Lck Induced Cellular Transformation Through STAT5b Activation And The Inhibition Effects Of SOCS1 And SOCS3

Lck is a Src family protein tyrosine kinase and is expressed predominantly in T cells. It is essential for normal T cell development and activation. Aberrant expression or activation of Lck kinase has been reported in both lymphoid and non-lymphoid malignancies. Like other Src family members, Lck kinase activity is negatively regulated by phosphorylation of a highly conserved tyrosine (Tyr505) located near the carboxy terminus of the protein. Intramolecular interaction between phosphorylated Tyr505 and the Src homology 2 (SH2) domain confers a closed conformation and excludes substrate binding to the kinase domain. A point mutation of Tyr505 to Phe locks Lck in an open conformation and results in a constitutively active kinase. The constitutively active Lck kinase is oncogenic and transforms fibroblasts in culture. Nevertheless, the molecular mechanisms of Lck-mediated tumorigenesis have not been fully characterized.Suppressor of cytokine signaling (SOCS) modulates Janus kinase-signal transducer and activator of transcription (JAK-STAT) signaling in a negative feedback manner. Upon cytokine stimulation, active JAK and downstream STAT proteins induce the expression of STAT-target genes, including the SOCS genes. Cytokine-induced SOCS1 and SOCS3 proteins bind to JAK directly or the JAK-proximal sites on cytokine receptors to inhibit JAK kinase activity. The physiological importance of SOCS1 and SOCS3 is demonstrated by the lethal phenotypes observed in knockout mice, Thus they contribute to the down-regulation of JAK-STAT signaling and the transient kinetics of JAK-STAT activation by cytokines and growth factors. It suggests that SOCS1 and SOCS3 may also target oncogenic protein tyrosine kinases and function as tumor suppressors.Constitutive activation of the JAK-STAT pathway is frequently associated with oncogenic protein tyrosine kinases and is.reported in a wide variety of human cancers. A causal relationship between STAT activation and tumorigenesis has also been established in distinct tumor models. These findings raise the possibility that the negative feedback control involving SOCS proteins may be defective in these malignant cells. The observation that fibroblasts lacking SOCS1 are more susceptible to transformation supports this hypothesis. Inhibition of SOCS3 activity in human hepatocellular carcinoma cells also promotes cell migration that contribute to metastasis. Further evidence comes from high frequencies of SOCS gene silencing by DNA hypermethylation in human cancers. SOCS1 gene silencing has been reported in both lymphoid and nonlymphoid malignancies, while SOCS3 gene silencing was observed in head and neck cancer and lung cancer. However, it is not clear whether simultaneous loss of multiple SOCS gene expression occurs in tumor cells.In this report, we investigate the potential involvement of STAT5b activation in Lck-mediated cellular transformation and further characterize the mechanisms of SOCS1 and SOCS3 dysregulation and their tumor suppressing activity in Lck-transformed cells.This paper includes two parts.PartⅠ:AIM:To investigate the potential involvement of STAT5b activation in Lck-mediated cellular transformation.METHODS:We establish a tetracycline-inducible system to study the biochemical and biological effects of a constitutively active Lck mutant with a point mutation at the negative regulatory tyrosine (Y505F). To determine the effects of the constitutively active Lck kinase on STAT5b phosphorylation, a STAT5b expression construct was also transiently transfected into both T-REx-293/Lck (Y505F) and the vector control cells.T-REx-293 cells were stably transfected with pcDNA5/TO without (vec) or with Lck (Y505F). Cells were either left untreated or treated with tetracycline (Tet) for various times. Normalized whole cell lysates were subjected to SDS-PAGE and subsequent immunoblotting with anti-Lck antibody to check Lck expression. T-REx-293/Lck (Y505F) and the vector control cells were transiently transfected with STAT5b expression construct, and then either left untreated or treated with tetracycline for 24 hours. Proteins in normalized whole cell lysates were immunoprecipitated (IP) with anti-STAT5b antibody. The immunoprecipitates were resolved by SDS-PAGE and analyzed by immunoblotting with anti-phosphotyrosine (pTyr) monoclonal antibody 4G10. The membrane was stripped and re-blotted with anti-STAT5 antibody to check STAT5 expression.We also performed electrophoretic mobility shift assay (EMSA) to study whether Lck (Y505F) activates STAT5b DNA binding activity. T-REx-293/Lck (Y505F) and the vector control cells were transfected with STAT5b expression construct. Nuclear extracts containing the same amount of total proteins were subjected to EMSA with a 32P-labeled STAT5 consensus probe, or with the absence or presence of 100-fold molar excess of unlabeled wild-type or mutant MGE, anti-STAT5b or control antibody. Nuclear extract prepared from IL-3-stimulated BaF3 cells was also analyzed by EMSA for comparison.In order to figure out whether Lck (Y505F) associates with STAT5b in cells, we performed reciprocal co-immunoprecipitation experiments in T-REx-293/Lck (Y505F) and the vector control cells which were transiently transfected with STAT5b expression construct or the vector control, whole cell lysates were analyzed by immunoblotting with anti-Myc and anti-STAT5 antibodies. Proteins were immunoprecipitated with anti-STAT5b antibody or anti-Lck antibody, immunoprecipitates were subjected to SDS-PAGE and subsequent immunoblotting with anti-Myc or anti-STAT5 antibody respectively.The effects of active Lck kinase on cell growth and IL-3-withdrawal-induced apoptosis in T-REx-BaF3 cells were further studied by cell counting, flow cytometry analyzing annexin V conjugate to phosphatidylserine from the inner to the outer leaflet of the plasma membrane in apoptotic cells and DNA fragmentation assay.Finally we transiently transfected T-REx-BaF3/Lck(Y505F) with expression constructs carrying wild-type STAT5b, STAT5b with a point mutation of Tyr699 to Phe, or the vector alone to analyze the effect of exogenous STAT5b on cell proliferation and cell death, the cell number was determined by trypan blue staining.RESULTS:Tetracycline-regulated expression of Lck (Y505F) strongly induced STAT5b tyrosine phosphorylation, as a negative control, tetracycline alone did not induce STAT5b phosphorylation in the vector control cells.Tetracycline-regulated expression of Lck (Y505F) specifically induced a distinct DNA binding activity. Lck-induced DNA binding activity could be specifically competed out by unlabeled MGE oligonucleotides, but not by unlabeled MGE oligonucleotides with mutations at consensus DNA binding sites, indicating that the binding was specific. The presence of active STAT5b was further confirmed by supershifting specifically with anti-STAT5b antibody, but not control antibody. These results clearly demonstrate that the constitutively active Lck kinase can activate STAT5b and increase DNA binding activity.Lck (Y505F) co-immunoprecipitated with STAT5b and STAT5b co-immunoprecipitated with Lck (Y505F), the interaction between Lck (Y505F) and STAT5b suggests that the constitutively active Lck kinase may directly phosphorylate STAT5b.T-REx-BaF3/Lck (Y505F) grew significantly faster than T-REx-BaF3 control cells at each time point, these results support the notion that the constitutively active Lck kinase promotes cell proliferation. Removal of IL-3 also caused cell death of BaF3 expressing Lck (Y505F) in the first three days, but the viability was significantly higher than the vector control. Nevertheless, T-REx-BaF3/Lck (Y505F) gradually recovered after day 3 and became IL-3-independent. The expression of Lck (Y505F) remained high in these cells after continuous passage in the absence of IL-3. Our findings clearly demonstrate that a constitutively active Lck kinase can support IL-3-independent growth of BaF3 cells. As compared to the vector control cells, expression of Lck (Y505F) significantly reduced the percentage of cells recognized by the annexin V conjugate and greatly reduced the levels of DNA fragmentation shown as a distinct ladder pattern of low molecular weight DNAs. These results further illustrate that the active Lck (Y505F) kinase can protect cells from apoptosis induced by cytokine withdrawal. The effect of exogenous STAT5b on cell proliferation was also analyzed. As compared to the vector control, transfection of wild-type STAT5b significantly accelerated the growth of BaF3 cells expressing Lck (Y505F). Our data suggest that elevated expression of wild-type STAT5b may cooperate with the active Lck kinase in promoting cell proliferation. In contrast, STAT5b (Y699F) functioned as a dominant-negative protein to attenuate Lck-induced cell proliferation. We also examined the effects of exogenous STAT5b on cell death of T-REx-BaF3/Lck (Y505F) after IL-3 deprivation. In comparison to the vector control, transfection of wild-type STAT5b significantly reduced the death of Lck (Y505F)-expressing BaF3 cells; while transfection of STAT5b (Y699F) augmented the death of Lck (Y505F)-expressing BaF3 cells. All together, these results suggest that STAT5b may function as an important effecter molecule downstream of the constitutively active Lck kinase.CONCLUSION:In this report, we establish a tetracycline-inducible system to study the biochemical and biological effects of a constitutively active Lck mutant with a point mutation at the negative regulatory tyrosine. Expression of the active Lck kinase induces both tyrosine phosphorylation and DNA binding activity of signal transducer and activator of transcription 5b (STAT5b), a STAT family member activated in a wide variety of tumor cells. The active Lck kinase interacts with STAT5b in cells, suggesting that Lck may directly phosphorylate STAT5b. Expression of the constitutively active Lck mutant in interleukin-3 (IL-3)-dependent BaF3 cells promotes cell proliferation. In addition, the active Lck kinase protects BaF3 cells from IL-3-withdrawal-induced apoptotic death and leads to IL-3-independent growth. These transforming properties of the oncogenic Lck kinase can be further augmented by expression of exogenous wild-type STAT5b, but attenuated by a dominant-negative form of STAT5b. All together, our results suggest the potential involvement of STAT5b in Lck-mediated cellular transformation.Part II:AIM:To further characterize the mechanisms of SOCS1 and SOCS3 dysregulation and their tumor suppressing activity in Lck-transformed cells.METHODS:Using SOCS1 or SOCS3 full-length cDNA probes, we examined SOCS1 and SOCS3 gene expression in LSTRA cells by Northern blot analysis. To verify whether STAT5 is functional in activating other target genes, we examined the expression of Bcl-xL, an anti-apoptotic gene induced by active STAT5.We also performed pervanadate (a potent tyrosine phosphatase inhibitor that strongly activates tyrosine kinase signal transduction) stimulation experiments in LSTRA cells and used Northern blot to detect SOCS1 and SOCS3 gene expression. LSTRA and U266 cells were either untreated (-) or treated with 5'aza-cytidine (5-AzaC) or vehicle, SOCS1 and SOCS3 mRNA expression levels was determined RT-PCP to confirm that SOCS1 and SOCS3 gene silence was cause by DNA hypermethylation.The effects of SOCS1 and SOCS3 on Lck-transformed BaF3 cell growth and IL-3-withdrawal-induced apoptosis in T-REx-BaF3 cells were further studied by cell counting, flow cytometry analyzing annexin V conjugate to phosphatidylserine from the inner to the outer leaflet of the plasma membrane in apoptotic cells and DNA fragmentation assay.Lck-transformed BaF3 cells were transfected with pEF (vector), pEF-FLAG-I/mSOCSl (SOCS1), or pEF-FLAG-Ⅰ/mSOCS3 (SOCS3). Proteins in .lysates were immunoprecipitated with anti-Lck antibody and subjected to in vitro kinase assay (IVK). Anti-Lck immunoprecipitates were subjected to SDS-PAGE and transferred to membrane, followed by autoradiography and immunoblotting with anti-Myc antibody.We also performed electrophoretic mobility shift assay (EMSA) to check whether exogenous SOCS1 and SOCS3 reduce STAT5b activity in Lck-transformed BaF3 cells. Nuclear extracts each containing 2μg of total proteins isolated from Lck-transformed BaF3 cells transfected with pEF (vector), pEF-FLAG-I/mSOCS1 (SOCS1), or pEF-FLAG-I/mSOCS3 (SOCS3) were subjected to EMSA with a 2P-labeled mammary gland element (MGE) that contains consensus STAT5-binding site. The effects of exogenous SOCS1 and SOCS3 on STAT5b activity in Lck-transformed BaF3 cells were studied by immunoprecipitation using anti-STAT5b antibody, immunoprecipitates were resolved by SDS-PAGE followed by anti-phosphotyrosine immunoblotting, the blot was stripped and then reprobed with anti-STAT5 antibody to check STAT5 expression.RESULTS:We failed to detect mRNA from either gene in LSTRA cells, that means SOCS1 and SOCS3 gene expression is undetectable in LSTRA cells. The results also demonstrate the levels of Bcl-xL transcripts is elevated in LSTRA cells, from these observations, we conclude that STAT5-target genes are differently regulated in LSTRA cells. While Bcl-xL expression is induced, two SOCS family members are not expressed.SOCS3 mRNA can be detected in LSTRA cells after stimulation with pervanadate for 1 and 2 hours. These results demonstrate that the machinery in activating SOCS3 gene expression is still intact in LSTRA cells. In contrast to SOCS3, SOCS1 expression cannot be detected during our time course of pervanadate treatment in LSTRA cells. It raises the possibility that SOCS1 gene may be silenced by DNA hypermethylation. Therefore, the relatively fast SOCS3 induction by pervanadate also suggests that the absence of SOCS3 expression in LSTRA. is independent of DNA hypermethylation. As shown by RT-PCR analysis, SOCS1 gene expression can be reactivated by 5'aza-cytidine treatment, a known DNA methyltransferase inhibitor which reverse the effects from DNA hypermethylation, but not in untreated or vehicle-treated cells. These results point to the existence of two distinct mechanisms underlying the loss of SOCS1 and SOCS3 gene expression in LSTRA cells. So in LSTRA leukemia, SOCS1 and SOCS3 genes are silenced by DNA methylation-dependent and independent mechanisms, respectively.There was a significant decrease in cell proliferation 24 hours after transfection of either SOCS1 or SOCS3 in comparison to vector-transfected cells. SOCS1 and SOCS3 expression also led to a significant increase in cell death over this same time period. These results suggest that enforced SOCS1 and SOCS3 expression could reverse the transformed phenotypes in Lck transformed BaF3 cells. We found that the percentage of AnnexinⅤ-positive cells is significantly higher in SOCS1-and SOCS3-transfected cells as compared to vector-transfected cells. Consistent with elevated AnnexinⅤstainning, enforced SOCS1 and SOCS3 expression results in significant DNA fragmentation.IVK results shows that Lck (Y505F) prepared from vector-transfected Lck-transformed BaF3 cells have high levels of 32P incorporation due to autophosphorylation. In contrast, Lck autophosphorylation was greatly reduced in cells expressing exogenous SOCS1 or SOCS3. We conclude from these experiments that Lck kinase activity is attenuated in cells expressing SOCS1 or SOCS3.Similar to LSTRA cells, Lck-transformed BaF3 nuclear extracts exhibit elevated DNA binding activity to a STAT5 consensus sequence in an electrophoretic mobility shift assays (EMSA). As compared to the vector control, STAT5b DNA-binding activity is greatly reduced in cells expressing either SOCS1 or SOCS3. It demonstrates that enforced SOCSl or SOCS3 expression can suppress STAT5b DNA binding activity in Lck-transformed BaF3 cells.STAT5b immunoprecipitates show high levels of tyrosine phosphorylation in vector-transfected cells. Similarly, STAT5b tyrosine phosphorylation is greatly reduced in cells expressing SOCS1 or SOCS3. This result illustrates that expression of SOCS1 or SOCS3 in Lck-transformed BaF3 cells also reduces STAT5b activity, which is downstream of Lck (Y505F).CONCLUSION:In summary, this is the first report confirming tumor suppressor activity of SOCS1 and SOCS3 toward oncogenic Lck kinase. Both SOCS1 and SOCS3 are not expressed in Lck transformed cells, highlighting the importance of silencing both genes to create an intracellular environment favorable for STAT activation and tumor progression. Nevertheless, loss of SOCS1 and SOCS3 gene expression is mediated by different mechanisms in Lck-transformed cells. Further characterization of SOCS gene expression in other tumor cells will provide important insights in understanding the roles of different SOCS family members in tumor progression.Our results clearly demonstrate that exogenous SOCS1 and SOCS3 inhibit oncogenic Lck kinase activity and downstream STAT5b functions. We showed previously that STAT5b activation is critical in Lck-mediated oncogenesis. Therefore, attenuation of Lck-STAT5b signaling may contribute to the subsequent biological effects, including reduced cell proliferation and augmented apoptosis, in Lck-transformed cells. Altogether, we conclude that introduction of SOCS1 and SOCS3 can reverse Lck-induced transformation in BaF3 cells by inactivating Lck and the downstream transcription factor STAT5b.