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 vario us 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 th e strength of MAP-2 for microtubules, we focused on understanding the effect of protein kinase C phosphorylation on MTBR binding to taxol-st abilized microtubules. We used bacterially expressed MAP-2 MTBR, conta ining newly introduced CNBr-cleavable methionyl residues, as well as m ass spectrometry and site-specific mutagenesis to locate and confirm a ssignments of critical phosphorylation sites. We report on the localiz ation and role of phosphoryls on two specific residues (Ser-1703 and S er-1711) in terms of kinase-mediated control of MTBR-stimulated tubuli n polymerization. Ser-1703 is situated in the so-called first inter-re peat, and Ser-1711 is located in the second nonidentical repeat. Upon more extensive protein kinase C action, microtubule binding can be abo lished by phosphorylation at Ser-1728, but this effect is conditionall y 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 st rategy presented in this report may facilitate future studies with oth er kinases.