Src Kinases Act Downstream Akt and Are Required for the Transforming Properties of Constitutively Activated Forms of STAT5A and STAT5B

Stat5A and Stat5B transcription factors play an important role in the control of hematopoietic cell proliferation and survival and are constitutively activated in number of hematological neoplasia. Moreover, expression of constitutively active Stat5 (caStat5) mutants in primary hematopoietic cells can lead to leukaemia. The transforming properties of CaStat5 have been previously shown to require Akt activation. However, the mechanisms by which CaStat5 mutants and Akt are activated remain enigmatic. In this study we aimed to determine the roles of src kinases in these processes,

For this purpose, we used murine Ba/F3 cells transfected or not with CaSta5A (Stat5A1*6) or CaStat5B (Stat51*B6) or with the oncogenic fusion proteins Tel-JAK2 and Tel-Abl as controls. Primary hematopoietic progenitors obtained from mouse bone marrow were also used in this sutdy. Cells were treated or not with the pan-src inhibitors PP1, PP2, and SU 6656 (and with PP3 an inactive analogue of PP1 as a control), imatinib (Abl inhibitor) and AG490 (JAK inhibitor). In some experiments cells were treated with U0126 (MEK inhibitor) or with the highly transducible TaT-dominant negative (dn) Akt fusion protein. Cell proliferation was assessed by cell counting. Cell death was evaluated using the trypan blue dye exclusion test. Cell apoptosis was assessed by analysing annexin V staining using flow cytometry and PARP cleavage using western blot. Expression of kinases and their phosphotylated forms were examined by western blot.

CaStat5A and CaStat5B cells were shown to express constitutively phosphorylated Lyn, Hck, Src kinases as well as Akt, IKK and ERK1/2. Stat1, Stat3, and JAK family members (TYK2 and JAK1-3) were not phosphorylated in these cells. Use of pan-Src inhibitors inhibited proliferation of CaStat5 cells and induced apoptosis of a fraction of these cells, in a dose-dependent manner. By contrast, imatinib and AG490 had no effect on the growth and survival of CaStat5 cells. Moreover, pan-src inhibitors prevented Erk but not Akt, IKK or STAT5 phosphorylation. By contrast, inhibition of Akt by means of TaT-dnAkt abolished Lyn, Src and ERK1/2 phosphorylation. Importantly, inhibition of MAPK did not interfere with Akt phosphorylation and did not interfere with CaStat5 cell survival.

Altogether our data indicate that Akt acts upstream in the CaStat5-associated signalling cascade, then activates src kinases and thereby promotes MAPK-dependent cell proliferation and MAPK-independent cell survival. These results shed light onto a hitherto undescribed role of Src kinases acting downstream Akt in the mechanisms of action of CaStat5.

No relevant conflicts of interest to declare.