Related Experiment Videos
Phosphorylation determines the binding of microtubule-associated protein 2 (MAP2) to microtubules in living cells
Abstract:
The influence of phosphorylation on the binding of microtubule-associated protein 2 (MAP2) to cellular microtubules was studied by microinjecting MAP2 in various phosphorylation states into rat-1 fibroblasts, which lack endogenous MAP2. Conventionally prepared brain MAP2, containing 10 mol of endogenous phosphate per mol (MAP2-P10), was completely bound to cellular microtubules within 2-3 min after injection. MAP2 prepared in the presence of phosphatase inhibitors, containing 25 mol/mol of phosphate (MAP2-P25), also bound completely. However, MAP2 whose phosphate content had been reduced to 2 mol phosphate per mol by treatment with alkaline phosphatase in vitro (MAP2-P2) did not initially bind to microtubules, suggesting that phosphorylation of certain sites in MAP2 is essential for binding to microtubules. MAP2-P10 was further phosphorylated in vitro via an endogenously bound protein kinase activity, adding 12 more phosphates, giving a total of 22 mol/mol. This preparation (MAP2-P10+12) also did not bind to microtubules. Assay of the binding of these preparations to taxol-stabilized tubulin polymers in vitro confirmed that their binding to tubulin depended on the state of phosphorylation, but the results obtained in microinjection experiments differed in some cases from in vitro binding. The results suggest that the site of phosphate incorporation rather than the amount is the critical factor in determining microtubule binding activity of MAP2. Furthermore, the interaction of MAP2 with cellular microtubules may be influenced by additional factors that are not evident in vitro.
Insights
Phosphorylation critically influences microtubule-associated protein 2 (MAP2) binding to cellular microtubules. Specific phosphorylation sites, not just the total phosphate amount, are essential for MAP2
Area of Science:
- Cell Biology
- Molecular Biology
- Biochemistry
Background:
- Microtubule-associated protein 2 (MAP2) plays a crucial role in microtubule stabilization and dynamics.
- The phosphorylation state of MAP2 is known to affect its function, but its precise impact on microtubule binding is not fully understood.
Purpose of the Study:
- To investigate the influence of varying phosphorylation levels and sites on MAP2's binding affinity to cellular microtubules.
- To compare in vitro tubulin binding with in vivo microtubule binding of MAP2 under different phosphorylation conditions.
Main Methods:
- Microinjection of MAP2 with defined phosphorylation states (MAP2-P10, MAP2-P25, MAP2-P2, MAP2-P10+12) into MAP2-deficient rat-1 fibroblasts.
- In vitro binding assays using taxol-stabilized tubulin polymers.
- Analysis of phosphate content and phosphorylation sites on MAP2 preparations.
Main Results:
- MAP2 with reduced phosphorylation (MAP2-P2) failed to bind to microtubules, indicating phosphorylation is essential for initial binding.
- Highly phosphorylated MAP2 (MAP2-P25 and MAP2-P10+12) also exhibited impaired or no binding, suggesting a critical range or specific sites are required.
- In vitro binding assays confirmed phosphorylation-dependent tubulin interaction, but results sometimes diverged from in vivo microinjection observations.
Conclusions:
- The specific sites of phosphorylation, rather than the overall phosphate stoichiometry, are critical determinants of MAP2's microtubule binding activity.
- Cellular microtubules may involve additional factors influencing MAP2 interaction that are not recapitulated in in vitro binding assays.