Phosphorylation and dephosphorylation of distinct isoforms of the heavy neurofilament protein NF-H.
1. Previous immunohistochemical studies led to the suggestion that distinctly phosphorylated neurofilament isoforms exist in different types of neurons. We have recently examined this hypothesis by direct biochemical experiments, which revealed that the heavy neurofilament protein NF-H of bovine ventral root cholinergic neurons is more acidic and markedly more phosphorylated than that of bovine dorsal root neurons. 2. In the present study we employed this system to study the degree to which distinctly phosphorylated NF-H isoforms differ in the extents to which they can be phosphorylated and dephosphorylated in vitro. This was performed utilizing alkaline phosphatase and protein kinase PK40ERK, which is specific to serines of Lys-Ser-Pro (KSP) repeats. The results obtained reveal that: 3. The more extensively phosphorylated ventral root NF-H is dephosphorylated more rapidly than dorsal root NF-H. 4. Ventral root NF-H and dorsal root NF-H in their native form are both poor substrates of PK40ERK. 5. Following dephosphorylation, ventral root and dorsal root NF-H are phosphorylated extensively and differentially by this kinase. Under these conditions, PK40ERK catalyzes the incorporation of, respectively, 4.2 +/- 1.3 and 2.8 +/- 0.6 mol of phosphate per molecule of ventral root NF-H and dorsal root NF-H. The ratio of phosphates incorporated into ventral root NF-H to those incorporated into dorsal root NF-H is 1.46 +/- 0.17. 6. These findings support the hypothesis that different classes of neurons contain distinctly phosphorylated neurofilaments and show that ventral root and dorsal root neurons are a useful model system for studying the distinct characteristics of neurofilament phosphorylation in different types of neurons.
Chertoff
R
R
Department of Biochemistry, Tel Aviv University, Ramat Aviv, Israel.
Soussan
L
L
Roder
H
H
Michaelson
D M
DM
D000469:Alkaline Phosphatase / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D002417:Cattle; D004591:Electrophoresis, Polyacrylamide Gel; D005727:Ganglia, Spinal; D015151:Immunoblotting; D019950:Mitogen-Activated Protein Kinase 1; D020928:Mitogen-Activated Protein Kinases / Q000378:metabolism; D008969:Molecular Sequence Data; D009419:Nerve Tissue Proteins / Q000378:metabolism; D016900:Neurofilament Proteins / Q000032:analysis / Q000378:metabolism*; D009474:Neurons / Q000166:cytology / Q000378:metabolism*; D010766:Phosphorylation; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism; D013126:Spinal Nerve Roots / Q000166:cytology / Q000378:metabolism*; D013379:Substrate Specificity
D009419:Nerve Tissue Proteins; D016900:Neurofilament Proteins; D017346:Protein Serine-Threonine Kinases; D019950:Mitogen-Activated Protein Kinase 1; D020928:Mitogen-Activated Protein Kinases; D000469:Alkaline Phosphatase