Multiple phosphorylation sites in mammalian neurofilament polypeptides

Area of Science:

• Neuroscience
• Biochemistry
• Cell Biology

Background:

• Neurofilaments are key structural proteins in neurons, essential for axonal integrity and function.
• The phosphorylation state of neurofilament subunits is known to influence their assembly and transport dynamics.

Purpose of the Study:

• To quantify the phosphate content of different neurofilament subunits.
• To identify the specific phosphorylated amino acid residues within these subunits.
• To investigate the functional impact of phosphorylation on neurofilament subunit properties.

Main Methods:

• Chemical phosphate analysis was employed to determine the molar ratio of phosphate to polypeptide for isolated neurofilament subunits (68,000, 145,000, and 200,000 daltons).
• Amino acid analysis was performed to identify phosphoserine and phosphothreonine residues.
• Isoelectric focusing and SDS-PAGE were used to assess changes in subunit properties before and after alkaline phosphatase treatment.

Main Results:

• The 68,000, 145,000, and 200,000 dalton neurofilament subunits contained approximately 3, 9, and 22 mol of phosphate per mole of polypeptide, respectively.
• Phosphoserine was detected in all three subunits, while phosphothreonine was also found in the 145,000 dalton subunit.
• The highly phosphorylated 200,000 dalton subunit exhibited significant shifts in isoelectric point and apparent molecular weight following dephosphorylation with alkaline phosphatase.

Conclusions:

• Rat neurofilament subunits are extensively phosphorylated, with varying degrees across different molecular weight classes.
• Phosphorylation, particularly in the 200,000 dalton subunit, plays a critical role in modulating neurofilament properties.
• These findings contribute to understanding the post-translational modifications regulating neurofilament structure and function in the central nervous system.