Functional analysis of the MAP2 repeat domain

Abstract

The neuronal microtubule-associated protein MAP2 binds to microtubules via a domain near its C terminus containing a set of 3 or 4 imperfect repeats of a 31 amino acid motif. Using naturally occurring and mutated forms of the molecule containing between 1 and 4 repeats we have examined the contribution that these repeats make to MAP2 function and explored the significance of their repetition. The experiments utilised the short 3- and 4-repeat splice variants MAP2c and MAP2d that are expressed in developing neurons and in glia respectively, and mutant 1- and 2-repeat versions that were produced by using in vitro mutagenesis to remove further 31 amino acid units while leaving the rest of the molecule unaltered. The properties of these MAP2 variants were compared both with respect to their influence on microtubules in transfected non-neuronal cells and their ability to promote microtubule assembly in vitro. We found that each of the known effects of MAP2, including the bundling of microtubules and induction of process formation in living cells, are expressed by the 1-repeat form MAP2c3, which contains only the third repeat (R3). A second 1-repeat form, MAP2c4, which contains only R4, interacts more weakly with tubulin in vitro and does not bind to microtubules in transfected cells. The microtubule-related properties of MAP2 thus arise mainly from a single predominant repeat unit, R3. In vitro assembly experiments showed that the primary effect of all the repeats is to lower the critical concentration of tubulin required for microtubule assembly but that they differ greatly in potency. The results did not reveal a separate function related to the repetition of the repeat motifs, but instead suggest that its purpose is to tailor the efficiency of MAP2 to the cellular environment in which it has to function.

The Phosphorylation State of Threonine-220, a Uniquely Phosphatase-Sensitive Protein Kinase A Site in Microtubule-Associated Protein MAP2c, Regulates Microtubule Binding and Stability

“…The importance of T 220 , nevertheless, is underlined by the observations that microtubule binding and stabilization are particularly sensitive to the phosphorylation status of this site: MAP2c phosphorylated at the other (two) sites, but not at T 220 , binds and stabilizes microtubules significantly stronger than either nonphosphorylated or fully PKA-phosphorylated MAP2c. This observation raises the possibility of site-specific bidirectional control of MAP2 action and represents a significant deviation from previous one-sided data that show only weakening of microtubule-MAP2 interaction upon phosphorylation by PKA (5,9,10) or by other kinases, such as MARK (6,7), PKC (16), or Pro-directed kinases (15) so far. Our result recalls the classical observation that the site of phosphate incorporation, rather than its amount, is critical in determining the microtubule-binding activity of MAP2 (24).…”

“…The primary function of MAP2, along with other structural MAPs, is to regulate microtubule function via a rather multifarious influence on its stability and dynamics. MAP2 is instrumental in nucleation, polymerization, stability, and bundling ( − ) but also plays a role in tethering other proteins to microtubules ( , ), cross-linking microtubules with other cytoskeletal components ( , ), and controlling microtubular transport ( , ). To fulfill such a complicated function, the level and pattern of MAP2 phosphorylation is under complex control by various protein kinases and phosphatases ( , ).…”

“…Protein kinase C-mediated phosphorylation at several sites (S 1703 , S 1711 , and S 1728 ) within the microtubule-binding region has a profound effect on microtubule binding (). Specific phosphorylation at some other, adjacent KXGS motifs (S 1682 and S 1713 ) by p110 microtubule-affinity regulating kinase (MARK) leads to the dissociation of MAP2 from microtubules and a pronounced increase in the dynamic instability of microtubules ( , ). MARK phosphorylates homologous sites within MAP4 ( , ) and tau (); the effect is invariably a stabilization of the microtubule−MAP binding.…”

“…Specific phosphorylation at some other, adjacent KXGS motifs (S 1682 and S 1713 ) by p110 microtubule-affinity regulating kinase (MARK) leads to the dissociation of MAP2 from microtubules and a pronounced increase in the dynamic instability of microtubules ( , ). MARK phosphorylates homologous sites within MAP4 ( , ) and tau (); the effect is invariably a stabilization of the microtubule−MAP binding. Similar consequences have been observed for the Pro-rich region nearby (T 1615 ), where specificity of phosphorylation by Pro-directed kinases (MAPK, GSK3, and cdc-2 kinase) () and of dephosphorylation by PP1 and PP2A has been reported ().…”

More than a marker: potential pathogenic functions of MAP2

“…MAP2 is comprised of five functionally distinct domains: the N-terminus, a projection domain, a proline-rich domain, the MT-binding domain, and the C-terminus (Figure 1A). The MT-binding domain in turn contains 3-4 MT binding imperfect repeats, though only the last of these seems to be necessary for the MT bundling activity of the protein (Ludin et al, 1996). Though this domain was originally identified as the critical domain for MT-binding functionality based on its affinity for bovine MTs (Lewis et al, 1988), subsequent data has suggested that it alone is not sufficient for full MT-binding of MAP2.…”