Figure 6. NPM mutant A acts as a... | American Society of Hematology

Figure 6.

$Figure 6. NPM mutant A acts as a dominant-negative on wild-type NPM. (A) Cotransfection of H1299 cells with pEGFP-C1-NPMmA and HA-NPM wild-type. About 30% of cells were transfected and in about 70% of transfected cells the NPM mutant causes partial recruitment of the wild-type NPM from nucleoli to the nucleoplasm and the cytoplasm. (B) Both FH-NPMwt and FH-NPM mutant A interact with the endogenous NPM in H1299 cells. FH-NPM wt and FH-NPM mutant A plasmids were stably transfected into H1299 cells. In the left panel, 5% of the whole-cell lysates from stably transfected FH-NPMwt, FH-NPM mutant A, or control H1299 cells were subjected to Western blot with either α-NPM or α-HA. In the right panel, the rest 95% of whole cell lysates were immunoprecipitated with α-flag (M2), followed by Western blot with either α-NPM or α-HA. (C) Endogenous wild-type NPM protein is dislocated in the cytoplasm of NPMc+ AML primary samples. In the top panel, nuclear and cytoplasmic extracts from either NPMc+ or NPMc– AML primary patient cells were subjected to Western blot analysis with either the anti-NPM mouse monoclonal antibody (clone FC-61991) from Invitrogen (anti-NPM wt) or the Sil-C antibody (anti-NPMm). A strong signal for NPMwt was evident in both the nuclear and cytoplasmic fraction of AML cells carrying NPM mutation but only in the nuclear fraction of NPMc–AML cells. The lower panel shows immunohistochemical study of 2 different AML samples with the anti-NPMwt antibody. The NPMc+ AML sample expresses the wild-type NPM both in the nucleus and cytoplasm (arrow). In contrast, the NPMc–AML sample shows the expected nucleus-restricted expression of wild-type NPM (arrow). Images were collected using an Olympus BX61 microscope and a UPlanFl 100 ×/1.3 NA objective. (D) Hypothetical mechanism of altered nucleo-cytoplasmic traffic of NPM mutant A and wild-type NPM. Red circles indicate tryptophan residues; black circles, mutated tryptophans; magenta boxes, NES motif; blue boxes, mutated NPM protein; and green boxes, wild-type NPM protein. To simplify, the scheme shows only the binding of Crm1 to the mutant NES but not to the physiologic one, nor to the full ternary complex (Crm1-NES-ranGTP). NLS indicates nuclear localization signal.$

NPM mutant A acts as a dominant-negative on wild-type NPM. (A) Cotransfection of H1299 cells with pEGFP-C1-NPMmA and HA-NPM wild-type. About 30% of cells were transfected and in about 70% of transfected cells the NPM mutant causes partial recruitment of the wild-type NPM from nucleoli to the nucleoplasm and the cytoplasm. (B) Both FH-NPMwt and FH-NPM mutant A interact with the endogenous NPM in H1299 cells. FH-NPM wt and FH-NPM mutant A plasmids were stably transfected into H1299 cells. In the left panel, 5% of the whole-cell lysates from stably transfected FH-NPMwt, FH-NPM mutant A, or control H1299 cells were subjected to Western blot with either α-NPM or α-HA. In the right panel, the rest 95% of whole cell lysates were immunoprecipitated with α-flag (M2), followed by Western blot with either α-NPM or α-HA. (C) Endogenous wild-type NPM protein is dislocated in the cytoplasm of NPMc⁺ AML primary samples. In the top panel, nuclear and cytoplasmic extracts from either NPMc⁺ or NPMc^– AML primary patient cells were subjected to Western blot analysis with either the anti-NPM mouse monoclonal antibody (clone FC-61991) from Invitrogen (anti-NPM wt) or the Sil-C antibody (anti-NPMm). A strong signal for NPMwt was evident in both the nuclear and cytoplasmic fraction of AML cells carrying NPM mutation but only in the nuclear fraction of NPMc–AML cells. The lower panel shows immunohistochemical study of 2 different AML samples with the anti-NPMwt antibody. The NPMc⁺ AML sample expresses the wild-type NPM both in the nucleus and cytoplasm (arrow). In contrast, the NPMc–AML sample shows the expected nucleus-restricted expression of wild-type NPM (arrow). Images were collected using an Olympus BX61 microscope and a UPlanFl 100 ×/1.3 NA objective. (D) Hypothetical mechanism of altered nucleo-cytoplasmic traffic of NPM mutant A and wild-type NPM. Red circles indicate tryptophan residues; black circles, mutated tryptophans; magenta boxes, NES motif; blue boxes, mutated NPM protein; and green boxes, wild-type NPM protein. To simplify, the scheme shows only the binding of Crm1 to the mutant NES but not to the physiologic one, nor to the full ternary complex (Crm1-NES-ranGTP). NLS indicates nuclear localization signal.

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