Differential phosphorylation of dynamin I isoforms in subcellular compartments demonstrates the hidden complexity of phosphoproteomes.
Large-scale comparative phosphoproteomics studies have frequently been done on whole cells or organs by conventional bottom-up mass spectrometry approaches, that is, at the phosphopeptide level. Using this approach, there is no way to know which protein isoforms the phosphopeptide signal originated from. Also, as a consequence of the scale of these studies, important information on the localization of phosphorylation sites in subcellular compartments is not surveyed. As a case study, we investigated whether the isoforms of dynamin I (dynI), at the whole brain and subcellular level, had differential phosphorylation. We first established that the dynI isoforms xa, xb, and xd were expressed in nerve terminals. Our investigation revealed that dynI xa was constitutively phosphorylated to a higher extent than the other isoforms despite identical sequences in the phosphorylated subdomains. DynI xa had a 10-fold higher stoichiometry of diphosphorylation at Ser-774 and Ser-778 than dynI xb and xd combined. Diphosphorylation was 2-fold enriched in nerve terminals relative to whole brain and was preferentially targeted for stimulus-dependent dephosphorylation. Phospho-Ser-851 and Ser-857 were depleted from nerve terminals. Our data reveals major differential phosphorylation of dynI phosphosites in different variants and in different neuronal compartments that would be completely imperceptible to a large-scale phosphoproteomics approach.
Chan
Ling-Shan
LS
Cell Signalling Unit, Children's Medical Research Institute, The University of Sydney, Westmead, Australia.
Hansra
Gurdip
G
Robinson
Phillip J
PJ
Graham
Mark E
ME
D000595:Amino Acid Sequence; D000818:Animals; D001921:Brain / Q000378:metabolism*; D002853:Chromatography, Liquid; D019295:Computational Biology; D034284:Dynamin I / Q000235:genetics / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods; D040461:Gene Components; D042541:Intracellular Space / Q000378:metabolism*; D007527:Isoenzymes / Q000378:metabolism; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D010766:Phosphorylation; D040901:Proteomics / Q000379:methods*; D051381:Rats; D013572:Synaptic Vesicles / Q000378:metabolism*
D007527:Isoenzymes; D034284:Dynamin I