TABLA DE MAYORIA

of the ABL1 tyrosine kinase activity in the fusion protein.

3.8 MECHANISM OF TRANSFORMATION USED BY THE

SHIP1/ABL1 FUSION

As explained earlier, in all the ABL1 fusions proteins examined so far the constitutive activation the ABL1 tyrosine kinase is due to a dimerization or oligomerization of the fusion protein mediated by a protein domain contributed by the fusion partner. Therefore, we asked the question whether the novel SHIP1/ABL1 fusion protein would also follow the same mechanism of activation: namely dimerization or oligomerization of the SHIP1/ABL1 mediated by the SHIP1 protein domain. To prove homo-dimerization of the SHIP1/ABL1 protein we constructed two SHIP1/ABL1 fusions bearing different 5’-epitope tags in eukaryotic expression vectors and performed co- immunoprecipitation experiments.

3.8.1 Cloning of epitope tagged full length SHIP1/ABL1 into

eukaryotic expression vectors

A FLAG or HA epitope tag was introduced in frame at the 5’ end of the SHIP1/ABL1 fusion by PCR using the pGEM-Teasy-SA(39-1508)-CLN9 clone as template. Three consecutive PCRs were performed in order to introduce the epitope tags and the necessary restriction enzyme sites at the 5’ end of the SHIP1/ABL1(38-1508) fragment. For the generation of Flag- SHIP1/ABL1(154-1508) fragments, the forward primers Hind3-Mun1-Flag- SHIP1No1 (for PCR no 1), Hind3-Mun1-Flag-SHIP1No2 (for PCR no 2) and

Hind3-Mun1-Flag-SHIP1No3 (for PCR no 3) and the reverse primer SHIP- ABL1-B1508-1489 (PCR no 1, 2 and 3) (2.1.13) were used (Figure 3.15). One microliter of a 1:100 dilution of PCR no 1 was used as template for PCR no 2, and similarly 1 µl of a 1:100 dilution of PCR no 2 was used as template in the third PCR. With each PCR, about 15 additional nucleotides were added to the 5’ end of the fragment, thus building up the sequence for the Flag epitope and the HindIII and Mun1 restriction sites. The HA-SHIP1/ABL1

fragments were obtained in a similar fashion in three consecutive PCRs by using the forward primers Hind3-Mun1-HA-SHIP1No1, Hind3-Mun1-HA- SHIP1No2 and Hind3-Mun1-HA-SHIP1No3 and the reverse primer SHIP- ABL1-B1508-1489 (2.1.13). The epitope-tagged products were cloned into pGEM-Teasy vector and the clones were sequence verified. The sequence- verified clones pGEM-Teasy-Flag-SA(154-1508)-CLN96 and pGEM-Teasy- HA-SA(154-1508)-CLN91 (Appendix II) were used for further cloning. The inserts from these pGEM-Teasy clones were excised by a HindIII/Kpn1 double-digest and inserted into the HindIII and KpnI sites of pcDNA-BCR/ABL replacing the BCR/ABL fusion from the HindIII site 5’ of the BCR ATG till the

Kpn1 site within ABL1 as described in section 3.4.

Figure 3.15: Introduction of epitope tags at the 5’ end of the SHIP1/ABL1 fusion. In the upper diagram the positions of the primers are shown in the pGEM-Teasy-S/A (39-1508)-CLN9 clone, which was used for the introduction of the Flag or HA epitope at the 5’ end of the SHIP1/ABL1 fusion by PCR. The lower diagram shows the resulting PCR products, which were cloned into the pGEM-Teasy vector.

The pcDNA clones with the tagged full-length SHIP1/ABL1 fusion, pcDNA- Flag-SA-96E and pcDNA-HA-SA-91E, were transiently transfected into HEK293T cells. The expression of the epitope-tagged SHIP1/ABL1 proteins was confirmed by Western blot analysis using antibodies against the Flag and HA (2.1.14) epitopes as well as against the ABL1 protein (Figure 3.16). As

controls for the anti Flag and anti HA antibodies plasmids expressing Flag- tagged AF10 protein and HA-tagged Ikaros protein were also transiently transfected into HEK293T cells.

Figure 3.16: Western blot analysis of the total cell lysates of the HEK293T cells transiently over-expressing SHIP1/ABL1, Flag-SHIP1/ABL1 or HA- SHIP1/ABL1. Blot1: Immunoblotting with an antibody against c-ABL1: All the ABL1 fusion proteins are detected; Blot 2: Immunoblotting with an anti-body against the Flag tag: Flag tagged AF10 and SHIP1/ABL1 are detected but HA- SHIP1/ABL1 is not detected; Blot 3: Immunoblotting with an antibody against the HA epitope: HA tagged IKAROS and SHIP1/ABL1 are detected but Flag- SHIP1/ABL1 is not detected.

3.8.2 Homo di- or oligomerization of the SHIP1/ABL1 fusion

protein

To demonstrate the dimerization or oligomerization of the SHIP1/ABL1 fusion protein, HEK293T cells were co-transfected (2.2.3.3) with the pcDNA-Flag- SA-96E and pcDNA-HA-SA-91E plasmids. For controls, HEK293T cells were also transfected with pcDNA-Flag-SA-CLN96E or pcDNA-HA-SA-CLN91E alone. After 48 hrs, total cell lysates were prepared (2.2.4.1) from transfected and non-transfected cultures. The lysates were then subjected to co- immunoprecipitation (CoIP) (2.2.4.4). During the CoIPs, protein complexes were precipitated with an anti–FLAG antibody, followed by immunoblotting with anti-HA antibodies. These CoIP experiments revealed that protein

complexes containing Flag-SHIP1/ABL1 (i.e., complexes precipitated with the anti-FLAG antibody) could also be detected with the anti–HA antibody, demonstrating the interaction between FLAG-tagged SHIP1/ABL1 proteins and HA-tagged SHIP1/ABL1 proteins (Figure 3.17). These results showed that the SHIP1/ABL1 fusion protein is present as a dimer or multimer in the cells, and strongly suggests that the activation of the ABL1 tyrosine kinase activity in SHIP1/ABL1 is mediated via homo di- or oligomerization. Homodimerization or oligomerization has also been demonstrated for the BCR/ABL1, ETV6/ABL1 and EML1/ABL1 fusions (McWhirter et al., 1993; Golub et al., 1996; De Keersmaecker et al., 2005).

Figure 3.17: Detection of a homotypic interaction of the SHIP1/ABL1 protein: (A) Diagram showing important domains of the SHIP1/ABL1 protein with the two different N-terminal epitope tags used in the co- immunoprecipitation experiments: Flag and HA indicates the Flag and HA epitope tags, SH2: Src homology 2, SH3: Src homology 3, TK: Tyrosine kinase domain, DB: DNA binding domain, AB: Actin binding domain; (B) Co- immunoprecipitation of Flag-SHIP1/ABL1 and HA-SHIP1/ABL1: The indicated Flag-tagged SHIP1/ABL1 and HA-tagged SHIP1/ABL1 proteins were transiently expressed in the HEK293T cells and immunoprecipitated with

antibody against the Flag epitope (lane 1-3). As a control, the protein lysates obtained from the Flag-SHIP1/ABL1+HA-SHIP1/ABL1 cotransfected cells, were immunoprecipitated with antibody against normal IGg (lane 4). The immunoprecipitates were separated by SDS-PAGE and immunoblotted with anti-HA antibody (lane 1-4). In addition, 5% amount of the total cell lysates were subjected to SDS-PAGE and immunoblotted with anti-HA antibody (lane 5-7).

3.9 IDENTIFICATION OF THE HOMO DIMERIZATION DOMAIN