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.
Department of Biochemistry and Molecular Biology, University of Florida College of Medicine, Gainesville 32610-0245.
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AM
Ainsztein
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Purich
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