A UNIQUE PHOSPHORYLATION-DEPENDENT MECHANISM FOR THE ACTIVATION OF CA2+ CALMODULIN-DEPENDENT PROTEIN-KINASE TYPE-IV/GR/

The activity of the Ca2+/calmodulin-dependent protein kinase IV/Gr (Ca MKIV/Gr) is shown to be strictly regulated by phosphorylation of three residues both in vitro and in response to antigen receptor-mediated s ignaling in lymphocytes. One residue, Thr-200, is indispensable for en hancement of Ca2+/calmodulin-dependent basal activity by CaMKIV/Gr kin ase. This event requires Ca2+/calmodulin in the full-length CaMKIV/Gr but is Ca2+/calmodulin-independent when a truncated version of CaMKIV/ Gr is used as a substrate (Delta CaMKIV/Gr(1-317) (Delta 1-317)). The other two residues, Ser(12) and Ser(13), are apparently autophosphoryl ated by the Ca2+/calmodulin-bound CaMKIV/Gr. Phosphorylation of neithe r Ser(12)-Ser(13) nor Thr(312) (the residue in a homologous position t o Thr(286) of CaMKII alpha influences the development of Ca2+/calmodul in-independent activity or any other property of CaMKIV/Gr examined. S imilarly, removal of the NH2-terminal 20 amino acids has no effect on the activation or function of CaMKIV/Gr. However, mutation of both Ser (12) and Ser(13) residues to Ala in Delta 1-317 completely abrogates a ctivity, while individual substitutions have no effect. These results indicate that the NH2-terminal Ser cluster mediates a novel type of in trasteric inhibition and suggest that three events are required for Ca MKIV/Gr activation: 1) Ca2+/calmodulin binding; 2) phosphorylation of the Ca2+/calmodulin-bound enzyme on Thr(200) by a Ca2+/calmodulin-depe ndent protein kinase kinase; and 3) autophosphorylation of Ser(12)-Ser (13). This three-step requirement is unique among the multifunctional Ca2+/calmodulin-dependent kinases.