Identification of interaction partners and substrates of the cyclin A1-CDK2 complex

Abstract

The CDK2-associated cyclin A1 is essential for spermatogenesis and contributes to leukemogenesis. The detailed molecular functions of cyclin A1 remain unclear, since the molecular networks involving cyclin A1-CDK2 have not been elucidated. Here, we identified novel cyclin A1/CDK2 interaction partners in a yeast triple-hybrid approach. Several novel proteins (INCA1, KARCA1, and PROCA1) as well as the known proteins GPS2 (G-protein pathway suppressor 2), Ku70, receptor for activated protein kinase C1/guanine nucleotide-binding protein β-2-like-1, and mRNA-binding motif protein 4 were identified as interaction partners. These proteins link the cyclin A1-CDK2 complex to diverse cellular processes such as DNA repair, signaling, and splicing. Interactions were confirmed by GST pull-down assays and co-immunoprecipitation. We cloned and characterized the most frequently isolated unknown gene, which we named INCA1 (inhibitor of CDK interacting with cyclin A1). The nuclear INCA1 protein is evolutionarily conserved and lacks homology to any known gene. This novel protein and two other interacting partners served as substrates for the cyclin A1-CDK2 kinase complex. Cyclin A1 and all interaction partners were highly expressed in testis with varying degrees of tissue specificity. The highest expression levels were observed at different time points during testis maturation, whereas expression levels in germ cell cancers and infertile testes decreased. Taken together, we identified testicular interaction partners of the cyclin A1-CDK2 complex and studied their expression pattern in normal organs, testis development, and testicular malignancies. Thereby, we establish a new basis for future functional analyses of cyclin A1. We provide evidence that the cyclin A1-CDK2 complex plays a role in several signaling pathways important for cell cycle control and meiosis.