Document Detail


Anti-phosphopeptide antibody, P-STM as a novel tool for detecting mitotic phosphoproteins: identification of lamins A and C as two major targets.
MedLine Citation:
PMID:  15597429     Owner:  NLM     Status:  MEDLINE    
Abstract/OtherAbstract:
A polyclonal, phospho-epitope-specific antibody (P-STM) was generated to detect the activated p21-activated kinase 2 (PAK2), based on the regulatory autophosphorylation site Thr(402) of PAK2 [Yu et al., 1998]. In this report, we show that this antibody can also recognize many phosphoproteins in mitotic HeLa and A431 cells. Signal of these phosphoproteins emerged after treating the cells with nocodazole and okadaic acid, and was highly detected in G2-M phase transition of HeLa cells released from double thymidine block. Immunofluorescence analysis revealed that P-STM strongly stained HeLa cells at prometaphase and metaphase, but not at interphase and anaphase. Interestingly, this staining pattern was almost identical to that obtained by staining with MPM2, a monoclonal antibody known to react with phosphoproteins in mitotic HeLa cells. However, the phosphoproteins detected by the two antibodies are quite different. Two-dimensional gel electrophoresis (2DE) and tryptic peptide fingerprint analysis by matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) mass spectrometry were employed to identify lamins A and C as two of the mitotic cell-specific phosphoproteins recognized by P-STM. Lamins A and C immunoprecipitated from nocodazole-treated cells, but not from untreated cells showed strong reactivity to P-STM, and this reactivity lost completely after protein phosphatase 2A treatment. In summary, our results show that P-STM represents a novel tool for detecting mitotic phosphoproteins, which are different from those recognized by MPM2, and that lamins A and C are the two prominent mitotic phosphoproteins detected by P-STM.
Authors:
I-Chen Tsai; Ya-Ju Hsieh; Ping-Chiang Lyu; Jau-Song Yu
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Publication Detail:
Type:  Journal Article; Research Support, Non-U.S. Gov't    
Journal Detail:
Title:  Journal of cellular biochemistry     Volume:  94     ISSN:  0730-2312     ISO Abbreviation:  J. Cell. Biochem.     Publication Date:  2005 Apr 
Date Detail:
Created Date:  2005-03-15     Completed Date:  2005-09-26     Revised Date:  2006-11-15    
Medline Journal Info:
Nlm Unique ID:  8205768     Medline TA:  J Cell Biochem     Country:  United States    
Other Details:
Languages:  eng     Pagination:  967-81     Citation Subset:  IM    
Copyright Information:
(c) 2005 Wiley-Liss, Inc.
Affiliation:
Department of Cell and Molecular Biology, Graduate Institute of Basic Medical Sciences, Medical College of Chang Gung University, Tao-Yuan, Taiwan, Republic of China.
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MeSH Terms
Descriptor/Qualifier:
Amino Acid Sequence
Antibodies / immunology*
Electrophoresis, Gel, Two-Dimensional
Hela Cells
Humans
Lamin Type A / chemistry,  metabolism*
Mitosis*
Molecular Sequence Data
Phosphopeptides / immunology*
Phosphoproteins / analysis*,  immunology
Sequence Homology, Amino Acid
Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
Chemical
Reg. No./Substance:
0/Antibodies; 0/Lamin Type A; 0/Phosphopeptides; 0/Phosphoproteins; 0/lamin C

From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine


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