Fig. 6: Activated p41Plk3 phosphorylates c-Fos at Thr164 and regulates a Plk3/c-Fos feed-forward pathway to promote apoptosis. | Nature Communications

Fig. 6: Activated p41Plk3 phosphorylates c-Fos at Thr164 and regulates a Plk3/c-Fos feed-forward pathway to promote apoptosis.

From: Nardilysin-regulated scission mechanism activates polo-like kinase 3 to suppress the development of pancreatic cancer

Fig. 6

a IP and IB analysis of the interaction between Plk3WT or mutants and c-Fos in PANC-1 cells. b–e qRT-PCR analysis of c-Fos–regulated pro-apoptotic or cell-cycle–related (b, c) and anti-apoptotic (d, e) gene expression in HPNE cells with inducible expression of p72Plk3 or p41Plk3. f In vitro kinase assay using p72Plk3-SFB (boxed, S protein-FLAG-streptavidin binding peptide, tagged at Plk3 C-terminus), Flag-p41Plk3 (boxed), or Flag-p72Plk3KD (kinase-dead K91R mutant) immunoprecipitated from 293T cells to incubate with purified recombinant GST–c-Fos with or without Plk inhibitor. P53 and c-Fos phosphorylation by recombinant GST-Plk3 (a mixture of p72Plk3 and p41Plk3) was used as positive control; phosphorylation was detected by α-ThioP (asterisk). g In vitro kinase assay showing Plk3-mediated phosphorylation of c-Fos caused c-Fos migration to a higher molecular weight position. Shifted and unshifted c-Fos bands (Lane 3) with (Lane 4) or without (Lane 3) Plk inhibitor treatment were analyzed by mass spectrometry to deduce phosphorylation sites. c-Fos alone (Lane 2) was analyzed by mass spectrometry to exclude the c-Fos auto-phosphorylation. Both GST-Plk3 and GST–c-Fos are purified proteins as described in (f). Gel was stained by Coomassie blue. h, i qRT-PCR (h) and IB (i) analysis of p72Plk3 expression in c-Fos–transfected PANC-1 cells. j Chromatin IP assay and real-time PCR comparing the ratio of anti–c-Fos antibody to IgG at the indicated Plk3 promoter region. k Effects of expressing c-Fos WT and T164A mutant on the activity of Plk3 reporter in inducible PATC148/ip41Plk3 cells. l–n Apoptosis-inducing activity (l, m) and immunoblot of cleaved PARP (n) in inducible148/ip41Plk3 cells stably transfected with c-Fos WT or T164A mutant. Data are representative of two independent experiments with similar results (a, f, g, i, n). Error bars, mean ± SEM (b–e, h, j, k, m), n = 3 independent experiments, two-tailed unpaired t-test (b–e, h, j, k, m), Source data are provided as a Source Data file.

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