of Hematology

Comparison of the full-length form of hMgcRacGAP and mMgcRacGAP Comparison of the full-length form of hMgcRacGAP and mMgcRacGAP.(A) The amino acid sequences of the full-length form of hMgcRacGAP (upper line) and mMgcRacGAP (lower line) were deduced from the open reading frames detected in cDNAs. Comparison of the full-length form of hMgcRacGAP and mMgcRacGAP.(A) The amino acid sequences of the full-length form of hMgcRacGAP (upper line) and mMgcRacGAP (lower line) were deduced from the open reading frames detected in cDNAs. Amino acid sequences that are related to the 3 consensus active site boxes of RhoGAPs are indicated by brackets. Boxed sequences in a dotted line indicate the cysteine-rich regions in which cysteine and histidine residues are conserved in similar regions of chimaerins and protein kinase C. Amino acid sequences that are related to a myosin-like motif are boxed in a straight line. (B) Alignment of the myosin-like domains of the full-length form of hMgcRacGAP, mMgcRacGAP, and tropomyosins (TPM). Homologous residues in myosin-like motif are boxed. Toshiyuki Kawashima et al. Blood 2000;96:2116-2124 ©2000 by American Society of Hematology

Tissue distribution of hMgcRacGAP mRNA Tissue distribution of hMgcRacGAP mRNA.Human multiple-tissue RNA blot (Clontech, Palo Alto, CA) containing poly(A)+ RNA from the indicated tissues was probed with the full-length hMgcRacGAP cDNA probe. Tissue distribution of hMgcRacGAP mRNA.Human multiple-tissue RNA blot (Clontech, Palo Alto, CA) containing poly(A)+ RNA from the indicated tissues was probed with the full-length hMgcRacGAP cDNA probe. A GAPDH cDNA was used as a control probe. Toshiyuki Kawashima et al. Blood 2000;96:2116-2124 ©2000 by American Society of Hematology

Enforced expression of hMgcRacGAP inhibits proliferation and induces macrophage differentiation of human myeloid leukemia HL-60 cells.(A) Proliferation of HL-60 cells overexpressing hMgcRacGAP. Enforced expression of hMgcRacGAP inhibits proliferation and induces macrophage differentiation of human myeloid leukemia HL-60 cells.(A) Proliferation of HL-60 cells overexpressing hMgcRacGAP. The Flag-tagged hMgcRacGAP cDNAs were transduced into HL-60 cells using retrovirus vector pMX-IRES-EGFP. GFP+ HL-60 cells infected with these viruses were sorted on FACS 2 days after virus infection (HL-60/pMX-MgcRacGAP-IRES-EGFP cells). GFP+ HL-60 cells transduced with a blank pMX-IRES-EGFP vector were similarly sorted and used as a negative control (HL-60/pMX-IRES-EGFP cells). Both transfectants were counted at the indicated time points after the sorting. The results shown are the averages ± SD of triplicate cultures. (B) Enforced expression of the full-length form of hMgcRacGAP induces macrophage differentiation of HL-60 cells. Differentiation of parental HL-60, HL-60/pMX-IRES-EGFP, and HL-60/pMX-MgcRacGAP-IRES-EGFP cells was evaluated using flow cytometric analysis. The x-axis indicates forward scatter. The y-axis indicates side scatter. (C) CD14 expression of HL-60/pMX-IRES-EGFP cells and HL-60/pMX-MgcRacGAP-IRES-EGFP cells. The cells were stained with PE-conjugated mouse antihuman CD14 antibody or PE-conjugated isotype-matched control mouse IgG2a. (D) Morphologic changes in HL-60 cells overexpressing hMgcRacGAP. May-Grunwald-Giemsa staining profiles of HL-60/pMX-IRES-EGFP and HL-60/pMX-MgcRacGAP-IRES-EGFP cells are shown. HL-60 cells treated with TPA (16 nmol/L) for 2 days are also shown as a positive control. Cells were centrifuged onto glass slides and stained with May-Grunwald-Giemsa stain. Photographs were taken at 400 × magnification. Toshiyuki Kawashima et al. Blood 2000;96:2116-2124 ©2000 by American Society of Hematology

Stable and ectopic expression of the MgcRacGAP transgene in HL-60 and M1 transfectants.(A) Northern blot analysis of mRNA from parental HL-60 cells, HL-60/pMX-IRES-EGFP cells, and HL-60/pMX-MgcRacGAP-IRES-EGFP cells (lanes 1, 2, and 3, respectively). Stable and ectopic expression of the MgcRacGAP transgene in HL-60 and M1 transfectants.(A) Northern blot analysis of mRNA from parental HL-60 cells, HL-60/pMX-IRES-EGFP cells, and HL-60/pMX-MgcRacGAP-IRES-EGFP cells (lanes 1, 2, and 3, respectively). (B) Western blot analysis of cell lysates from control M1 cells (lanes 1,3) and M1 cells with the introduced MgcRacGAP transgene (lanes 2,4) (3 × 107per lane). Toshiyuki Kawashima et al. Blood 2000;96:2116-2124 ©2000 by American Society of Hematology

Down-regulation of the endogenous MgcRacGAP expression along with differentiation of HL-60 cells and M1 cells into macrophages.Poly(A)+ RNA was harvested from HL-60 (A) or M1 (B) cells before and after TPA (A) or IL-6 (B) stimulation, respectively, at the i... Down-regulation of the endogenous MgcRacGAP expression along with differentiation of HL-60 cells and M1 cells into macrophages.Poly(A)+ RNA was harvested from HL-60 (A) or M1 (B) cells before and after TPA (A) or IL-6 (B) stimulation, respectively, at the indicated time points. Poly(A)+ RNA (2 μg) from each condition was subjected to the Northern blot analysis, hybridizing with a human (A) or a mouse (B) MgcRacGAP cDNA probe. A GAPDH cDNA was used as a control probe. Toshiyuki Kawashima et al. Blood 2000;96:2116-2124 ©2000 by American Society of Hematology

A conserved arginine residue in the first homology box of GAP domains in all members of the Rho-GAP family and also GTPase-stimulating activity of the wild-type hMgcRacGAP and the mutant R385A*MgcRacGAP.(A) Homology between members of RhoGAP family is shown. A conserved arginine residue in the first homology box of GAP domains in all members of the Rho-GAP family and also GTPase-stimulating activity of the wild-type hMgcRacGAP and the mutant R385A*MgcRacGAP.(A) Homology between members of RhoGAP family is shown. Sequence alignments were performed on the original GenBank entries for the indicated proteins using the Clustal algorithm of the DNA Star program. ABR indicates Bcr-related gene product. (B) The GTP-hydrolyzing activity of several recombinant GTPases was measured in the presence or absence of recombinant GAP domain of the wild-type hMgcRacGAP or the mutant R385A*MgcRacGAP. The amount of [γ-32P] GTP remaining bound to GTPases after 3 minutes was determined by a filter-binding assay and expressed as a percentage of the initial amount of GTP bound to each protein. The results shown are the averages ± SD of 3 independent experiments. Toshiyuki Kawashima et al. Blood 2000;96:2116-2124 ©2000 by American Society of Hematology

Structures of the mutants of hMgcRacGAP Structures of the mutants of hMgcRacGAP.Structures of the wild-type MgcRacGAP, R385A*MgcRacGAP (R385A*), and 2 deletion mutants lacking the myosin-like domain (ΔMyo) and the cysteine-rich domain (ΔCys) are shown together with the ability to induce growth su... Structures of the mutants of hMgcRacGAP.Structures of the wild-type MgcRacGAP, R385A*MgcRacGAP (R385A*), and 2 deletion mutants lacking the myosin-like domain (ΔMyo) and the cysteine-rich domain (ΔCys) are shown together with the ability to induce growth suppression and differentiation of HL-60 cells. Toshiyuki Kawashima et al. Blood 2000;96:2116-2124 ©2000 by American Society of Hematology

Functional analysis of the mutants of hMgcRacGAP Functional analysis of the mutants of hMgcRacGAP.(A) Suppression of growth of HL-60 cells overexpressing the wild-type or mutated hMgcRacGAPs. Functional analysis of the mutants of hMgcRacGAP.(A) Suppression of growth of HL-60 cells overexpressing the wild-type or mutated hMgcRacGAPs. Each of the wild-type, the mutant R385A*, and 2 deletion mutants was transduced into HL-60 cells using the retrovirus vector pMX-IRES-EGFP. GFP+ HL-60 cells transduced with these viruses were sorted on FACS 2 days after virus infection. GFP+ HL-60 cells, transduced with a blank pMX-IRES-EGFP vector, were sorted as a negative control. The cell number of each transfectant was plotted against time. The results shown are the averages ± SD of triplicate cultures. (B) Expression of the Flag-tagged wild-type and mutant hMgcRacGAPs in HL-60 transfectants. Cell lysates from control HL-60 cells (lane 5) and HL-60 transfectants expressing the wild-type (lane 1), ΔCys (lane 2), ΔMyo (lane 2), or R385A* (lane 4) MgcRacGAP (3 × 107 per lane) were examined by Western blotting using the anti-Flag M2 monoclonal antibody. (C) Differentiation of HL-60 cells overexpressing the wild-type or mutated hMgcRacGAPs. Morphologic differentiation of control HL-60 cells and HL-60 cells transduced with the wild-type, R385A*, or deletion mutants of hMgcRacGAP is shown. Cells were centrifuged onto glass slides and stained with May-Grunwald-Giemsa stain. Photographs were taken at 400  × magnification. Toshiyuki Kawashima et al. Blood 2000;96:2116-2124 ©2000 by American Society of Hematology