Orthogonal tools for controlling protein function by post-translational modifications open up new possibilities for protein circuit engineering in synthetic biology. Phosphoregulation is a key mechanism of signal processing in all kingdoms of life, but tools to control the involved processes are very limited. Here, we repurpose components of bacterial two-component systems (TCSs) for chemically induced phosphotransfer in mammalian cells. TCSs are the most abundant multi-component signal-processing units in bacteria, but are not found in the animal kingdom. The presented phosphoregulated orthogonal signal transduction (POST) system uses induced nanobody dimerization to regulate the trans-autophosphorylation activity of engineered histidine kinases. Engineered response regulators use the phosphohistidine residue as a substrate to autophosphorylate an aspartate residue, inducing their own homodimerization. We verify this approach by demonstrating control of gene expression with engineered, dimerization-dependent transcription factors and propose a phosphoregulated relay system of protein dimerization as a basic building block for next-generation protein circuits. Phosphoregulation is a key mechanism of signal processing. Here the authors build a phosphoregulated relay system in mammalian cells for orthogonal signal transduction.