A Unique Phosphorylation-dependent Mechanism for the Activation of Ca/Calmodulin-dependent Protein Kinase Type IV/GR*

The activity of the Ca/calmodulin-dependent protein kinase IV/Gr (CaMKIV/Gr) is shown to be strictly regulated by phosphorylation of three residues both in vitro and in response to antigen receptor-mediated signaling in lymphocytes. One residue, Thr-200, is indispensable for enhancement of Ca/calmodulin-dependent basal activity by CaMKIV/Gr kinase. This event requires Ca/calmodulin in the full-length CaMKIV/Gr but is Ca/calmodulin-independent when a truncated version of CaMKIV/Gr is used as a substrate (DCaMKIV/ Gr1–317 (D1–317)). The other two residues, Ser 12 and Ser, are apparently autophosphorylated by the Ca/ calmodulin-bound CaMKIV/Gr. Phosphorylation of neither Ser-Ser nor Thr (the residue in a homologous position to Thr of CaMKIIa influences the development of Ca/calmodulin-independent activity or any other property of CaMKIV/Gr examined. Similarly, removal of the NH2-terminal 20 amino acids has no effect on the activation or function of CaMKIV/Gr. However, mutation of both Ser and Ser residues to Ala in D1– 317 completely abrogates activity, while individual substitutions have no effect. These results indicate that the NH2-terminal Ser cluster mediates a novel type of intrasteric inhibition and suggest that three events are required for CaMKIV/Gr activation: 1) Ca/calmodulin binding; 2) phosphorylation of the Ca/calmodulinbound enzyme on Thr by a Ca/calmodulin-dependent protein kinase kinase; and 3) autophosphorylation of Ser-Ser. This three-step requirement is unique among the multifunctional Ca/calmodulin-dependent kinases.