Phosphorylation of the amino-terminal head domain of the middle molecular mass 145-kDa subunit of neurofilaments. Evidence for regulation by second messenger-dependent protein kinases.
To begin to understand the regulation and roles of neurofilament phosphorylation, we localized the phosphorylated domains on the 140-145-kDa neurofilament subunit (NF-M) and identified the protein kinases that may specifically phosphorylate the sites within these domains in vivo. Mouse retinal ganglion cells were labeled in vivo by injecting mice intravitreally with [32P]orthophosphate, and neurofilament-enriched fractions were obtained from the optic axons. Two-dimensional phosphopeptide map analysis of NF-M after digestion with alpha-chymotrypsin and trypsin revealed seven major (M8-M14) and at least eight minor (M1-M7 and M15) phosphopeptides. Two-dimensional phosphopeptide map analyses of NF-M phosphorylated in vitro by individual purified or endogenous axonal cytoskeleton-associated protein kinases showed that five peptides (M9-M13) were substrates for the heparin-sensitive second messenger-independent protein kinase(s). Protein kinase A and/or protein kinase C phosphorylated eight other peptides (M1-M8). Two alpha-chymotryptic peptides (C1 and C2) that were phosphorylated by protein kinase A but not by the endogenous independent kinase(s) were isolated by high performance liquid chromatography on a reverse-phase C8 column. Partial sequence analysis of peptides C1 (S R V S G P S ...) and C2 (S R G S P S T V S ...) showed that the peptides were localized on the head domain of NF-M at 25 and 41 residues from the amino terminus, respectively. Tryptic digest of peptide C1 (less than 12 kDa) generated the phosphopeptides M1-M6. Peptide C2 was a breakdown product of peptide C1. Since the polypeptide sites targeted by second messenger-independent kinase(s) associated with neurofilaments are localized on the carboxyl-terminal domain, separate aspects of NF-M function appear to be regulated by separate kinase systems that selectively phosphorylate head or tail domains of the polypeptide.
Sihag
R K
RK
Ralph Lowell Laboratories, Mailman Research Center, McLean Hospital, Belmont, Massachusetts 02178.
Nixon
R A
RA
D000595:Amino Acid Sequence; D000818:Animals; D002851:Chromatography, High Pressure Liquid; D002918:Chymotrypsin; D003599:Cytoskeleton / Q000378:metabolism*; D015180:Electrophoresis, Gel, Two-Dimensional; D007381:Intermediate Filament Proteins / Q000302:isolation & purification / Q000378:metabolism*; D007382:Intermediate Filaments / Q000378:metabolism*; D046911:Macromolecular Substances; D051379:Mice; D008810:Mice, Inbred C57BL; D008969:Molecular Sequence Data; D008970:Molecular Weight; D016900:Neurofilament Proteins; D010449:Peptide Mapping; D010710:Phosphates / Q000378:metabolism; D010748:Phosphopeptides / Q000302:isolation & purification; D010766:Phosphorylation; D011487:Protein Conformation; D011494:Protein Kinases / Q000378:metabolism*; D012160:Retina / Q000378:metabolism*; D012165:Retinal Ganglion Cells / Q000378:metabolism*; D015290:Second Messenger Systems
D007381:Intermediate Filament Proteins; D046911:Macromolecular Substances; D016900:Neurofilament Proteins; D010710:Phosphates; D010748:Phosphopeptides; D011494:Protein Kinases; D002918:Chymotrypsin