Updated publication reference for PubMed record(s): 29666759. 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.