Figure 7
Figure 7. Liar regulates Epo-induced signaling. (A) Altered intracellular signaling caused by overexpression of Liar and LiarΔNLS. Lysates of J2E cells expressing Liar (MSCV-Liar) or LiarΔNLS (MSCV-LiarΔNLS) were analyzed after Epo stimulation by Western blot analysis (i) with anti-phosphotyroine, anti–phospho-STAT5, anti-STAT5, anti-Lyn, anti–phospho-Erk2, and anti-Erk2 (loading control) antibodies. Phosphotyrosine proteins reduced in Liar overexpressing cells are indicated by open arrowheads, compared with an unchanged protein indicated by a closed arrowhead. Quantitation of phospho-STAT5 (ii) and phospho-Erk2 (iii) levels relative to total STAT5, Erk2. (B) Overexpression of Liar or LiarΔNLS alters Epo-induced Lyn subcellular localization. J2E cells described in panel A were analyzed by confocal microscopy and immunofluorescence staining using anti-myc antibodies over 60 minutes of Epo-stimulation (i). A representative cell from each time point is illustrated, depicting the nuclear localization of Lyn and Liar in control (MSCV) cells, nuclear colocalizing spots in MSCV-Liar cells, and membrane colocalization in MSCV-LiarΔNLS cells. Changes in Epo-induced Liar/Lyn colocalization in nuclear spots in the Liar-expressing cells were enumerated (ii), as was the relative membrane colocalization of Lyn and LiarΔNLS (iii). (C) Model of potential Liar interactions and its effects upon shuttling/signaling. (i) Endogenous Liar dynamically interacts with Lyn and HS1 and potentially other molecules (Vav1, Hip55, LASP1, ESE2L), allowing normal nuclear:cytoplasmic shuttling and signaling to HS1 pathways (eg, Arp2/3/F-actin) and facilitating Akt activation. (ii) Overexpression of wild-type Liar enhances the nuclear accumulation of Liar/Liar complexes and reduces global Epo-induced signaling events and inhibiting Akt activation. (iii) Overexpression of NLS-mutated Liar (LiarΔNLS) enhances the cytoplasmic accumulation of Liar/Liar complexes altering Epo-induced erythroid signaling including inhibiting Akt activation. The NLS (red N), NES (blue N), and ankyrin repeats (Ank) are depicted.

Liar regulates Epo-induced signaling. (A) Altered intracellular signaling caused by overexpression of Liar and LiarΔNLS. Lysates of J2E cells expressing Liar (MSCV-Liar) or LiarΔNLS (MSCV-LiarΔNLS) were analyzed after Epo stimulation by Western blot analysis (i) with anti-phosphotyroine, anti–phospho-STAT5, anti-STAT5, anti-Lyn, anti–phospho-Erk2, and anti-Erk2 (loading control) antibodies. Phosphotyrosine proteins reduced in Liar overexpressing cells are indicated by open arrowheads, compared with an unchanged protein indicated by a closed arrowhead. Quantitation of phospho-STAT5 (ii) and phospho-Erk2 (iii) levels relative to total STAT5, Erk2. (B) Overexpression of Liar or LiarΔNLS alters Epo-induced Lyn subcellular localization. J2E cells described in panel A were analyzed by confocal microscopy and immunofluorescence staining using anti-myc antibodies over 60 minutes of Epo-stimulation (i). A representative cell from each time point is illustrated, depicting the nuclear localization of Lyn and Liar in control (MSCV) cells, nuclear colocalizing spots in MSCV-Liar cells, and membrane colocalization in MSCV-LiarΔNLS cells. Changes in Epo-induced Liar/Lyn colocalization in nuclear spots in the Liar-expressing cells were enumerated (ii), as was the relative membrane colocalization of Lyn and LiarΔNLS (iii). (C) Model of potential Liar interactions and its effects upon shuttling/signaling. (i) Endogenous Liar dynamically interacts with Lyn and HS1 and potentially other molecules (Vav1, Hip55, LASP1, ESE2L), allowing normal nuclear:cytoplasmic shuttling and signaling to HS1 pathways (eg, Arp2/3/F-actin) and facilitating Akt activation. (ii) Overexpression of wild-type Liar enhances the nuclear accumulation of Liar/Liar complexes and reduces global Epo-induced signaling events and inhibiting Akt activation. (iii) Overexpression of NLS-mutated Liar (LiarΔNLS) enhances the cytoplasmic accumulation of Liar/Liar complexes altering Epo-induced erythroid signaling including inhibiting Akt activation. The NLS (red N), NES (blue N), and ankyrin repeats (Ank) are depicted.

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