STAT3 is a substrate of SYK tyrosine kinase in B-lineage leukemia/lymphoma cells exposed to oxidative stress

Abstract

We provide unprecedented genetic and biochemical evidence that the antiapoptotic transcription factor STAT3 serves as a substrate for SYK tyrosine kinase both in vitro and in vivo. Induction of SYK in an ecdysone-inducible mammalian expression system results in STAT3 activation, as documented by tyrosine phosphorylation and nuclear translocation of STAT3, as well as amplified expression of several STAT3 target genes. STAT3 activation after oxidative stress (OS) is strongly diminished in DT40 chicken B-lineage lymphoma cells rendered SYK-deficient by targeted disruption of the syk gene. Introduction of a wild-type, C-terminal or N-terminal SH2 domain-mutated, but not a kinase domain-mutated, syk gene into SYK-deficient DT40 cells restores OS-induced enhancement of STAT-3 activity. Thus, SYK plays an important and indispensable role in OS-induced STAT3 activation and its catalytic SH1 domain is critical for this previously unknown regulatory function. These results provide evidence for the existence of a novel mode of cytokine-independent cross-talk that operates between SYK and STAT3 pathways and regulates apoptosis during OS. We further provide experimental evidence that SYK is capable of associating with and phosphorylating STAT3 in human B-lineage leukemia/lymphoma cells challenged with OS. In agreement with a prerequisite role of SYK in OS-induced STAT3 activation, OS does not induce tyrosine phosphorylation of STAT3 in SYK-deficient human proB leukemia cells. Notably, inhibition of SYK with a small molecule drug candidate prevents OS-induced activation of STAT3 and overcomes the resistance of human B-lineage leukemia/lymphoma cells to OS-induced apoptosis.

Fig. 1.

Activation of STAT3 in an ecdysone-inducible mammalian expression system for SYK. (A) AntiSYK WBA of SYK immune complexes from whole cell lysates of untransfected vs. transfected U373 cells before and after exposure to the ecdysone-analogue Pon-A (10 μM). (B) Antiactin WBA of actin IC from the same lysates used in (A). (C) Anti-phospho-STAT3^Y705 WBA of STAT3 immune complexes from whole cell lysates of transfected uninduced vs. induced U373 cells. Lysates from syk-transfected U373 cells were prepared before (CON) and at various time points after addition of Pon-A (10 μM) as indicated. Some samples were exposed to PV as well where indicated. (D) Anti-STAT3 WBA of the STAT3 immune complexes shown in (C). (E) Confocal image depicting the cytoplasmic localization of native STAT3 in a representative syk-transfected U373 cell before exposure to Pon-A (10 μM). (F) Confocal image showing substantial nuclear (in addition to cytoplasmic) localization of native STAT3 in a representative syk-transfected U373 cell 6-h after exposure to Pon-A (10 μM). (G) Pon-A (10 μM)-induced SYK induction leads to upregulation of STAT3-responsive genes. The heat map represents the color-coded expression value reported as fold change relative to the average expression levels in the control samples (key shows from 0.6- to 3.6-fold change, Blue to Red, respectively). The two-way dendrogram depicts the similarity of expression pattern for all probes (39 probesets for 29 genes) across the 6 treatments and the 6 treatments across the 39 probesets arranged in rows and columns respectively.

Fig. 2.

Role of SYK in oxidative stress-induced activation of STAT3 in DT40 chicken lymphoma B-cells (7, 9). EMSAs in (A) and (B) were performed with end-labeled SIE probe and nuclear extracts prepared from wild-type DT40 cells, SYK-deficient DT40 cells (SYK^-), SYK-deficient DT40 cells reconstituted with wild-type SYK (SYK^-, rSYK [WT]), catalytic kinase domain-mutant of SYK (SYK^-, rSYK [K^-], and SH2 domain mutants of SYK (SYK^-, rSYK [mSH2-C] and SYK^-, rSYK [mSH2-N]). Cells were left untreated (no PV) or treated with 400 μM PV for 15 min, 30 min, or 60 min, as indicated. The nuclear extracts were preincubated and then the labeled probe was added (A, Lanes 2–13). In unlabeled competition reactions, 100-fold excess unlabeled homologous SIE (A, Lanes 14 and 16) or nonhomologous AP-1 probe (A, Lanes 15 and 17) was added prior to the preincubation. Controls included samples containing only the SIE probe without any nuclear extract (A, Lane 1; B, Lane 7). Mobility shifts were determined by electrophoresis as described in Materials and Methods. Following electrophoresis, gels were dried and subjected to autoradiography on film. Shifted bands are indicated by arrows.

Fig. 3.

Role of SYK in oxidative stress-induced tyrosine phosphorylation of STAT3 in human B-lineage lymphoid cells. (A and B) RAMOS Burkitt’s leukemia/lymphoma cells were either left untreated or treated with the oxidative agent PV at a 400 μM concentration in the presence or absence of SYKINH-61 (1 nM, 10 nM, 50 nM, 100 nM) or 100 μM PCT. Cells were lysed using Nonidet-P40 buffer after 30 min exposure to PV, PV + SYKINH-61, or PV + PCT and lysates were immunoprecipitated with antiSYK antibodies, as indicated. The SYK immune complexes were resolved by SDS-PAGE and examined by APT (A) or antiSYK (B) Western blot analysis. (C and D) BCL-1 cells were either left untreated or treated with 100 mM H₂O₂ as the oxidative agent in the presence or absence of 100 nM SYKINH-61. Cells were lysed using Nonidet-P40 buffer after 1 min, 5 min, or 10 min exposure to H₂O₂ or H₂O₂ + SYKINH-61 and lysates were immunoprecipitated with APT antibodies. The immune complexes were resolved by SDS-PAGE and examined by APT (C) or antiSYK (D) WBA. (E and F) RAMOS cells were left untreated (CON, -H₂O₂) (Lane 4) or treated with 100 mM H₂O₂ for 10 min in the absence (Lane 5) or presence of SYK kinase inhibitors SYKINH-61 (100 nM) (Lane 6) and PCT (100 μM) (Lane 8), JAK1,2,3 inhibitor AG-490 (100 μM) (Lane 1), JAK3 kinase inhibitor JANEX-1 (100 μM), or BTK inhibitors compound 1 (CP-1)/HI-12 (100 μM) (Lane 3), and compound 2 (CP-2/HI-86) (100 μM) (Lane 7). STAT3 immune complexes from whole cell lysates of these cells were subjected to WBA with antiphospho STAT3^Y705 (E) or anti-STAT3 (F) antibodies. (G and H) RAMOS Burkitt’s leukemia/lymphoma cell and ProB#4 cells (27) were left untreated (CON) (Lanes 1 and 2) or treated with 400 μM PV for 30 min (PV) (Lanes 3 and 4). Controls were treated with an anti-CD19 antibody homoconjugate (1 μg/mL) (Lanes 5 and 6) to stimulate the CD19-linked signaling pathway. STAT3 immune complexes from whole cell lysates were subjected to WBA with antiphospho STAT3^Y705 (G) or anti-STAT3 (H) antibodies.

Fig. 4.

SYKINH-61 promotes oxidative stress-induced apoptosis in primary B-lineage ALL cells. (A) Cells from two EBV-transformed lymphoblastoid cell lines BCL-1 and BCL-2, Burkitt’s leukemia/lymphoma cell line RAMOS, as well as primary leukemic cells from two B-lineage ALL patients were either left untreated or treated with 100 mM H₂O₂, 50 nM SYKINH-61, or 100 mM H₂O₂ + 50 nM SYKINH-61 for 30 min at 37 °C. TUNEL assays were used after 24 hr to determine the percentage of apoptotic cells after treatment. (B) Cells from a B-lineage ALL patient in postSCT relapse were either left untreated or treated with 100 mM H₂O₂, 50 nM SYKINH-61, or 100 mM H₂O₂ + 50 nM SYKINH-61 for 30 min at 37 °C. After 24 hr of culture, cells were costained with a rabbit polyclonal antitubulin antibody (Green Fluorescence) and the DNA-specific dye Toto-3 (Blue Fluorescence), and examined by laser scanning confocal microscopy (7, 8, 48).