CALCIUM CALMODULIN-DEPENDENT PROTEIN-KINASE KINASE - IDENTIFICATION OF REGULATORY DOMAINS/

We recently cloned a calmodulin-dependent protein kinase kinase (CaM-K K) which phosphorylates and activates CaM-KI and CaM-KIV [Tokumitsu, H ., Enslen, H., and Soderling, T. R. (1995) J. Biol. Chem. 270, 19320-1 9324]. In the present study, we have identified its regulatory CaM-bin ding and autoinhibitory domains (CBD and AID, respectively) using a se ries of COOH-terminal truncations and site-directed mutants expressed in COS-7 cells. Truncation mutant CaM-KK1-463 activated CaM-KIV and bo und CaM similar to wild-type enzyme (CaM-KK1-505); CaM-KK1-448 did not bind CaM and was largely inactive; and CaM-KK1-434 also did not bind CaM but activated a CaM-independent mutant of CaM-KIV in the absence o f Ca2+/CaM. Substitution of triple negative charges (Asp) at positions (RKR)-R-455, (ILV)-I-448, or (SWT)-S-443 blocked CaM binding and supp ressed by 70-90% CaM-KK activities. Mutants (VKL)-V-438 and (KNS)-K-43 5 to DDD exhibited partial Ca2+/CaM-independent activities. These resu lts identify overlapping AID and CBD between residues 430 and 460 in C aM-KK, similar to other CaM-Ks. Consistent with this assignment, the s ynthetic peptide corresponding to residues 438-463 bound CaM in a Ca2-dependent manner with a K-d in the low nanomolar range. Furthermore, phosphorylation by cAMP-kinase of Ser(458) at the COOH-terminus of the CBD in CaM-KK, which suppresses subsequent CaM binding [Wayman, G., T okumitsu, H., and Soderling, T. R. (1997) J. Biol. Chem. 272, 16073-16 076], was blocked by prior binding of Ca2+/CaM to CaM-KK.