Site-specific phosphorylation by protein kinase C inhibits assembly-promoting activity of microtubule-associated protein 4.
We have examined the phosphorylation of bovine microtubule-associated protein 4 (MAP4), formerly named MAP-U, by protein kinase C (PKC). When MAP4 was incubated with PKC, about 1 mol of phosphate was incorporated/mol of MAP4. Phosphorylation of MAP4 caused a remarkable decrease in the ability of the MAP to stimulate microtubule assembly. MAP4 consists of an amino-terminal projection domain and a carboxyl-terminal microtubule-binding domain. The carboxyl-terminal domain is subdivided into a Pro-rich region and an assembly-promoting (AP) sequence region containing four tandem repeats of AP sequence that is conserved in MAP4, MAP2, and tau [Aizawa et al. (1990) J. Biol. Chem. 265, 13849-13855]. In order to identify the site of MAP4 phosphorylated by PKC, a series of expressed MAP4 fragments was prepared and treated with the kinase. A fragment corresponding to the Pro-rich region (P fragment) was phosphorylated, while fragments corresponding to the projection domain and the AP sequence region were not. In addition, chymotryptic digestion of an authentic MAP4 prephosphorylated by PKC revealed that phosphate was incorporated almost exclusively into a 27-kDa fragment containing the carboxyl-terminal half of the Pro-rich region. We investigated the phosphorylation site in MAP4 using the P fragment and found that Ser815 was phosphorylated almost exclusively. We conclude that the phosphorylation of a single Ser residue in the Pro-rich region negatively regulates the assembly-promoting activity of MAP4.
Mori
A
A
Department of Biophysics and Biochemistry, Faculty of Science, University of Tokyo, Japan.
Aizawa
H
H
Saido
T C
TC
Kawasaki
H
H
Mizuno
K
K
Murofushi
H
H
Suzuki
K
K
Sakai
H
H
D000818:Animals; D002417:Cattle; D066298:In Vitro Techniques; D008869:Microtubule-Associated Proteins / Q000737:chemistry / Q000378:metabolism*; D008870:Microtubules / Q000378:metabolism* / Q000648:ultrastructure; D010449:Peptide Mapping; D010750:Phosphoproteins / Q000737:chemistry / Q000378:metabolism; D010766:Phosphorylation; D010768:Phosphoserine / Q000378:metabolism; D011493:Protein Kinase C / Q000378:metabolism*; D014404:Tubulin / Q000378:metabolism
D008869:Microtubule-Associated Proteins; D010750:Phosphoproteins; D014404:Tubulin; D010768:Phosphoserine; D011493:Protein Kinase C