Proline‐Directed Protein Kinase (p34cdc2/p58cyclin A) Phosphorylates Bovine Neurofilaments

Abstract

Proline‐directed protein kinase (PDPK), a complex of p34^cdc2 and p58^{cyclin A}, phosphorylates bovine neurofilaments (NFs) in vitro. Incubation of intact filaments with PDPK led to strong labeling of the heavy (NF‐H) and middle (NF‐M) molecular weight NF proteins and weaker labeling of the low molecular weight protein (NF‐L). All three proteins were phosphorylated in solution, with the best substrate being NF‐H. Proteins that had been dephosphorylated by enzymatic treatment were better substrates than native proteins—as many as 6 mol of phosphate were incorporated per mole of NF‐H. Partial proteolytic cleavage experiments combined with two‐dimensional peptide mapping indicated that NF‐H and NF‐M were phosphorylated predominantly in the tail domains, with some phosphate also appearing in the heads. Soluble NF‐L is phosphorylated on the head domain peptide L‐3, whereas NF‐L within intact filaments is phosphorylated only on the tail domain peptide L‐1. Phosphorylation does not lead to filament disassembly. A possible role for PDPK in NF phosphorylation in vivo is discussed.