Analysis of connexin phosphorylation sites.
Most connexins, the proteins that form gap junction channels, are phosphoproteins. Connexin phosphorylation has been thought to regulate gap junctional protein trafficking, gap junction assembly, channel gating, and turnover. Connexin phosphorylation has been investigated in a variety of ways. Some connexins show mobility shifts in sodium dodecyl sulfate-polyacrylamide gel electrophoresis on phosphorylation. Kinase modulators can change the level of connexin phosphorylation and affect gap junctional communication levels. Metabolic labeling of cultured cells has allowed both phosphoamino acid identification and generation of phosphotryptic peptide maps. However, identification of the location of phosphorylated residues within the connexin sequence has required either targeted peptide synthesis, in vitro phosphorylation of known sites, and two-dimensional comigration studies or liquid chromatographic separation and N-terminal sequencing of peptides. In addition to these conventional methods, we discuss new applications of mass spectrometry to the identification of phosphorylated peptides and the specific residues phosphorylated within the connexin-derived peptide.
Cooper
C D
CD
Fred Hutchinson Cancer Research Center, 1100 Fairview Avenue N, DE-320, Seattle, Washington 98109-1024, USA.
Solan
J L
JL
Dolejsi
M K
MK
Lampe
P D
PD
D000595:Amino Acid Sequence; D000818:Animals; D001331:Automation; D002450:Cell Communication; D018929:Cell Culture Techniques / Q000379:methods; D002460:Cell Line; D002851:Chromatography, High Pressure Liquid; D018031:Connexin 43 / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D017629:Gap Junctions / Q000502:physiology*; D007313:Insecta; D007668:Kidney; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry; D010748:Phosphopeptides / Q000737:chemistry; D010766:Phosphorylation; D051381:Rats; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D014162:Transfection
D018031:Connexin 43; D010446:Peptide Fragments; D010748:Phosphopeptides; D011994:Recombinant Proteins