Phosphoproteomic analysis of distinct tumor cell lines in response to nocodazole treatment

Abstract

Here, we report for the first time a comparative phosphoproteomic analysis of distinct tumor cell lines in the presence or absence of the microtubule-interfering agent nocodazole. In total, 1525 phosphorylation sites assigned to 726 phosphoproteins were identified using LC-MS-based technology following phosphopeptide enrichment. Analysis of the amino acid composition surrounding the identified in vivo phosphorylation sites revealed that they could be classified into two motif groups: pSer-Pro and pSer-Asp/Glu. Phosphoproteomic change resulting from nocodazole treatment varied among cell lines in terms of the numbers of total phosphopeptides identified, motif groups, and functional annotation groups; however, the cell lines were equally sensitive to nocodazole. The identified phosphoproteome subset contained major signaling proteins and proteins known to be involved in mitosis, but did not always exhibit the same changes in the tumor cells from nocodazole treatment. In spite of the complex changes observed in the phosphorylation of many of the proteins, possible common features induced by nocodazole were found, including phosphorylation of nucleophosmin (NPM) S254 and coatomer protein complex, subunit alpha (COPA) S173, suggesting that the events are not cell-type specific but events generally occurring in mitosis or induced by a microtubule-interfering agent. Further, temporal analysis of phosphoproteome change revealed that phosphorylation of NPM S254 and COPA S173 was observed from the early (6 h) and late (24 h) time point after nocodazole treatment, respectively, suggesting that NPM S254 may be involved in the induction of M-phase arrest by nocodazole, whereas COPA S173 may be caused as a result of M-phase arrest.

Mitotic Modulation of Translation Elongation Factor 1 Leads to Hindered tRNA Delivery to Ribosomes

“…2A). Similar results were obtained upon reprobing with 32 P-labeled DNA oligonucleotide specific to arginyl tRNA and glutamyl tRNA (data not shown). Electrophoresis of tRNA samples from non-synchronized and mitotic cells under acidic conditions, which preserve aminoacylation, ruled out possible differences in the overall levels of charged aa-tRNA in mitotic cells (Fig.…”

Phosphorylation of eIF4GII and 4E-BP1 in response to nocodazole treatment: A reappraisal of translation initiation during mitosis

“…This comprehensive study did not identify any phosphopeptides in eIF4GI, although an earlier independent study 42 did find phosphorylation of this paralog at both known sites (Ser 1186 and Ser 1188), together with another at Ser 1232. All these sites reside in the C-FAG fragment of eIF4GI and again could reflect the alteration in intensity of the 2 bands observed in Figure 5B, comparing lanes 5 and 6.…”

Enhanced Antitumor Activity of an Anti-5T4 Antibody–Drug Conjugate in Combination with PI3K/mTOR inhibitors or Taxanes

“…More proteins were modulated by MMAF-Ome, 5T4-ADC, or PF-384 alone, with only some showing cooperative effects by combined treatment with 5T4-ADC/ PF-384 or MMAF-OMe/PF-384. This comports with other largescale phosphoproteomic studies that identified a number of initiation factors differentially phosphorylated in response to the nocodazole treatment (32,33). Whereas targeting of translational components by auristatin-based agents is a novel finding, the effects of PF-384 are quite expected, given the known role of PI3K and mTOR kinases in the control of protein synthesis.…”