Phosphoproteomics of DIA1-overexpressing HEK cells and protemics of probable DIA1 interactors

Many kinases are still “orphans”, i.e. knowledge about their substrates, and often also about the processes they regulate, is lacking. Here, DIA1/C3orf58, a member of a novel predicted kinase-like family is subjected to phosphoproteomics analysis in order to cast light on its signalling pathways. LC MS/MS proteomics approach with Ti phosphopeptide enrichment is applied to membrane fractions of DIA1-overexpressing and control HEK cells, and phosphosites dependent on DIA1 presence are elucidated. Most of The these phosphosites belonged to CK2- and proline-directed kinase types. In parallel, proteomics of proteins immunoprecipitated with DIA1 provided its probable interactors. Intersection of the DIA1-affected phosphoproteins and interactors provides putative DIA1 substrates, including calnexin . This pilot study data, mapped on literature, provides basis for deeper studies of DIA1 signalling.