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Abstract Protein kinases are critical components of a myriad biological processes and strongly associated with various diseases. While kinase research has been a point of focus in biomedical research for several decades, a large portion of the kinome is still considered understudied or “dark,” because prior research is targeted towards a subset of kinases with well-established roles in cellular processes. We present an empirical and in-silico hybrid workflow to extend the functional knowledge of understudied kinases. Utilizing multiplex peptide activity arrays and robust in-silico analyses, we extended the functional knowledge of five dark tyrosine kinases (AATK, EPHA6, INSRR, LTK, TNK1) and explored their roles in schizophrenia, Alzheimer’s dementia (AD), and major depressive disorder (MDD). Using this hybrid approach, we identified 195 novel kinase-substrate interactions with variable degrees of affinity and linked extended functional networks for these kinases to biological processes that are impaired in psychiatric and neurological disorders. Biochemical assays and mass spectrometry were used to confirm a putative substrate of EPHA6, an understudied dark tyrosine kinase. We examined the EPHA6 network and knowledgebase in schizophrenia using reporter peptides identified and validated from the multi-plex array with high affinity for phosphorylation by EPHA6. Identification and confirmation of putative substrates for understudied kinases provides a wealth of actionable information for the development of new drug treatments as well as exploration of the pathophysiology of disease states using signaling network approaches.

Despite the absence of a confirmed exogenously replicating retrovirus in Canis lupus familiaris (C. familiaris), past retroviral infections are evident in the genomes of living animals via the presence of endogenous retroviruses (ERVs). Although gammaretrovirus-like transcripts and enzyme activities were previously reported to be present in canine leukemias and lymphomas, those findings were not further explored. Initial analysis of the C. familiaris reference genome revealed a minor subset of one ERV lineage, classified as CfERV-Fc1(a), or Fc1(a) here, with features characteristic of recent integration, including the presence of ORFs and identical or nearly identical LTRs. Our previous analysis of whole genome sequence data belonging to extant Canidae revealed a burst of past infections in Canis ancestors resulting in numerous young, polymorphic, and highly intact loci now segregating in dogs. Here, we demonstrate the expression of full-length Fc1(a) proviruses in tissues collected from healthy animals and from animals with cancer. We observed significantly higher expression in samples of dogs with various cancer diagnoses when compared to samples from healthy dogs. Genotyping of insertionally polymorphic Fc1(a) loci identified candidate expressed proviruses and delineated distributions over sample groups. Collectively, the data show that Fc1(a) proviruses retain biological activity in the domestic dog and provides a means to examine potential genetic links with disease states in this species.