A NEUROFILAMENT-ASSOCIATED KINASE PHOSPHORYLATES ONLY A SUBSET OF SITES IN THE TAIL OF CHICKEN MIDSIZE NEUROFILAMENT PROTEIN

Although neurofilaments are among the most highly phosphorylated prote ins extant, relatively little is known about the kinases involved in t heir phosphorylation. The majority of the phosphates present on the tw o higher-molecular-mass neurofilament subunits are added to multiply r epeated sequence motifs in the tail. We have examined the specificity of a neurofilament-associated kinase (NFAK) partially purified from ch icken spinal cord that selectively phosphorylates the middle-molecular -mass neurofilament subunit, NF-M. Two-dimensional phosphopeptide mapp ing of P-32-labeled NF-M shows that, in vitro, NFAK phosphorylates a s ubset of peptides phosphorylated in vivo in cultured neurons. The abse nce of a complete complement of labeled phosphopeptides following in v itro phosphorylation, compared with phosphorylation in vivo, is not du e to a lack of availability of phosphorylation sites because the same maps are obtained when enzymatically dephosphorylated NF-M is used as an in vitro substrate. Phosphopeptide maps from in vitro-phosphorylate d NF-M and those from a recombinant fusion protein containing only a s egment of the tail piece of chicken NF-M reveal identical labeled pept ides. The fusion protein lacks a segment containing 17 KXX(S/T)P putat ive phosphorylation sites contained in the tail of chicken NF-M but co ntains a segment that includes four KSPs and a KSD site also present i n the intact tail. These results suggest (a) that NFAK mediates the ph osphorylation of some, but not all, potential phosphorylation sites wi thin the tail of NF-M and (b) that multiple kinases are necessary for complete phosphorylation of the NF-M tail.