TY - JOUR
T1 - Partial characterization of the MPM-2 phosphoepitope
AU - Ding, Min
AU - Feng, Yang
AU - Vandré, Dale D.
N1 - Funding Information:
We thank Yunhi Choi for providing MAP4 samples. This work was supported by NIH Grant NS31777 to D.D.V.
PY - 1997/2/25
Y1 - 1997/2/25
N2 - The MPM-2 monoclonal antibody recognizes a distinctive group of proteins that are associated with structural components of the mitotic apparatus. These proteins become phosphorylated and MPM-2 reactive during M- phase and appear to be required for both the onset and completion of M- phase. Based upon the analysis of reported MPM-2 reactive sequences, we have developed a model for the essential elements that comprise the MPM-2 epitope. This model was tested by employing a series of synthetic phosphopeptides. We show here that a 14 amino acid synthetic phosphopeptide, derived from a potential MPM-2 site on human DNA topoisomerase II, is recognized by the MPM2 antibody. This phosphopeptide was sufficient to compete for MPM-2 antibody recognition of (1) an isolated native mitotic MPM-2 antigen on dot blots, (2) proteins on immunoblots of mitotic cell lysates, and (3) specific immunostaining of mitotic cells. These results indicated that the topoisomerase peptide contained all of the essential elements of the MPM-2 epitope. By substituting selected amino acids with alanine, we were able to examine the contribution of different amino acids to the binding between the MPM-2 antibody and the epitope. Changing the amino acid that was adjacent to the phosphorylated threonine residue on the C-terminal side (the +1 position) had no effect on MPM-2 antibody binding. However, substitution of aromatic amino acids at either the -2 or +2 positions reduced antibody recognition. The aromatic amino acid at the -2 position appeared to be the most critical residue of those tested that influenced antibody binding. These results provide information required for the molecular definition of the MPM-2 epitope and should aid in the identification of potential MPM-2 reactive sites on other mitotic phosphoproteins.
AB - The MPM-2 monoclonal antibody recognizes a distinctive group of proteins that are associated with structural components of the mitotic apparatus. These proteins become phosphorylated and MPM-2 reactive during M- phase and appear to be required for both the onset and completion of M- phase. Based upon the analysis of reported MPM-2 reactive sequences, we have developed a model for the essential elements that comprise the MPM-2 epitope. This model was tested by employing a series of synthetic phosphopeptides. We show here that a 14 amino acid synthetic phosphopeptide, derived from a potential MPM-2 site on human DNA topoisomerase II, is recognized by the MPM2 antibody. This phosphopeptide was sufficient to compete for MPM-2 antibody recognition of (1) an isolated native mitotic MPM-2 antigen on dot blots, (2) proteins on immunoblots of mitotic cell lysates, and (3) specific immunostaining of mitotic cells. These results indicated that the topoisomerase peptide contained all of the essential elements of the MPM-2 epitope. By substituting selected amino acids with alanine, we were able to examine the contribution of different amino acids to the binding between the MPM-2 antibody and the epitope. Changing the amino acid that was adjacent to the phosphorylated threonine residue on the C-terminal side (the +1 position) had no effect on MPM-2 antibody binding. However, substitution of aromatic amino acids at either the -2 or +2 positions reduced antibody recognition. The aromatic amino acid at the -2 position appeared to be the most critical residue of those tested that influenced antibody binding. These results provide information required for the molecular definition of the MPM-2 epitope and should aid in the identification of potential MPM-2 reactive sites on other mitotic phosphoproteins.
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U2 - 10.1006/excr.1996.3439
DO - 10.1006/excr.1996.3439
M3 - Article
C2 - 9056407
AN - SCOPUS:0031585864
SN - 0014-4827
VL - 231
SP - 3
EP - 13
JO - Experimental Cell Research
JF - Experimental Cell Research
IS - 1
ER -