1. TEL-JAK2 constitutively activates the extracellular signal–regulated kinase (ERK), stress-activated protein/Jun kinase (SAPK/JNK), and p38 signaling pathways
by Jenny M.-Y. Ho, Melody H.-H. Nguyen, Jamil K. Dierov, Karla M. Badger, Bryan K. Beattie, Piero Tartaro, Rizwan Haq, Brent W. Zanke, Martin P. Carroll, and Dwayne L. Barber
Blood
Volume 100(4):
August 15, 2002
©2002 by American Society of Hematology

2. TEL-JAK2 fusion proteins are constitutively tyrosine phosphorylated in Ba/F3 cells.(A) The characterized TEL-JAK2 fusions and the wild-type forms of TEL and JAK2.
TEL-JAK2 fusion proteins are constitutively tyrosine phosphorylated in Ba/F3 cells.(A) The characterized TEL-JAK2 fusions and the wild-type forms of TEL and JAK2. The breakpoints involved in the TEL-JAK2 chromosomal translocations are indicated by arrows. The TEL-JAK2(4-17) translocation fused nucleotide (nt) 463 of TEL to nt 2126 of JAK2, whereas TEL-JAK2(5-19) and TEL-JAK2(5-12) resulted in the fusion of TEL nt 1009 to JAK2 nt 2426 and JAK2 nt 1506, respectively. The 3 fusion proteins also contain a Myc-tag at the carboxy (C-) terminus. (B) Ba/F3 cells expressing the indicated TEL-JAK2 constructs were depleted of cytokine and then stimulated in the presence (+) or absence (−) of 10 ng/mL IL-3. IPs were performed with a pan-JAK antibody, and tyrosine-phosphorylated proteins were detected by IB with pTyr antibody.
Jenny M.-Y. Ho et al. Blood 2002;100:
©2002 by American Society of Hematology

3. TEL-JAK2 expression results in constitutive tyrosine phosphorylation of Ship-1 and association with the SH2 domain of Ship-1.(A) Ba/F3 and Ba/F3-TEL-JAK2 cells lines were depleted of cytokine and then stimulated in the presence (+) or absence (−) of IL-3.
TEL-JAK2 expression results in constitutive tyrosine phosphorylation of Ship-1 and association with the SH2 domain of Ship-1.(A) Ba/F3 and Ba/F3-TEL-JAK2 cells lines were depleted of cytokine and then stimulated in the presence (+) or absence (−) of IL-3. IPs were performed using a Ship-1 antibody, and tyrosine-phosphorylated proteins were detected by pTyr IB (upper panel). Total Ship-1 was detected by reprobing with a Ship-1 antibody (lower panel). (B) Ba/F3 and Ba/F3-TEL-JAK2(5-19) cells were depleted of cytokine and then stimulated in the presence (+) or absence (−) of IL-3. GST in vitro mixes were performed using 5 μg GST (lanes 1-4), GST–Ship-1 SH2 domain (lanes 5-8), or GST–Ship-1 Arg34Gln (lanes 9-12). Ba/F3 lysates are shown (lanes 13, 14). Tyrosine-phosphorylated proteins were detected by IB with a pTyr antibody.
Jenny M.-Y. Ho et al. Blood 2002;100:
©2002 by American Society of Hematology

4. TEL-JAK2 expression results in constitutive tyrosine phosphorylation of Shc and association with the SH2 domain of Shc.(A) Ba/F3 and Ba/F3-TEL-JAK2 cell lines were depleted of cytokine and then stimulated in the presence (+) or absence (−) of IL-3.
TEL-JAK2 expression results in constitutive tyrosine phosphorylation of Shc and association with the SH2 domain of Shc.(A) Ba/F3 and Ba/F3-TEL-JAK2 cell lines were depleted of cytokine and then stimulated in the presence (+) or absence (−) of IL-3. IPs were performed using a Shc antibody, and tyrosine-phosphorylated proteins were detected by pTyr IB (upper panel). Total Ship-1, Grb2, and Shc proteins were detected upon reprobing with Ship-1, Grb2, and Shc antibodies, respectively (lower panels). (B) Ba/F3 and Ba/F3-TEL-JAK2(5-19) cells were depleted of cytokine and then stimulated in the presence (+) or absence (−) of IL-3. GST in vitro mixes were performed using 5 μg GST (lanes 1-4), GST-Shc PTB domain (lanes 5-8), GST-Shc CH1 domain (lanes 9-12), or GST-Shc SH2 domain (lanes 13-16). Tyrosine-phosphorylated proteins were detected by pTyr IB.
Jenny M.-Y. Ho et al. Blood 2002;100:
©2002 by American Society of Hematology

5. Grb2 constitutively associates with TEL-JAK2 fusions through the SH2 domain of Grb2.(A) Ba/F3 and Ba/F3-TEL-JAK2 cell lines were depleted of cytokine and then stimulated in the presence (+) or absence (−) of IL-3.
Grb2 constitutively associates with TEL-JAK2 fusions through the SH2 domain of Grb2.(A) Ba/F3 and Ba/F3-TEL-JAK2 cell lines were depleted of cytokine and then stimulated in the presence (+) or absence (−) of IL-3. IPs were performed using a Grb2 antibody, and tyrosine-phosphorylated proteins were detected by pTyr IB (upper panel). Total Ship-1, Shc, and Grb2 proteins were detected upon reprobing with Ship-1, Shc, and Grb2 antibodies, respectively (lower panels). (B) Ba/F3 and Ba/F3-TEL-JAK2 cell lines were depleted of cytokine and stimulated in the presence (+) or absence (−) of IL-3. GST in vitro mixes were performed using 5 μg GST (lanes 1-8) or GST-Grb2 SH2 domain (lanes 9-16). Tyrosine-phosphorylated proteins were detected by IB with a pTyr antibody.
Jenny M.-Y. Ho et al. Blood 2002;100:
©2002 by American Society of Hematology

6. TEL-JAK2 expression results in constitutive phosphorylation of ERK1 and ERK2.(A) Ba/F3 and Ba/F3-TEL-JAK2 cells were depleted of cytokine and then stimulated in the presence (+) or absence (−) of IL-3.
TEL-JAK2 expression results in constitutive phosphorylation of ERK1 and ERK2.(A) Ba/F3 and Ba/F3-TEL-JAK2 cells were depleted of cytokine and then stimulated in the presence (+) or absence (−) of IL-3. Phosphorylated ERK1 and ERK2 were detected by IB lysates with an antibody specific for phosphorylated ERK1/2 (pERK1/2) (upper panel). Total ERK1/2 was detected upon reprobing with an ERK1/2 antibody (lower panel). (B) U0126 inhibits IL-3 and TEL-JAK2–mediated proliferation. Ba/F3 cells growing in IL-3 (closed squares) and Ba/F3-TEL-JAK2(5-19) cells growing in the presence of IL-3 (closed circles) or in the absence of IL-3 (open circles) were incubated in increasing concentrations of U0126. An XTT assay was then performed.
Jenny M.-Y. Ho et al. Blood 2002;100:
©2002 by American Society of Hematology

7. TEL-JAK2 expression results in constitutive phosphorylation of p38
TEL-JAK2 expression results in constitutive phosphorylation of p38.Ba/F3 and Ba/F3-TEL-JAK2 cell lines were depleted of cytokine and then stimulated in the presence (+) or absence (−) of IL-3.
TEL-JAK2 expression results in constitutive phosphorylation of p38.Ba/F3 and Ba/F3-TEL-JAK2 cell lines were depleted of cytokine and then stimulated in the presence (+) or absence (−) of IL-3. Phosphorylated p38 was detected by IB lysates with a phosphorylation-specific p38 antibody (pp38) (upper panel). Total p38 was detected upon reprobing with a p38 antibody (lower panel). (B) A total of 2000 Ba/F3 cells (closed squares) and Ba/F3-TEL-JAK2(5-19) cells in the absence of IL-3 (open circles) or Ba/F3-TEL-JAK2(5-19) in the presence of IL-3 (closed circles) were seeded into 96-well plates containing media of increasing SB concentration. The cells were incubated for 48 hours at 37°C prior to the addition of XTT and PMS. Absorbance was read at 450 nm following 4 hours of incubation at 37°C.
Jenny M.-Y. Ho et al. Blood 2002;100:
©2002 by American Society of Hematology

8. Expression of TEL-JAK2 results in constitutive activation of SAPK/JNK activity.Ba/F3 and Ba/F3-TEL-JAK2 cell lines were depleted of cytokine and then stimulated in the presence (+) or absence (−) of IL-3.
Expression of TEL-JAK2 results in constitutive activation of SAPK/JNK activity.Ba/F3 and Ba/F3-TEL-JAK2 cell lines were depleted of cytokine and then stimulated in the presence (+) or absence (−) of IL-3. SAPK was immunoprecipitated, and an in vitro kinase assay was performed using GST–c-Jun as substrate. Reaction mixtures were resolved by SDS-PAGE, and radiolabeled proteins were detected by PhosphorImager analysis (upper panel). Total SAPK was detected by IB with a SAPK antibody (lower panel). (B) A total of 2000 Ba/F3 cells (closed squares) and Ba/F3-TEL-JAK2(5-19) cells in the absence of IL-3 (open circle) or Ba/F3-TEL-JAK2(5-19) in the presence of IL-3 (closed circles) were seeded into 96-well plates containing media of increasing SP concentration. The cells were incubated for 48 hours at 37°C prior to the addition of XTT and phenazine methosulfate (PMS). Absorbance was read at 450 nm following 4 hours of incubation at 37°C.
Jenny M.-Y. Ho et al. Blood 2002;100:
©2002 by American Society of Hematology

9. Expression of TEL-JAK2(5-19)Tyr314Phe in Ba/F3 cells does not affect factor-independent growth.(A) Ba/F3 cells expressing the indicated TEL-JAK2 constructs were depleted of cytokine and then stimulated in the presence (+) or absence (−) of 10 ng/mL IL-3.
Expression of TEL-JAK2(5-19)Tyr314Phe in Ba/F3 cells does not affect factor-independent growth.(A) Ba/F3 cells expressing the indicated TEL-JAK2 constructs were depleted of cytokine and then stimulated in the presence (+) or absence (−) of 10 ng/mL IL-3. IPs were performed with a pan-Jak antibody, and tyrosine-phosphorylated proteins were detected by IB with pTyr antibody. (B) A total of 2000 Ba/F3 cells (closed squares) and Ba/F3 cells expressing TEL-JAK2(4-17) (open diamonds), TEL-JAK2(5-19) (open circles), and TEL-JAK2(5-19)Tyr314Phe (open triangles) constructs were seeded into 96-well plates containing media of increasing IL-3 concentration. The cells were incubated for 48 hours at 37°C prior to the addition of XTT and PMS. Absorbance was read at 450 nm following 4 hours of incubation at 37°C.
Jenny M.-Y. Ho et al. Blood 2002;100:
©2002 by American Society of Hematology

10. TEL-JAK2(5-19)Tyr314 represents a major binding site for Grb2
TEL-JAK2(5-19)Tyr314 represents a major binding site for Grb2.(A) Ba/F3 cells, Ba/F3-TEL-JAK2(4-17), TEL-JAK2(5-19), and TEL-JAK2(5-19)Tyr314Phe cells were depleted of cytokine and then stimulated in the presence (+) or absence (−) of IL-3.
TEL-JAK2(5-19)Tyr314 represents a major binding site for Grb2.(A) Ba/F3 cells, Ba/F3-TEL-JAK2(4-17), TEL-JAK2(5-19), and TEL-JAK2(5-19)Tyr314Phe cells were depleted of cytokine and then stimulated in the presence (+) or absence (−) of IL-3. IPs were performed using a Grb2 antibody, and tyrosine-phosphorylated proteins were detected by pTyr IB (upper panel). (B) Ba/F3, TEL-JAK2(4-17), TEL-JAK2(5-19), and TEL-JAK2(5-19)Tyr314Phe cells were depleted of cytokine and stimulated with (+) or without (−) IL-3. GST in vitro mixes were performed with 5 μg GST (lanes 1-8) or GST fused to the SH2 domain of Grb2 (lanes 9-16). Tyrosine phosphorylation was assayed by IB with pTyr antibody.
Jenny M.-Y. Ho et al. Blood 2002;100:
©2002 by American Society of Hematology

11. TEL-JAK2(5-19)Tyr314Phe displays impaired Ras activation
TEL-JAK2(5-19)Tyr314Phe displays impaired Ras activation.Ba/F3 and the indicated Ba/F3-TEL-JAK2 cell lines were cultured in RPMI containing 1% bovine serum albumin (upper panel) or RMPI supplemented with 10% fetal bovine serum (lower panel).
TEL-JAK2(5-19)Tyr314Phe displays impaired Ras activation.Ba/F3 and the indicated Ba/F3-TEL-JAK2 cell lines were cultured in RPMI containing 1% bovine serum albumin (upper panel) or RMPI supplemented with 10% fetal bovine serum (lower panel). Ba/F3 cells were depleted of cytokine and incubated in the presence or absence of IL-3 prior to lysis. Lysates were mixed with a GST-Raf fusion protein, and an anti-Ras Western blot was performed.
Jenny M.-Y. Ho et al. Blood 2002;100:
©2002 by American Society of Hematology

12. TEL-JAK2(5-19)Tyr314Phe does not alter constitutive activation of ERK1/2, p38, or SAPK/JNK.(A) Ba/F3 and the indicated Ba/F3-TEL-JAK2 cell lines were depleted of cytokine and then stimulated in the presence (+) or absence (−) of IL-3.
TEL-JAK2(5-19)Tyr314Phe does not alter constitutive activation of ERK1/2, p38, or SAPK/JNK.(A) Ba/F3 and the indicated Ba/F3-TEL-JAK2 cell lines were depleted of cytokine and then stimulated in the presence (+) or absence (−) of IL-3. Phosphorylated ERK1 and ERK2 were detected by IB lysates with an antibody specific for phosphorylated ERK1/2 (pERK1/2) (upper panel). Total ERK1/2 was detected upon reprobing (lower panel). (B) The indicated cell lines were depleted of cytokine and then stimulated in the presence (+) or absence (−) of IL-3. Phosphorylated p38 was detected by IB lysates with a phosphorylation-specific p38 antibody (pp38) (upper panel). Total p38 was detected upon reprobing (lower panel). (C) The indicated cell lines were depleted of cytokine and then stimulated in the presence (+) or absence (−) of IL-3. SAPK was immunoprecipitated, and an in vitro kinase assay was performed using GST–c-Jun as substrate. Reaction mixtures were resolved by SDS-PAGE, and radiolabeled proteins were detected by PhosphorImager analysis (upper panel). Total SAPK was detected by IB with a SAPK antibody (lower panel).
Jenny M.-Y. Ho et al. Blood 2002;100:
©2002 by American Society of Hematology