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

Abstract

Pre-phosphorylation of the microtubule-associated protein MAP2 with the co-purifying CAMP-independent protein kinase (a) decrease the affinity of MAP2 for taxol-stabilised microtubules, (b) increases the dissociation rate constant for microtubule polymerisation, each of which is dependent upon the level of phosphorylation, but (c) has no effect on the association rate constant. Microtubule assembly has no effect on the kinetics of phosphorylation, whereas phosphorylation of pre-assembled microtubules causes their immediate depolymerisation at a rate which is proportional to the initial rate of phosphorylation. The results suggest that the modulated phosphorylation of MAP2 may regulate microtubule length in vivo.The microtubule-associated protein MAP2 interacts in the reassembled microtubule with 12 tubulin dimers and can be phosphorylatated in vitro by a CAMP-independent protein kinase to the extent of 12 mol.mol-' [I -31. Ten of these phosphorylation sites lie within the known tubulin-binding domain [3] suggesting that phosphorylation may regulate the MAP2 : tubulin interaction. Indeed, phosphorylation has been reported to affect the rate constants for assembly and disassembly [4, S ] , although the precise extent of phosphorylation was not determined.The kinetics of microtubule assembly in vitro have been described in considerable detail (e.g. [6, 71). Following transfer of the microtubule protein to assembly conditions, for example by addition of GTP and/or raising the temperature, there is an initial lag phase followed by a period of rapid polymerisation to an equilibrium plateau value. The lag phase is due to the requirement to form nucleation sites, and it can be abolished by appropriate seeding, while the plateau value is directly proportional to the active subunit concentration above a critical concentration. Analysis of the kinetics shows that the rate of assembly conforms to a simple linear condensation model for polymerisation, with the pseudo-first-order rate being determined by the concentrations of free subunits and nucleation sites. When the number of nucleation sites is maintained constant, a plot of the rate of assembly as a function of the subunit concentration yields a straight line passing through the critical concentration and with a slope equal to the product of the association rate constant ( k , and the concentration of nucleation sites. The intercept at zero protein concentration of this plot equals the product of the dissociation rate constant (k1) and the concentration of nucleation sites. The critical concentration is therefore merely k.. l / k + Under conditions in which the critical concentration is different at the two ends of the microtubule, subunits will preferentially associate at one end (the + end) and preferentially dissociate at the other (the -end) [7, 81. There can therefore, at steady state, be a flux of subunits through the microtubule, a process known as treadmilling. The nonAbbreviution. MAP2, microtubule-associated protein 2.equivalence of the two ends is probably c...

Potentiation of antitumour activity of docetaxel by combination with trastuzumab in a human prostate cancer xenograft model and underlying mechanisms

“…Taxanes block tubulin depolymerisation, resulting in impairment of cellular mitosis (Horwitz, 1994), generating nuclear abnormalities and multipolar divisions, ultimately leading to cell death (Abal et al, 2003). Taxane-induced tubulin alterations may also affect microtubule-associated proteins (MAPS) (Burns et al, 1984, Haycock et al, 1992, potentially leading to multiple cellular dysfunctions. Microtubule-associated proteins are among the protein clients of heat-shock proteins (HSP70 and HSP90), themselves associated with and stabilising numerous proteins such as HER2, AKT and the AR (Georget et al, 2002).…”

The 93 kDa protein gephyrin and tubulin associated with the inhibitory glycine receptor are phosphorylated by an endogenous protein kinase

“…For the microtubul¢-assoeiated protein 2 (MAP 2), it has been shown that phosphorylation by a co-purifying cyclic AMP-independent kinase reduces its affinity to mierotubules [25]. Conversely, phosphorylation by calmodulin-dependent kinase reversibly renders tubulin incapable of assembly and binding to microtubule-associated proteins [18].…”

Phosphorylation of Microtubule-associated Proteins MAP2 and MAP4 by the Protein Kinase p110mark

“…MAPs isolated from tissue or cells are phosphoproteins (Sloboda et al, 1975;Vallee, 1980;Burns et al, 1984;Tsuyama et al, 1986;Brugg and Matus, 1991;Watanabe et al, 1993), MAPs are good substrates for many protein kinases in vitro (Theurkauf and Vallee, 1983;Lindwall and Cole, 1984;Mori et al, 1991;Drewes et al, 1992), and phosphorylation interferes with their microtubule stabilizing capacity (Brugg and Matus, 1991;Shiina et al, 1992;Drechsel et al, 1992;Biernat et al, 1993;Brandt et al, 1994;Ookata et al, 1995;Trinczek et al, 1995). In the case of tau protein, phosphorylation has been extensively studied, because aberrantly phosphorylated tau is involved in the neurofibrillar pathology of Alzheimer's disease (reviewed by Goedert (1993), Mandelkow and Mandelkow (1993), and Trojanowski and Lee (1994)).…”