Stimulation of tubulin polymerization by MAP-2. Control by protein kinase C-mediated phosphorylation at specific sites in the microtubule-binding region.
Microtubule-associated protein-2 (MAP-2) is extensively phosphorylated on serine and threonine residues, and such modifications affect various cellular processes, including microtubule dynamics. Although MAP-2 phosphorylation has been studied both in vitro and in vivo, nothing is known about the exact location of phosphorylated sites influencing the strength of MAP-2 binding to microtubules. Because the microtubule-binding region (MTBR) retains virtually all of the binding affinity of intact MAP-2 for microtubules, we focused on understanding the effect of protein kinase C phosphorylation on MTBR binding to taxol-stabilized microtubules. We used bacterially expressed MAP-2 MTBR, containing newly introduced CNBr-cleavable methionyl residues, as well as mass spectrometry and site-specific mutagenesis to locate and confirm assignments of critical phosphorylation sites. We report on the localization and role of phosphoryls on two specific residues (Ser-1703 and Ser-1711) in terms of kinase-mediated control of MTBR-stimulated tubulin polymerization. Ser-1703 is situated in the so-called first inter-repeat, and Ser-1711 is located in the second nonidentical repeat. Upon more extensive protein kinase C action, microtubule binding can be abolished by phosphorylation at Ser-1728, but this effect is conditionally dependent on the phosphorylation state at Ser-1703 and/or Ser-1711. Our results suggest that in vivo binding of MAP-2 to microtubules may be controlled by protein kinase C action at these residues, and the strategy presented in this report may facilitate future studies with other kinases.
Ainsztein
A M
AM
Department of Biochemistry and Molecular Biology, University of Florida College of Medicine, Gainesville 32610-0245.
Purich
D L
DL
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D001921:Brain / Q000201:enzymology / Q000378:metabolism*; D002417:Cattle; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism; D007700:Kinetics; D008869:Microtubule-Associated Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D008870:Microtubules / Q000378:metabolism*; D008954:Models, Biological; D008969:Molecular Sequence Data; D009206:Myocardium / Q000201:enzymology; D010766:Phosphorylation; D010768:Phosphoserine / Q000378:metabolism; D011493:Protein Kinase C / Q000378:metabolism*; D051381:Rats; D014404:Tubulin / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*
D008869:Microtubule-Associated Proteins; D014404:Tubulin; D010768:Phosphoserine; D017868:Cyclic AMP-Dependent Protein Kinases; D011493:Protein Kinase C