Calcium/calmodulin-dependent protein kinase kinase: identification of regulatory domains.
We recently cloned a calmodulin-dependent protein kinase kinase (CaM-KK) which phosphorylates and activates CaM-KI and CaM-KIV [Tokumitsu, H., Enslen, H., and Soderling, T. R. (1995) J. Biol. Chem. 270, 19320-19324]. In the present study, we have identified its regulatory CaM-binding and autoinhibitory domains (CBD and AID, respectively) using a series of COOH-terminal truncations and site-directed mutants expressed in COS-7 cells. Truncation mutant CaM-KK1-463 activated CaM-KIV and bound 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 of Ca2+/CaM. Substitution of triple negative charges (Asp) at positions 455RKR, 448ILV, or 443SWT blocked CaM binding and suppressed by 70-90% CaM-KK activities. Mutants 438VKL and 435KNS to DDD exhibited partial Ca2+/CaM-independent activities. These results identify overlapping AID and CBD between residues 430 and 460 in CaM-KK, similar to other CaM-Ks. Consistent with this assignment, the synthetic peptide corresponding to residues 438-463 bound CaM in a Ca2+-dependent manner with a Kd in the low nanomolar range. Furthermore, phosphorylation by cAMP-kinase of Ser458 at the COOH-terminus of the CBD in CaM-KK, which suppresses subsequent CaM binding [Wayman, G., Tokumitsu, H., and Soderling, T. R. (1997) J. Biol. Chem. 272, 16073-16076], was blocked by prior binding of Ca2+/CaM to CaM-KK.
Tokumitsu
H
H
Vollum Institute, Oregon Health Sciences University, 3181 SW Sam Jackson Park Road, Portland, Oregon 97201, USA.
Wayman
G A
GA
Muramatsu
M
M
Soderling
T R
TR
D000595:Amino Acid Sequence; D000818:Animals; D001224:Aspartic Acid; D001483:Base Sequence; D001665:Binding Sites; D001921:Brain / Q000201:enzymology; D019556:COS Cells; D007700:Kinetics; D020929:Mitogen-Activated Protein Kinase Kinases; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009838:Oligodeoxyribonucleotides; D010446:Peptide Fragments / Q000737:chemistry / Q000378:metabolism; D010766:Phosphorylation; D010957:Plasmids; D011494:Protein Kinases / Q000737:chemistry* / Q000378:metabolism*; D051381:Rats; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D017384:Sequence Deletion; D014162:Transfection
D009838:Oligodeoxyribonucleotides; D010446:Peptide Fragments; D011994:Recombinant Proteins; D001224:Aspartic Acid; D011494:Protein Kinases; D020929:Mitogen-Activated Protein Kinase Kinases