DOMAINS OF TAU-PROTEIN, DIFFERENTIAL PHOSPHORYLATION, AND DYNAMIC INSTABILITY OF MICROTUBULES

The dynamic instability of microtubules is thought to be regulated by MAPs and phosphorylation. Here we describe the effect of the neuronal microtubule-associated protein tau by observing the dynamics of single microtubules by video microscopy. We used recombinant tau isoforms an d tau mutants, and we phosphorylated tau by the neuronal kinases MARK (affecting the KXGS motifs within tau's repeat domain) and cdk5 (phosp horylating Ser-Pro motifs in the regions flanking the repeats). The va riants of tau can be broadly classified into three categories, dependi ng on their potency to affect microtubule dynamics. ''Strong'' tau var iants have four repeats and both flanking regions. ''Medium'' variants have one to three repeats and both flanking regions. ''Weak'' variant s lack one or both of the flanking regions, or have no repeats; with s uch constructs, microtubule dynamics is not significantly different fr om that of pure tubulin. N- or C-terminal tails of tau have no influen ce on dynamic instability. The two ends of microtubules (plus and minu s) showed different activities but analogous behavior. These results a re consistent with the ''jaws'' model of tau where the flanking region s are considered as targeting domains whereas the addition of repeats makes them catalytically active in terms of microtubule stabilization. The dominant changes in the parameters of dynamic instability induced by tau are those in the dissociation rate and in the catastrophe rate (up to 30-fold). Other rates change only moderately or not at all (as sociation rate increased up to twofold, rates of rescue or rapid shrin kage decreased up to similar to twofold). The order of repeats has lit tle influence on microtubule dynamics (i.e., repeats can be re-arrange d or interchanged), arguing in favor of the ''distributed weak binding '' model proposed by Butner and Kirschner (1991); however, we confirme d the presence of a ''hotspot'' of binding potential involving Lys274 and Lys281 observed by Goode and Feinstein, 1994. Phosphorylation of S er-Pro motifs by cdk5 (mainly Ser 202, 235, and 404) in the flanking r egions had a moderate effect on microtubule dynamics while phosphoryla tion at the ''Alzheimer''-site Ser262 by MARK eliminated tau's interac tions with microtubules. In both cases the predominant effects of phos phorylation are on the rates of tubulin dissociation and catastrophe w hereas the effects on the rates of association or rescue are comparati vely small.