Figure 5.

BTK links BCR-ABL1 to PLCγ1-mediated Ca²⁺ signals in pre–B lymphoblastic leukemia cells. Cytoplasmic Ca²⁺ levels were measured in single leukemia cells by confocal laser microscopy. Before Ca²⁺ measurement, cells were kept under control conditions or treated for 24 h with STI571 or LFM-A13 (A). Pre–B cell receptor engagement by addition of μ-heavy chain–specific antibodies (arrows) did not change cytoplasmic Ca²⁺ concentrations. Although PLCγ1^Y783 is phosphorylated in the presence—but not in the absence (+ STI571)—of BCR-ABL1 kinase activity (B, left panels), PLCγ2 was not tyrosine phosphorylated by BCR-ABL1 (not depicted). As a control for nonspecific side effects of STI571, murine B lymphoid cells carrying a doxycycline-inducible BCR-ABL1 transgene were analyzed in the presence or absence of 1 μg/ml doxycycline (DOX). These cells remain viable in the absence of BCR-ABL1 expression if the cell culture fluid is supplemented with 2 ng/ml murine recombinant IL-3. Protein extracts from these cells were subjected to Western blot using human- and mouse-reactive antibodies (B, right). Tyrosine phosphorylation of PLCγ1 was studied in BCR-ABL1 ⁺ pre–B lymphoblastic leukemia cells in the presence or absence of STI571 or LFM-A13. EIF-4E was used as a loading control in both Western blots (B, C). Expression of full-length BTK was silenced by RNA interference in a BCR-ABL1 ⁺ pre–B lymphoblastic leukemia cell line (IX in Table I). Sorted cells carrying fluorescein-labeled siRNAs (green) were permeabilized, stained for PLCγ1^pY783 (red), and analyzed by confocal laser microscopy (C, bottom). As a negative control, nontargeting siRNA duplices were used. BCR-ABL1–binding proteins were coimmunoprecipitated using an antibody against BCR. The immunoprecipitation was controlled by a Western blot using an ABL1-specific antibody (D).