Protein phosphorylation and dephosphorylation is one of the major mechanisms of signal integration in eukaryotic cells. It can be achieved through the phosphorylation of proteins that control the levels of second messengers, through the phosphorylation of protein kinases and phosphatases themselves, or through the reversible phosphorylation of their substrates. This article also highlights the important role of 'multi-site phosphorylation' in signal integration, in which different protein kinases produce additive, synergistic and antagonistic effects on activity by phosphorylating distinct Ser or Thr residues in a single protein.