A Requirement for SOCS-1 and SOCS-3 Phosphorylation in Bcr-Abl-Induced Tumorigenesis

Abstract

Suppressors of cytokine signaling 1 and 3 (SOCS-1 and SOCS-3) are inhibitors of the Janus tyrosine kinase (JAK)/signal transducers and activators of transcription (STAT) pathway and function in a negative feedback loop during cytokine signaling. Abl transformation is associated with constitutive activation of JAK/STAT-dependent signaling. However, the mechanism by which Abl oncoproteins bypass SOCS inhibitory regulation remains poorly defined. Here, we demonstrate that coexpression of Bcr-Abl with SOCS-1 or SOCS-3 results in tyrosine phosphorylation of these SOCS proteins. Interestingly, SOCS-1 is highly tyrosine phosphorylated in one of five primary chronic myelogenous leukemia samples. Bcr-Abl-dependent tyrosine phosphorylation of SOCS-1 and SOCS-3 occurs mainly on Tyr 155 and Tyr 204 residues of SOCS-1 and on Tyr 221 residue of SOCS-3. We observed that phosphorylation of these SOCS proteins was associated with their binding to Bcr-Abl. Bcr-Abl-dependent phosphorylation of SOCS-1 and SOCS-3 diminished their inhibitory effects on the activation of JAK and STAT5 and thereby enhanced JAK/STAT5 signaling. Strikingly, disrupting the tyrosine phosphorylation of SOCS-1 or SOCS-3 impaired the expression of Bcl-X(L) protein and sensitized K562 leukemic cells to undergo apoptosis. Moreover, selective mutation of tyrosine phosphorylation sites of SOCS-1 or SOCS-3 significantly blocked Bcr-Abl-mediated tumorigenesis in nude mice and inhibited Bcr-Abl-mediated murine bone marrow transformation. Together, these results reveal a mechanism of how Bcr-Abl may overcome SOCS-1 and SOCS-3 inhibition to constitutively activate the JAK/STAT-dependent signaling, and suggest that Bcr-Abl may critically requires tyrosine phosphorylation of SOCS-1 and SOCS-3 to mediate tumorigenesis when these SOCS proteins are present in cells.

eIF4B Phosphorylation by Pim Kinases Plays a Critical Role in Cellular Transformation byAblOncogenes

“…The 32D myeloid cells stably expressing wild-type (WT) Bcr-Abl or Bcr-Abl-T315I were kind gifts from Dr. Guangbiao Zhou (Institute of Zoology, Chinese Academy of Science, Beijing, China). Pim-or eIF4B-overexpressing Abl transformants (K562, NS2, and W44) were generated by stably infecting the cells with retroviruses or lentiviruses encoding Pim or eIF4B using viral vectors pMSCV-GFP or pNL-GFP (Addgene) as previously described (35). Short hairpin RNA (shRNA)-expressing stable Abl transformants were generated by infection of the cells with lentiviruses expressing specific shRNA in pSIH-H1-GFP vector (System Biosciences) as described previously (36).…”

“…GST pull-down experiment and in vitro kinase assay Glutathione S-transferase (GST) fusion proteins were expressed and purified as previously described (6,35). Pull-down assays were conducted by incubating aliquots of purified GST fusion protein beads with cell lysates at 4 C overnight.…”

“…Nude-mouse injection was carried out as described previously (35). Tumor growth was monitored and measured in volume (length  height  width) at the indicated time points after inoculation.…”

“…vAbl-and Bcr-Abl-mediated bone marrow transformation was conducted as previously described (6,35). Transformation efficiency was scored by counting the number of the wells containing the transformed cell clones showing cytokine-independent growth 3 weeks after infection.…”

Silencing of Suppressor of Cytokine Signaling-3 due to Methylation Results in Phosphorylation of STAT3 in Imatinib Resistant BCR-ABL Positive Chronic Myeloid Leukemia Cells

“…The JAK/STAT signaling pathway, is subject to negative regulation by three protein families; the SH2-containing phosphataes (SHP), the suppressors of cytokine signaling (SOCS) and the protein inhibitors of activated STATs (PIAS) (Yoshikawa et al, 2001;Roman-Gomez et al, 2004;Qiu et al, 2012;Furqan et al, 2013). Aberrant DNA methylation of promoter CpG dinucleotides is associated with the silencing of many proteins in human malignancies, including the negative regulators, SOCS-1 and SOCS-3.…”