Roles of paxillin phosphorylation in IL-3 withdrawal-induced Ba/F3 cell apoptosis
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Ba/F3, a mouse pro-B cell line, is dependent on IL-3 for its survival and proliferation. IL-3 withdrawal causes cells to round, stop in G1 phase, then undergo apoptosis. Additionally, IL-3 is known to induce tyrosine phosphorylation of paxillin, a scaffold and signaling protein. We previously determined that overexpression of paxillin prohibited Ba/F3 cell apoptosis induced by IL-3 withdrawal.
Address whether phosphorylation is essential for the anti-apoptotic effect of overexpressed paxillin.
Mutations were introduced into paxillin cDNA at five phosphorylation sites—Y31F, Y40F, Y118F, Y181F, S273A, or S273D. After overexpression of paxillin mutants in Ba/F3 cells, the apoptotic proportions of cell populations were measured by an annexin V conjugation assay while cells were undergoing IL-3 withdrawal.
The anti-apoptotic effect of paxillin overexpression was abolished by site-directed mutagenesis replacing Y31, Y40, Y118, and Y181 with phenylalanine, and S273 with aspartic acid. In contrast, the mutation replacing S273 with alanine had no effect on the anti-apoptotic effect.
The above results suggest that paxillin-mediated phosphorylation at Y31, Y40, Y118, and Y181 is essential for the anti-apoptotic effect of paxillin overexpression in Ba/F3 cells and contributes to the cell survival signaling pathway triggered by IL-3. Conversely, phosphorylation at S273 is involved in the negative regulation of the anti-apoptotic action of overexpressed paxillin.
KeywordsPaxillin Phosphorylation Survival Apoptosis IL-3 B cell
This research was supported by Basic Science Research Program through the National Research Foundation of Korea (NRF)—funded by the Ministry of Education, Science and Technology (2011-0025479).
Compliance with ethical standards
Conflict of interest
Nah, A. S. and Chay, K.O. declare that they have no conflicts of interest.
Research involving human and animal rights
This article does not contain any studies with human subjects or animals performed by any of the authors.
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