Functional determinants in the autoinhibitory domain of calcium/calmodulin-dependent protein kinase II. Role of His282 and multiple basic residues.
Important determinants in the autoinhibitory domain of calcium/calmodulin-dependent protein kinase II (CaMK-II), corresponding to residues 281-302 of the kinase alpha-subunit sequence, were identified. Replacement of Thr286 with Ala (CaMK-(281-302 Ala286)) had no effect on either the potency (IC50 = 2 MicroM) or inhibitory mechanism (competitive with ATP) using the catalytic fragment of CaMK-II. Single replacement of charged residues in CaMK-(281-302, Ala286) identified His282, Arg283, Lys291, Arg297, and Lys298 as important determinants (greater than 10-fold increase in IC50) for potent inhibition of CaMK-II. Glu285, Asp288, Lys291, Arg296, and Lys300 were not as essential (less than 4-fold change in IC50) for potent CaMK-II inhibition. Replacement of either Arg283, Lys291, or Arg297, and Lys298 with Ala did not alter the ATP-competitive mechanism of inhibition although the Ki values increased 16-530-fold. However, replacement of His282 with Ala decreased the IC50 by 20-fold and altered the mechanism of inhibition to noncompetitive with respect to ATP. The non-protonated form of His282 was functionally active since decreasing the pH from 7.5 to 5.5 increased the IC50 of CaMK-(281-302, Ala286) almost 20-fold. Histidine protonation also appeared to disrupt the autoinhibitory domain of intact forms of CaMK-II since preincubation of non-proteolyzed rat brain CaMK-II with calcium/calmodulin (in the absence of ATP) at pH 5.5 generated up to 16% calcium-independent activity when assayed at pH 5.5. Similarly, the level of calcium-independent activity of a baculovirus-expressed Asp286 mutant CaMK-II ((D286)mCaMK alpha) increased to almost 80% calcium independence when assayed at pH 5.5 compared to only 20% when assayed at pH 7.5. The levels of calcium-independent activity of both the (D286)mCaMK alpha (at pH 5.5 and 7.5) and the rat brain CaMK-II (at pH 5.5) were sensitive to the concentrations of both ATP and peptide substrate (syntide-2) in the assays. These data suggest that the basic residues Arg283, Lys291, Arg297, and Lys298 are important for potent inhibition of CaMK-II and that the non-protonated form of His282 may play a unique role in the ATP-directed mechanism of inhibition by the CaMK-II autoinhibitory domain.
Smith
M K
MK
Department of Molecular Physiology and Biophysics, Vanderbilt University School of Medicine, Nashville, Tennessee 37232.
Colbran
R J
RJ
Brickey
D A
DA
Soderling
T R
TR
D000595:Amino Acid Sequence; D000818:Animals; D001667:Binding, Competitive; D001921:Brain / Q000201:enzymology; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D002942:Circular Dichroism; D006639:Histidine; D007700:Kinetics; D008969:Molecular Sequence Data; D010455:Peptides / Q000138:chemical synthesis / Q000494:pharmacology*; D011487:Protein Conformation; D047428:Protein Kinase Inhibitors; D011494:Protein Kinases / Q000737:chemistry / Q000235:genetics; D051381:Rats; D013329:Structure-Activity Relationship
D010455:Peptides; D047428:Protein Kinase Inhibitors; D006639:Histidine; D011494:Protein Kinases; D017871:Calcium-Calmodulin-Dependent Protein Kinases