Localization of the phosphorylation sites for different kinases in the microtubule-associated protein MAP2.
The phosphorylation of microtubule-associated protein 2 (MAP2) by four different kinases was studied in vitro to determine whether MAP2 is phosphorylated in its tubulin binding region or in the microtubule projection portion. Fragments corresponding to both regions of MAP2 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 MAP2. The position of these two functional domains was determined with respect to the carboxy terminal of the molecule, by labeling MAP2 exclusively at the carboxy terminal and subjecting it to pepsin digestion. The results suggested that the projection region of MAP2 contained the carboxy terminal of the protein. A phosphorylation map was constructed by subjecting phosphorylated MAP2 to enzymatic digestion using Staphylococcus aureus V8 protease or to chemical cleavage using N-chlorosuccinimide. The results indicated that all four kinases phosphorylated MAP2 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 MAP2.
D000818:Animals; D001665:Binding Sites; D001921:Brain / Q000201:enzymology; D002118:Calcium / Q000494:pharmacology; D002147:Calmodulin / Q000494:pharmacology; D002918:Chymotrypsin; D000242:Cyclic AMP / Q000494:pharmacology; D010450:Endopeptidases; D007328:Insulin / Q000494:pharmacology; D008869:Microtubule-Associated Proteins / Q000378:metabolism*; D008870:Microtubules / Q000201:enzymology*; D010434:Pepsin A; D010446:Peptide Fragments / Q000378:metabolism; D010743:Phospholipids / Q000494:pharmacology; D010766:Phosphorylation; D011494:Protein Kinases / Q000378:metabolism*; D012697:Serine Endopeptidases; D013388:Succinimides; D013552:Swine; D014357:Trypsin; D014364:Tryptophan; D014404:Tubulin / Q000378:metabolism
D002147:Calmodulin; D007328:Insulin; D008869:Microtubule-Associated Proteins; D010446:Peptide Fragments; D010743:Phospholipids; D013388:Succinimides; D014404:Tubulin; C015752:N-chlorosuccinimide; D014364:Tryptophan; D000242:Cyclic AMP; D011494:Protein Kinases; D010450:Endopeptidases; D012697:Serine Endopeptidases; D002918:Chymotrypsin; C028344:glutamyl endopeptidase; D014357:Trypsin; D010434:Pepsin A; D002118:Calcium