Using quantitative proteomic method to analyze the P-STM antibody recognizable phosphorylation site on lamins A/C in mitotic HeLa S3 cells
| Autor: | Jeng-Ting Chen, 陳政廷 |
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| Rok vydání: | 2008 |
| Druh dokumentu: | 學位論文 ; thesis |
| Popis: | 96 P-STM, a phosphoepitope specific antibody was generated against the autophosphorylation site of p21-activated kinase 2 (PAK2), can also recognize many phosphoproteins including lamins A and C in mitotic HeLa S3 cells. The P-STM recognizable phosphoepitope on lamins A/C may serve as good targets to monitor the phosphorylation change of lamins A/C during cell cycle. The immunoprecipitated lamins A/C from unsynchronized cells could be in vitro phosphorylated by p34cdc2 and created the specific phosphoepitope to P-STM. The p34cdc2-catalyzed phosphorylation site may represent the same one recognized by P-STM from cells treated with nocodazole in vivo. In vitro phosphorylation and immunoblot analyses of recombinant proteins containing different region of lamin C and site-directed mutagenesis at potential phosphorylation sites, as well as dot blot analysis of various synthetic phosphopeptides based on the common amino sequence (S12GAQASSTPLSPTR25) from tryptic phosphorylated lamins A/C, revealed that phosphorylated Thr-19 represents the sole phosphoepitope to P-STM. CE-ESI-MS/MS and LC-ESI-MS/MS were applied to separate and determine the phosphopeptide isomers from the tryptic phosphorylated lamin C. Moreover, we have used the SILAC method to quantify the phosphorylation level of lamins A/C between interphase and mitotic phase. By this way, we showed that Thr-19 and Ser-22 are the two major sits phosphorylated in lamins A/C in mitotic HeLa cells. |
| Databáze: | Networked Digital Library of Theses & Dissertations |
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