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Site-specific phosphorylation by protein kinase C inhibits assembly-promoting activity of microtubule-associated
1Department of Biophysics and Biochemistry, Faculty of Science, University of Tokyo, Japan.
Abstract:
We have examined the phosphorylation of bovine microtubule-associated protein 4 (MAP4), formerly named MAP-U, by protein kinase C (PKC). When MAP4 was incubated with PKC, about 1 mol of phosphate was incorporated/mol of MAP4. Phosphorylation of MAP4 caused a remarkable decrease in the ability of the MAP to stimulate microtubule assembly. MAP4 consists of an amino-terminal projection domain and a carboxyl-terminal microtubule-binding domain. The carboxyl-terminal domain is subdivided into a Pro-rich region and an assembly-promoting (AP) sequence region containing four tandem repeats of AP sequence that is conserved in MAP4, MAP2, and tau [Aizawa et al. (1990) J. Biol. Chem. 265, 13849-13855]. In order to identify the site of MAP4 phosphorylated by PKC, a series of expressed MAP4 fragments was prepared and treated with the kinase. A fragment corresponding to the Pro-rich region (P fragment) was phosphorylated, while fragments corresponding to the projection domain and the AP sequence region were not. In addition, chymotryptic digestion of an authentic MAP4 prephosphorylated by PKC revealed that phosphate was incorporated almost exclusively into a 27-kDa fragment containing the carboxyl-terminal half of the Pro-rich region. We investigated the phosphorylation site in MAP4 using the P fragment and found that Ser815 was phosphorylated almost exclusively. We conclude that the phosphorylation of a single Ser residue in the Pro-rich region negatively regulates the assembly-promoting activity of MAP4.
Insights
Protein kinase C (PKC) phosphorylates bovine microtubule-associated protein 4 (MAP4), reducing its microtubule assembly-promoting ability. This phosphorylation occurs at Ser815 within the Pro-rich region, negatively regulating MAP4 function.
Area of Science:
- Biochemistry
- Cell Biology
- Molecular Neuroscience
Background:
- Microtubule-associated protein 4 (MAP4) is crucial for microtubule assembly.
- Protein kinase C (PKC) is involved in various cellular signaling pathways.
- Understanding MAP4 regulation is key to comprehending microtubule dynamics.
Purpose of the Study:
- To investigate the effect of PKC phosphorylation on bovine MAP4.
- To identify the specific site of MAP4 phosphorylation by PKC.
- To determine how phosphorylation impacts MAP4's microtubule assembly-promoting activity.
Main Methods:
- Incubation of bovine MAP4 with PKC.
- Analysis of phosphate incorporation into MAP4.
- Preparation and treatment of expressed MAP4 fragments with PKC.
- Chymotryptic digestion of phosphorylated MAP4.
- Identification of the phosphorylated residue using MAP4 fragments.
Main Results:
- PKC incorporated approximately 1 mole of phosphate per mole of MAP4.
- Phosphorylation significantly decreased MAP4's ability to stimulate microtubule assembly.
- Phosphorylation occurred within the Pro-rich region of MAP4, specifically at Ser815.
- MAP4 fragments corresponding to the projection domain and assembly-promoting region were not phosphorylated.
Conclusions:
- PKC-mediated phosphorylation of a single serine residue (Ser815) in the Pro-rich region of MAP4 negatively regulates its microtubule assembly-promoting activity.
- This finding elucidates a novel regulatory mechanism for microtubule dynamics.
- The study identifies a specific phosphorylation site that impacts MAP4 function.