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Localization of the Phosphorylation Sites for Different Kinases in the Microtubule‐Associated Protein MAP 2
Author(s) -
Hernández Maria Angeles,
Wandosell Francisco,
Avila Jesús
Publication year - 1987
Publication title -
journal of neurochemistry
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.75
H-Index - 229
eISSN - 1471-4159
pISSN - 0022-3042
DOI - 10.1111/j.1471-4159.1987.tb13130.x
Subject(s) - phosphorylation , kinase , mapk14 , trypsin , microtubule , biochemistry , protease , biology , chymotrypsin , peptide , microtubule associated protein , tubulin , chemistry , enzyme , microbiology and biotechnology , protein kinase a , mitogen activated protein kinase kinase
The phosphorylation of microtubule‐associated protein 2 (MAP 2 ) by four different kinases was studied in vitro to determine whether MAP 2 is phosphorylated in its tubulin binding region or in the microtubule projection portion. Fragments corresponding to both regions of MAP 2 were produced not only by chymotrypsin or trypsin digestion, but also using pepsin, a broad chain‐specificity protease, a result supporting previous notions of the two‐domain structure of MAP 2 . The position of these two functional domains was determined with respect to the carboxy terminal of the molecule, by labeling MAP 2 exclusively at the carboxy terminal and subjecting it to pepsin digestion. The results suggested that the projection region of MAP 2 contained the carboxy terminal of the protein. A phosphorylation map was constructed by subjecting phosphorylated MAP 2 to enzymatic digestion using Staphylococcus aureus V8 protease or to chemical cleavage using N ‐chlorosuccinimide. The results indicated that all four kinases phosphorylated MAP 2 in a 42‐kilodalton peptide that contained the tubulin binding region but differed in the level and localization of the sites at which they phosphorylated the projection of MAP 2 .