Dissecting the Phospho-Regulatory Landscape of Protein Kinase N1 (PKN1) and Its Downstream Signaling: Functional Insights into the Activity-Dependent and Disease-Relevant Phosphosites

Protein Kinase N1 (PKN1) is a PKC-related serine/threonine kinase of the AGC group within the eukaryotic protein kinase superfamily (ePK) that orchestrates oncogenic, metabolic, and cytoskeletal signaling. Despite these critical roles, the phosphorylation-dependent regulatory network of PKN1 remains largely undefined. We performed a large-scale phosphoproteomic data integration of publicly available human datasets (892 profiling datasets and 191 differential datasets) to identify recurrent PKN1 phosphorylation sites. This analysis identified two predominant PKN1 phosphosites, S562 and S916, that were frequently observed and differentially regulated across studies. The S916 maps to a turn motif (TM) in the AGC group of kinases, which is evolutionarily conserved among PKN paralogs, while S562 is non-conserved and appears to be PKN1-specific. Co-regulation and enrichment analyses suggest that S916 is associated with insulin/AMPK signaling and metabolic pathways, whereas S562 co-occurs with phosphosites involved in cell division, cytoskeletal regulation, and microtubule cytoskeleton organization. Integrating predicted and experimentally validated kinases, substrates, and interactors, we reconstructed a phospho-regulatory network that positions PKN1 at the crossroads of cytoskeleton organization and metabolic signaling. To assess the disease relevance of these phosphorylation events, we integrated transcriptomic and phosphoproteomic data from the hepatocellular carcinoma database (HCCDB). PKN1 was markedly up-regulated in HCC, and its phosphorylation at S916 was positively co-regulated with multiple oncogenic and proliferation-associated protein phosphosites. These results predict S562 and S916 as potential sites for targeted biochemical validation and functional experiments. The identification of S562 and S916 as key regulatory sites provides new mechanistic insight into PKN1 activation and highlights potential avenues for therapeutic targeting.