TEL-JAK2 mediates constitutive activation of the phosphatidylinositol 3'-kinase/protein kinase B signaling pathway.

A subset of chromosomal translocations that participate in leukemia involve activated tyrosine kinases. The ets transcription factor, TEL, undergoes translocations with several distinct tyrosine kinases including JAK2. TEL-JAK2 transforms cell lines to factor independence, and constitutive tyrosine kinase activity results in the phosphorylation of several substrates including STAT1, STAT3, and STAT5. In this study we have shown that TEL-JAK2 can constitutively activate the phosphatidylinositol 3'-kinase (PI 3'-kinase) signaling pathway. The regulatory subunit of PI 3'-kinase, p85, associates with TEL-JAK2 in immunoprecipitations, and this was shown to be mediated by the amino-terminal SH2 domain of p85 but independent of a putative p85-binding motif within TEL-JAK2. The scaffolding protein Gab2 can also mediate the association of p85. TEL-JAK2 constitutively phosphorylates the downstream substrate protein kinase B/AKT. Importantly, the pharmacologic PI 3'-kinase inhibitor, LY294002, blocked TEL-JAK2 factor-independent growth and phosphorylation of protein kinase B. However, LY294002 did not alter STAT5 tyrosine phosphorylation, indicating that STAT5 and protein kinase B activation mediated by TEL-JAK2 are independent signaling pathways. Therefore, activation of the PI 3'-kinase signaling pathway is an important event mediated by TEL-JAK2 chromosomal translocations.


INTRODUCTION
Chromosomal translocations play a central role in the development of leukemia. The participating genes generally fall into three groups involving tyrosine kinases, transcription factors or factors that modify transcriptional activation (1). The prototypical tyrosine kinase is the BCR-ABL translocation which is the causative agent in chronic myelogenous leukemia (2).
Many studies have focused on the mechanism of constitutive activation mediated by BCR-ABL. Substrates that are activated downstream of BCR-ABL include STAT1 (13)(14)(15), STAT3 (15), STAT5 (13)(14)(15)(16) and STAT6 (15). Grb2-Sos can be recruited to BCR-ABL either directly or indirectly through other adaptor proteins including Ship1 (17), Shp2 (18), Shc (19)(20)(21)(22)(23) and Cbl (24-28). BCR-ABL has also been shown to stimulate activation of Ras (29) and the related family member Rac (30).  (42). However, a constitutively activated form of STAT5 resulted in only a myeloproliferative disease (42). In summation, these elegant studies have shown that signaling pathways distinct from STAT5a/b activation play a role in leukemogenesis mediated by TEL-JAK2. The goal of this study is to characterize PI 3' kinase-dependent signaling mitigated by TEL-JAK2. Immunoprecipitations Immunoprecipitations were performed with 1.5 mg of protein lysates. Primary antibody was added for 1 hr, followed by 1 hr incubation with Protein A-Sepharose (Amersham Pharmacia Biotech). Alternatively, primary antibody and Protein A-Sepharose was added together and incubations were performed overnight. Bead-bound immune complexes were washed 3 times with ice cold lysis buffer, eluted by boiling for 5 minutes in Laemmli sample buffer containing 100 µM DTT, and separated by SDS-PAGE and transferred to PVDF transfer membrane for immunoblotting.

In vitro Mixes -GST fusion protein binding experiments
GST fusion proteins (2.5 µg) expressing the amino, carboxy, or amino and carboxy terminal SH2 domains of p85 (generously provided by Dr Ben Margolis, U of M, Ann Arbor, MI) or GST alone immobilized to Glutathione sepharose 4B beads (Amersham Pharmacia Biotech), were incubated with 1.5 mg of protein lysates. After a 1 hour incubation at 4°C, the precipitate was washed 3 times with ice cold lysis buffer. Samples were boiled for 5 minutes in Laemmli sample buffer with 100 µM DTT to elute proteins before separation on SDS-PAGE gels and transfer to PVDF transfer membrane.
Following two washes in TBST (TBS, 0.1% Tween-20), membranes were incubated with the by guest on March 24, 2020 http://www.jbc.org/ Downloaded from appropriate dilution of primary antibody solution for 1 hour at room temperature. Membranes were then washed four times in TBST and incubated with the relevant HRP-conjugated secondary antibody (1:5000 dilution in TBST) for 30 minutes. Following four washes in TBST, reactive proteins were visualized by enhanced chemiluminescence (ECL) (Amersham Pharmacia Biotech) with autoradiographic film (Amersham Life Science).
PVDF membranes for phospho-PKB and PKB immunoblots were blocked in 5% skim milk in TBST for 1 hour at room temperature, washed once in primary antibody dilution buffer and incubated with primary antibody (1:1000 dilution in 1% BSA in TBST) overnight at 4°C. After 6 washes in TBST, the membrane was incubated with HRP-ProteinA (1:2000 dilution in 2.5% skim milk in TBST) for 1 hour at room temperature. The membrane was washed 6 times in TBST and visualized by ECL.
Membranes for phospho-STAT5 immunoblots were blocked in 5% milk in TBST for 1 hour at room temperature, washed 2 times in TBST and incubated with primary antibody (1:1000 dilution in 3% BSA in TBST) for 3 hours at room temperature. After 4 washes in TBST, the membrane was incubated with HRP-Protein A (1:5000 dilution in 2.5% BSA in TBST) for 1 hour at room temperature. The membrane was washed 4 times in TBST prior to visualization by ECL. For reprobing, membranes were stripped in 62.5 mM Tris-HCl (pH6.8), 2% SDS and 0.1M β-mercaptoethanol for 30 minutes at 50°C, and rinsed twice in TBST.

Apoptosis assays
Annexin V, 7AAD and 10X binding buffer were purchased from PharMingen. Briefly, at distinct time points, untreated and treated cells were washed in 1X binding buffer (10 mM Hepes buffer and incubated with 2 µL Annexin V antibody conjugated to PE for 10 minutes at room temperature. Samples were adjusted to a final volume of 1 mL prior to FACS analysis (Becton Dickinson). Acquisition and analysis were performed using the CellQuest software.

PI 3' Kinase Activation is Required for TEL-JAK2 Mediated Cell Proliferation/Survival
To determine whether TEL-JAK2 mediated factor-independent growth was dependent on PI 3' kinase, we performed XTT assays in the absence or presence of IL-3 (100 pg/mL) and
The p85 subunit of PI 3' kinase contains two SH2 domains and one SH3 domain. We next determined whether the TEL-JAK2-p85 interaction was SH2 dependent. An in vitro mixing experiment was performed using cell lysates from Ba/F3 and TEL-JAK2 (5-19) expressing

The Scaffolding Protein Gab2 is a Possible Mediator of PI 3' Kinase Association with TEL-JAK2
The involvement of adaptor molecules is widespread in linking signaling pathways. With the suggestion that direct recruitment of p85 to TEL-JAK2 via the YMIM motif is not significant, we next investigated the possibility of adaptor molecules mediating the association between TEL-JAK2 and p85. Gab2 (37) has been shown to contain multiple consensus p85 binding sites thereby mediating PI 3' kinase signaling downstream of cytokine, growth factor and antigen receptor activation (37,38).

Expression of TEL-JAK2 Protects Ba/F3 Cells from Apoptosis
We have demonstrated an association between TEL-JAK2 and PI 3' kinase and the significance of this pathway in factor-independent growth. However, PI 3' kinase and PKB have also been implicated in modulating protection from programmed cell death (55)(56)(57)(58)(59). We were interested in assessing whether TEL-JAK2 expression can lead to decreased apoptosis of cells, thereby contributing to an increase in cell number. To compare the number of cells undergoing apoptosis when depleted of IL-3, we performed Annexin V and 7AAD staining (Figure 9).
Annexin V serves as a marker for apoptosis in the early phase while 7AAD stains the DNA of late apoptotic or necrotic cells whose membrane integrity has been compromised (60,61). The The importance of STAT5 in TEL-JAK2 signaling and leukemogenesis has been demonstrated in vitro (10,12,40) and in vivo (12,42). However, bone marrow transplants performed with a constitutively active STAT5 or a STAT5 target gene, failed to recapitulate the phenotype of TEL-JAK2 transplanted mice (42). This suggests that TEL-JAK2 activates signaling targets distinct from STAT5. Therefore, we examined whether pretreatment of TEL-JAK2 expressing cells with LY294002 would affect STAT5 tyrosine phosphorylation ( Figure   10). IL-3 (lanes 5-8) and TEL-JAK2 (5-19) (lanes 13 to 16) mediated PKB phosphorylation was decreased by LY294002 pretreatment. However, there was no diminution of constitutive or IL-3 induced STAT5 phosphorylation as determined by immunoblotting with a phospho-specific STAT5 antibody in the presence of either 10 or 20 µM LY294002 (third panel). Our results indicate that PI 3' kinase is involved in PKB phosphorylation, and that this signaling pathway is distinct from STAT5 phosphorylation and activation.

DISCUSSION
The pathways of hematopoietic cell transformation mediated by TEL-JAK2 have not been extensively characterized. TEL-JAK2 has been shown to be oncogenic in vivo (12) and capable of transforming cells to factor independence in vitro (10,12,40). The constitutive activation of STAT1, STAT3 and STAT5 is observed downstream of TEL-JAK2 activation, however, it is known that pathways distinct from STAT5 are required in vivo for leukemic progression (42).  (79). BCR-ABL appears to down-regulate Ship1 expression in Ba/F3-BCR-ABL transfectants (80), however, TEL-JAK2 does not alter Ship1 protein levels.
Deciphering the role of the lipid phosphatases, PTEN, Ship1 and the related gene, Ship2, in PKB activation will be a subject of particular interest. Future studies will be required to determine what targets upstream and downstream of PKB are regulated by TEL-JAK2 expression, potentially contributing to its transforming ability.
The expression of TEL-JAK2 confers IL-3 independent growth in vitro (10,12,40). Use of the PI 3' kinase inhibitor, LY294002, has allowed us to examine the importance of PI 3' kinase activity in TEL-JAK2-mediated factor-independent cell growth/survival. In addition, expression of the fusion protein protects cells from apoptosis in the absence of IL-3. This would suggest that TEL-JAK2 is capable of inducing both proliferation and survival. However, it remains to be determined if protection from programmed cell death is PI 3' kinase dependent.
Recent studies indicate that in the context of IL-3 signaling, the class I PI 3' kinases are required for cell proliferation and the phosphorylation of PKB and BAD but not for protection from apoptosis (48). If signals activated by TEL-JAK2 are a subset of those activated by IL-3, particularly with respect to signals downstream of PI 3' kinase, then it may also be true that the PI 3' kinase/PKB pathway activated by TEL-JAK2 predominantly targets cell proliferation. In support of this, it has been shown using LY294002 that PI 3' kinase is not absolutely required for the protection of cells expressing BCR-ABL from apoptosis (81). A recent study of PI 3' kinase and Raf pathways in BCR-ABL signaling alludes to the importance of both pathways acting independently but overlapping in their anti-apoptotic activity (32). Future studies examining cell cycle and utilizing dominant negative mutants of p85, PKB as well as downstream substrates including BAD may help distinguish the cell proliferation and survival signals activated by TEL-JAK2.
The leukemogenic potential of TEL-JAK2 has been demonstrated in mice. Bone marrow transplants have illustrated that TEL-JAK2 induces a fatal myelo-and lymphoproliferative disease (12). Interestingly, using STAT5a/b deficient mice, it was revealed that there was no onset of disease (42). However, only a myeloproliferative disease results when bone marrow cells are transduced with constitutively active STAT5a (42). This transplantation model and studies of other oncogenic tyrosine kinases, such as BCR-ABL, would imply that activation of multiple signaling pathways is necessary for cellular transformation and disease induction. Our results indicate that TEL-JAK2 activates the PI 3' kinase pathway and two additional pathways: the Ras/MEK/MAPK (43) pathway and STAT5 (42). We have shown that the tyrosine phosphorylation status of STAT5 is unaffected by the PI 3' kinase inhibitor, suggesting that the PI 3' kinase/PKB pathway can act in parallel with STAT5 activation downstream of TEL-JAK2.
The MEK/MAPK pathway has also been implicated in regulating apoptosis (82,83). However, the relevance of PI 3' kinase in TEL-JAK2 leukemogenesis will remain to be determined using murine bone marrow transplant models.
In summary, this study demonstrates the importance of PI 3' kinase in TEL-JAK2 mediated factor independent cell proliferation and phosphorylation of PKB. The association of TEL-JAK2 and the p85 subunit of PI 3' kinase is most likely mediated by adaptor proteins such as Gab2 and IRS-2. The putative p85 binding site in JAK2 is dispensable for this interaction.
Activation of the PI 3' kinase/PKB pathway is a common element observed in oncogenic progression. The requirement of this pathway for the onset of disease by TEL-JAK2 will be a subject of future investigation.