Background: Network motifs within biological networks show non-random abundances in systems at different scales. Large directed protein networks at the cellular level are now well defined in several diverse species. We aimed to compare the nature of significantly observed two-and three-node network motifs across three different kingdoms (Arabidopsis thaliana for multicellular plants, Saccharomyces cerevisiae for unicellular fungi and Homo sapiens for animals). Results: 'Two-node feedback' is the most significant motif in all three species. By considering the sign of each two-node feedback interaction, we examined the enrichment of the three types of two-node feedbacks [positive-positive (PP), negative-negative (NN) and positive-negative (PN)]. We found that PN is enriched in the network of A.thaliana, NN in the network of S.cerevisiae and PP and NN in the network of H.sapiens. Each feedback type has characteristic features of robustness, multistability and homeostasis. Conclusions: We suggest that amplification of particular network motifs emerges from contrasting dynamical and topological properties of the motifs, reflects the evolutionary design principles selected by the characteristic behavior of each species and provides a signature pointing to their behavior and function.