The major neuronal post-translational modification of tubulin, polyglutamylation, can act as a molecular potentiometer to modulate microtubule-associated proteins (MAPs) binding as a function of the polyglutamyl chain length. The relative affinity of Tau, MAP2, and kinesin has been shown to be optimal for tubulin modified by ϳ3 glutamyl units. Using blot overlay assays, we have tested the ability of polyglutamylation to modulate the interaction of two other structural MAPs, MAP1A and MAP1B, with tubulin. MAP1A and MAP2 display distinct behavior in terms of tubulin binding; they do not compete with each other, even when the polyglutamyl chains of tubulin are removed, indicating that they have distinct binding sites on tubulin. Binding of MAP1A and MAP1B to tubulin is also controlled by polyglutamylation and, although the modulation of MAP1B binding resembles that of MAP2, we found that polyglutamylation can exert a different mode of regulation toward MAP1A. Interestingly, although the affinity of the other MAPs tested so far decreases sharply for tubulins carrying long polyglutamyl chains, the affinity of MAP1A for these tubulins is maintained at a significant level. This differential regulation exerted by polyglutamylation toward different MAPs might facilitate their selective recruitment into distinct microtubule populations, hence modulating their functional properties. Microtubules (MTs)1 are dynamic polymers, which are essential for a large variety of cellular functions such as cell morphology and polarity, cell motility, intracellular trafficking, and cell division. They are made up of ␣-and ␤-tubulin heterodimers, the two related subunits displaying a large isoform polymorphism due to the expression of multiple genes whose products are substrates for several post-translational modifications (for review, see Refs.

High molecular weight microtubule-associated proteins MAP1A and MAP2 form thin projections from microtubule surfaces and have been implicated in crosslinking microtubules and other cytoskeletal components. We have purified native MAP1A from bovine brain and have studied its interaction with G- and F-actin. Using a solid-phase immunoassay we show that MAP1A binds in a dose-dependent manner to both G-actin and F-actin. Addition of MAP1A to F-actin causes gelation of F-actin and SDS-PAGE analysis shows that MAP1A co-sediments with the gelled network, under conditions where F-actin alone does not pellet. The low apparent viscosity of F-actin is markedly increased in the presence of MAP1A, suggesting that MAP1A can crosslink F-actin. Co-incubation experiments indicate that MAP1A and MAP2 may bind to common or overlapping sites on the actin molecule. The widespread distribution of MAP1A and its interaction with microtubules, actin, and intermediate filaments suggests that it may constitute an important determinant of neuronal and non-neuronal cellular morphology.

. We now show that phosphorylation affects the in vitro binding of MAP1B with microfilaments. Native MAP1B does not bind to microfilaments but after treatment with alkaline phosphatase the dephosphorylated MAP1B binds and cosediments with microfilaments. Dephosphorylation kinetics suggest that the PDPK site, but not CKII sites, may negatively regulate the interaction with F-actin. The ability of dephosphorylated MAP1B to crosslink microfilaments was also examined and showed that MAP1B exhibits only a weak crosslinking of F-actin when compared with MAP2.Key words." High-molecular weight MAPs; MAP1B; Phosphorylation; Microfilaments; Cytoskeleton MAP1B is known to be phosphorylated by several prolinedirected protein kinases (PDPK) and casein kinase II (CKII; [13,14]). MAP1B phosphorylation is also regulated during development: PDPK phosphorylation is abundant in axons at early neuronal development stages and restricted to growth cones at later stages [14] while CKII phosphorylation is present in both axons and dendrites even in late stages of neuronal maturation [14,15], suggesting a different function in neurogenesis for these two phosphorylation modes.We have recently characterised the phosphorylation state of purified MAP1B and shown that phosphorylation at specific residues can modulate the affinity of MAP1B for microtubules: phosphorylation at the PDPK sites weakens its interaction with microtubules while the phosphorylation at the casein kinase II sites does not apparently alter MAP1B:microtubule interaction [2]. Consequently, in this report we have further examined the affect of phosphorylation on the interaction of MAP1B with actin. Our data suggests that dephosphorylated MAP1B can bind to actin filaments and that its interaction is modulated by the state of phosphorylation.

Prion diseases are marked by the cerebral accumulation of conformationally modified forms of the cellular prion protein (PrPC), known as PrPres. The region comprising the residues 106-126 of human PrP seems to have a key role in this conformational conversion, because a synthetic peptide homologous with this sequence (PrP106-126) adopts different secondary structures in different environments. To investigate the molecular determinants of the physicochemical characteristics of PrP106-126, we synthesized a series of analogues including PrP106-126 HD, PrP106-126 A and PrP106-126 K, with L-His → D-His, His → Ala and His → Lys substitutions respectively at position 111, PrP106-126 NH2 with amidation of the C-terminus, PrP106-126 V with an Ala → Val substition at position 117, and PrP106-126 VNH2 with an Ala → Val substitution at position 117 and amidation of the C-terminus. The analysis of the secondary structure and aggregation properties of PrP106-126 and its analogues showed the following. (1) His111 is central to the conformational changes of PrP peptides. (2) Amidation of the C-terminal Gly126 yields a predominantly random coil structure, abolishes the molecular polymorphism and decreases the propensity of PrP106-126 to generate amyloid fibrils. (3) PrP106-126 V, carrying an Ala → Val substitution at position 117, does not demonstrate a fibrillogenic ability superior to that of PrP106-126. However, the presence of Val at position 117 increases the aggregation properties of the amidated peptide. (4) Amyloid fibrils are not required for neurotoxicity because the effects of PrP106-126 NH2 on primary neuronal cultures were similar to those of the wild-type sequence. Conversely, astroglial proliferation is related to the presence of amyloid fibrils, suggesting that astrogliosis in prion encephalopathies without amyloid deposits is a mediated effect rather than a direct effect of disease-specific PrP isoforms.

In a recent study, we have shown that sulfonate buffers affect microtubule assembly and alter microtubule protein composition (Pedrotti et al., 1993). In particular, we noted that PIPES buffer leads to removal of MAP1 from the microtubule surface without affecting the association of MAP2 with microtubules. This observation has been exploited to develop a simple purification procedure for MAP1A using twice-cycled microtubule protein prepared from whole bovine brain. A single chromatographic step on an ion-exchange column results in > 90% pure MAP1A. Using purified MAP1A, we now show that MAP1A (a) binds in a dose-dependent manner to unpolymerized tubulin and assembled microtubules, (b) binds 13-15 mol of tubulin dimers in assembled microtubules, (c) promotes both nucleation and elongation of tubulin, and (d) promotes incorporation of tubulin dimers at low GTP concentrations and of tubulin dimers and oligomers at high GTP concentrations. MAP1A lowers the critical concentration for assembly, and MAP1A-promoted incorporation of dimers has an association rate constant (K+1) of 39.3 x 10(6) M-1s-1 and a dissociation rate constant (K-1) of 15 s-1; both constants are about 2-3-fold higher compared with MAP2.