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. 2002 Sep 2;196(5):667–678. doi: 10.1084/jem.20020519

Figure 3.

Figure 3.

Figure 3.

Figure 3.

Figure 3.

Figure 3.

Figure 3.

Figure 3.

Defect of SDF-1–induced Lyn/PI3-kinase pathway in chronic myeloid leukemia. (A and B) Representative time course of Lyn and p85-dependent PI3-kinase phosphorylation in SDF-1–stimulated BCR/ABL-positive leukemic blasts (CML). Lyn tyrosine kinase activity (A) and PI3-kinase activity (B) are not increased after SDF-1 stimulation. This is in contrast to time courses observed in normal CD34+ cells and BCR/ABL-negative HL-60 cells, which are presented in Fig. 1, 2, and 3, E and F. Results are representative of four experiments. (C) Expression of exogenous BCR/ABL in HL-60 cells. Infected cells were analyzed for protein levels of exogenous BCR/ABL by Western blot and for BCR/ABL tyrosine kinase activity by immune complex in vitro kinase assays, as described in Materials and Methods. P56/53-kD tyrosine phosphorylated protein is detectable in BCR/ABL precipitates from BCR/ABL-infected Mig210 cells, but not from cells infected with control vector (MigR1). (D) Expression of endogenous Lyn in BCR/ABL-infected HL-60 cells. P210-kD tyrosine phosphorylated protein is coimmunoprecipitated with anti-Lyn antibody in Mig210, but not MigR1 cells. This protein comigrates with endogenous BCR/ABL in K562 cells (positive control). The phosphorylated 210-kD protein was identified as BCR/ABL by secondary p210 BCR/ABL immunoprecipitations on primary Lyn precipitates. The secondary IPs were performed after the disruption of protein complexes by disruption buffer (see Material and Methods). The position of BCR/ABL is indicated by the arrow. The relatively weak signal from secondary IPs is explained by the loss of precipitated proteins during disruption and reprecipitation. Results are representative of two experiments (D-a). (E and F) Comparison of SDF-1–stimulated Lyn and PI3-kinase activities and protein levels in BCR/ABL-negative HL-60 cells and BCR/ABL-positive HL-60 cells. Lyn autophosphorylation is inducible by SDF-1 in MigR1, but not in Mig210 cells (E). P85 subunit-dependent PI3-kinase activity is stimulated by SDF-1 in control cells, but not in Mig210 cells (F).