Inhibition of microtubule assembly by phosphorylation of microtubule-associated proteins

Abstract

32P labeling of microtubular protein by endogenous protein kinase activity is shown to result from a net increase in protein-bound phosphate and is not the result of a phosphate exchange reaction between ATP and phosphoprotein. Protein phosphorylation is maximal in the presence of 0.5 mM Mg2+ and 0.25 mM ATP, resulting in approximately 2.8 nmol of phosphate/mg of protein. However, phosphorylation can be increased two-to threefold by cAMP. The protein substrates for phosphorylation either the absence or presence of cAMP are the microtubule-associated proteins which copurify with tubulin and promote microtubule assembly. Phosphorylation of microtubule-associated proteins inhibits both the rate and extent of microtubule assembly when the protein is exposed to conditions which result in dissociation of rings. These results are taken to indicate that phosphorylation modifies MAPs so that they have a reduced ability to form an assembly-competent complex with tubulin.

Protein‐kinase‐C‐catalyzed phosphorylation of the microtubule‐binding domain of microtubule‐associated protein 2 inhibits its ability to induce tubulin polymerization

“…4). Therefore, it is likely that the phosphorylation of the microtubule-binding domain (1 -2 niol phosphate/mol domain) [23], phosphorylation of MAP2 by any of these three kinases inhibits the ability of MAP2 to promote tubulin polymerization [8,10,14,18,231. Furthermore, in the phosphorylation reaction catalyzed by each of these three kinases the close relationship was commonly observed between the amount of phosphate incorporated into MAP2 and the extent of decrease in MAP2 activity.…”

“…Phosphorylation of MAP2 catalyzed by these kinases equally results in reduction of abilities of MAP2 to promote tubulin polymerization and to cross-link actin filaments [8,10,14,[16][17][18][19], On digestion with trypsin or chymotrypsin, MAP2 is divided into two domains [9]. The larger domain, about 240 kDa in size, appears as a projection on the surface of microtubules and is called the projection domain [9].…”

The multiple phosphorylation of the microtubule-associated protein MAP2 controls the MAP2: tubulin interaction

“…However, ATP can also bind to tubulin and induce assembly [I I] so that the effect of ATP on treadmilling could be mediated by either phosphorylation or by binding to tubulin. Similarly, the effect of guanosine S'-[p,ymethyleneltriphosphate on treadmilling could be due to either mechanism since GTP can also act as a phosphoryl donor [4].We have therefore sought to determine whether the phosphorylation of MAP2 changes the MAP2: tubulin interaction, and if so whether it influences the kinetics of microtubule assembly. The results show that phosphorylation reduces the affinity of MAP2 for tubulin in proportion to the level of phosphorylation, and that this reduced affinity induces an equivalent effect on the dissociation rate constant.…”

“…However, ATP can also bind to tubulin and induce assembly [I I] so that the effect of ATP on treadmilling could be mediated by either phosphorylation or by binding to tubulin. Similarly, the effect of guanosine S'-[p,ymethyleneltriphosphate on treadmilling could be due to either mechanism since GTP can also act as a phosphoryl donor [4].…”

Effects of microtubule‐associated proteins on network formation by neurofilament‐induced polymerization of tubulin

“…Here, it should be noted that the activity of neurofilaments to stimulate tubulin polymerization was totally suppressed when the neurofilament preparation was first treated with phosphatase to be dephosphorylated [ 141. Because dephosphorylation of MAPS would enhance the nucleating ability for tubulin assembly [25,26], if MAPS contaminated the <purified neurofilament preparation and promoted the gelling reaction, dephosphorylation reaction should have occurred in MAPS as well and thus caused promotion of gelation. However, this was not the case.…”