Phosphorylation sites

Mass-spec analysis reveals phosphorylation sites in Nav1.2

Berendt and co-workers published a manuscript in the Journal of Proteome Research that looked at phosphorylation in brain Nav1.x channels using affinity purification, tryptic digestion, and mass spec of the generated peptides (J Proteome Res, EPub 23Feb2010, PMID: 20131913). The antigenicity of the antibodies was such that one was highly selective for Nav1.2 (96 % selective vs other Nav's), while the second antibody detected all Nav channels. Fifteen reversible phosphorylation sites were identified in peptides unique to Nav1.2; three were found in Nav1.1 fragments, all of which analogous to sites in Nav1.2. Some sites identified in Nav1.2 may have been missed, as the peptide coverage did not entirely overlap with all candidate sites in Nav1.1 as determined by sequence alignment with Nav1.2 (specially marked below).

Earlier biochemical work assessing Nav phosphorylation

The Berendt et al. study confirms sites identified in earlier biochemical work by the Catterall group (Murphy & Catterall, JBC 267:16129, 1992 and Murphy et al., JBC 268:27355, 1993) looking at protein kinase C and A, respectively. A more recent analysis also identified several Fyn kinase sites in Nav1.2 (Beacham et al, J Neurosci 27:11543, 2007). There is furthermore a report by Brechet et al. (J Cell Biol 183:1101, 2008) that suggests phosphorylation of three sites via casein kinase II.

Nav1.1 phosphorylation sites not associated with epilepsy

To date, none of residues of interest has been associated with epilepsy. None of the phosphorylation sites lies within the Nav1.1 variable splicing region.

Phosphorylation sites in Nav1.1

The following list identifies the phosphorylation sites in Nav1.1 (GenBank number P35498). Only sites that were either directly confirmed in the Berendt study or candidate sites indicated either by comparison to Nav1.2 in the same publication or in analogy to older studies as indicated but not covered the Berendt study are included. Three-letter amino acid code followed by residue position with the starting methionine = 1.
  • Ser470
  • Ser480*
  • Ser525*
  • Ser551
  • Ser570* (deduced from JBC 267:16129 and JBC 286:27355)
  • Ser607
  • Ser1122* (deduced from JCB 183:1101, 2008)
  • Ser1516* (deduced from JBC 267:16129)
Residues in bold are confirmed in vivo phosphorylation sites by the Berendt study. A single asterisk indicates possible phosphorylation based on paralog data, and lack of peptide coverage in the Berendt study, so the question remains whether or not these are phosphorylated in Nav1.1 or not.