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Dephosphorylated but not phosphorylated microtubule associated protein MAP1B binds to microfilaments
1Lepetit Research Center, Marion Merell Dow Research Institute, Gerenzano (VA), Italy.
Abstract:
We have reported that purified native MAP1B interacts with microtubules but not with microfilaments [Pedrotti and Islam, Cell Motil. Cytoskel. (1995) 30, 301-309]. However, MAP1B can be phosphorylated at multiple sites by casein kinase 11 (CKII) and proline-directed protein kinases (PDPK) and immunoblotting studies show that purified native MAP1B is phosphorylated at least at two CKII sites and at one PDPK site [Pedrotti et al., Biochemistry (1996) 35, 3016-3023]. 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.
Insights
Microtubule-associated protein 1B (MAP1B) phosphorylation regulates its interaction with microfilaments. Dephosphorylated MAP1B binds F-actin, suggesting proline-directed protein kinase sites negatively regulate this interaction.
Area of Science:
- Cell Biology
- Molecular and Structural Biology
- Biochemistry
Background:
- Microtubule-associated protein 1B (MAP1B) is known to interact with microtubules.
- MAP1B is subject to phosphorylation by casein kinase II (CKII) and proline-directed protein kinases (PDPK).
Purpose of the Study:
- To investigate the effect of MAP1B phosphorylation on its interaction with microfilaments.
- To determine which phosphorylation sites regulate MAP1B binding to F-actin.
Main Methods:
- In vitro binding assays using purified native MAP1B.
- Treatment of MAP1B with alkaline phosphatase to induce dephosphorylation.
- Cosedimentation assays to assess binding with microfilaments (F-actin).
- Analysis of dephosphorylation kinetics.
Main Results:
- Native MAP1B does not bind to microfilaments.
- Dephosphorylated MAP1B, after alkaline phosphatase treatment, binds and cosediments with microfilaments.
- Dephosphorylation kinetics suggest that PDPK sites, not CKII sites, negatively regulate MAP1B's interaction with F-actin.
- Dephosphorylated MAP1B shows weak F-actin crosslinking compared to MAP2.
Conclusions:
- Phosphorylation state critically regulates MAP1B's interaction with microfilaments.
- PDPK-mediated phosphorylation at specific sites appears to inhibit MAP1B binding to F-actin.
- MAP1B's role in microfilament dynamics may be modulated by its phosphorylation status.