Lyn kinase is activated following thrombopoietin stimulation of the megakaryocytic cell line B1647.
OBJECTIVE
B1647 is a cell line derived from bone marrow cells of a patient with acute myeloid leukemia (M2) with a complete erythro-megakaryocytic phenotype and bears both k and p isoforms of c-mpl. Interestingly, spontaneous B1647 cell proliferation is significantly potentiated by thrombopoietin (TPO).


METHODS
We aimed to evaluate the proliferative signal transduction events following the activation of c-mpl and we stimulated B1647 cells with TPO 40 ng/mL for 3, 7, 15 and 30 minutes; cells were then lysed and whole lysates were immunoprecipitated with anti-phosphotyrosine antibodies.


RESULTS
In our hands, TPO stimulation induced phosphorylation of several substrate proteins in B1647 cells. The increase in tyrosine phosphorylation from background spontaneous activation was transient, maximal after 10 minutes and declined to reach constitutive levels after 30 minutes. In particular, protein substrates between 50 and 140 kDa appeared to be selectively phosphorylated by TPO. We demonstrated that Jak2, Stat3 and Shc were activated in B1647 cells after TPO, as already shown for different cell lines by other authors. Moreover, Lyn kinase activation was detected. Grb2 co-immunoprecipitated with phosphorylated proteins. The phosphorylation of Syk kinase was not demonstrated, whereas Vav was activated by TPO.


CONCLUSIONS
The pattern of protein phosphorylation determined in B1647 cells by TPO testifies the role of this cytokine in sustaining cell growth and indicates Lyn tyrosine kinase as a possible target protein in transduction of the TPO proliferative signal.
Santini
Valeria
V
Dept. of Hematology, University of Florence, Policlinico di Careggi, viale Morgagni 85, Italy. santini@unifi.it
Scappini
Barbara
B
Grossi
Alberto
A
Gozzini
Antonella
A
Bonsi
Laura
L
Pagliai
Gabriella
G
Rossi Ferrini
Pierluigi
P
Bagnara
Gian Paolo
GP
D004789:Enzyme Activation / Q000187:drug effects; D006801:Humans; D008533:Megakaryocytes / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism; D010766:Phosphorylation / Q000187:drug effects; D015398:Signal Transduction; D013926:Thrombopoietin / Q000494:pharmacology*; D014407:Tumor Cells, Cultured; D019061:src-Family Kinases / Q000378:metabolism*
D013926:Thrombopoietin; C060902:lyn protein-tyrosine kinase; D019061:src-Family Kinases