Use of DNA sequence and mutant analyses and antisense oligodeoxynucleotides to examine the molecular basis of nonmuscle myosin light chain kinase autoinhibition, calmodulin recognition, and activity.
The first primary structure for a nonmuscle myosin light chain kinase (nmMLCK) has been determined by elucidation of the cDNA sequence encoding the protein kinase from chicken embryo fibroblasts, and insight into the molecular mechanism of calmodulin (CaM) recognition and activation has been obtained by the use of site-specific mutagenesis and suppressor mutant analysis. Treatment of chicken and mouse fibroblasts with antisense oligodeoxynucleotides based on the cDNA sequence results in an apparent decrease in MLCK levels, an altered morphology reminiscent of that seen in v-src-transformed cells, and a possible effect on cell proliferation. nmMLCK is distinct from and larger than smooth muscle MLCK (smMLCK), although their extended DNA sequence identity is suggestive of a close genetic relationship not found with skeletal muscle MLCK. The analysis of 20 mutant MLCKs indicates that the autoinhibitory and CaM recognition activities are centered in distinct but functionally coupled amino acid sequences (residues 1,068-1,080 and 1,082-1,101, respectively). Analysis of enzyme chimeras, random mutations, inverted sequences, and point mutations in the 1,082-1,101 region demonstrates its functional importance for CaM recognition but not autoinhibition. In contrast, certain mutations in the 1,068-1,080 region result in a constitutively active MLCK that still binds CaM. These results suggest that CaM/protein kinase complexes use similar structural themes to transduce calcium signals into selective biological responses, demonstrate a direct link between nmMLCK and non-muscle cell function, and provide a firm basis for genetic studies and analyses of how nmMLCK is involved in development and cell proliferation.
Department of Pharmacology, Vanderbilt University, Nashville, Tennessee 37232.
M O
MO
Shoemaker
W
W
Lau
R L
RL
Shattuck
A P
AP
Kwiatkowski
P E
PE
Matrisian
L
L
Guerra-Santos
E
E
Wilson
T J
TJ
Lukas
L J
LJ
Van Eldik
D M
DM
Watterson
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002147:Calmodulin / Q000378:metabolism*; D002642:Chick Embryo; D004247:DNA; D004252:DNA Mutational Analysis; D016373:DNA, Antisense; D004789:Enzyme Activation; D005347:Fibroblasts / Q000378:metabolism; D007700:Kinetics; D008969:Molecular Sequence Data; D009219:Myosin-Light-Chain Kinase / Q000235:genetics* / Q000378:metabolism*; D011485:Protein Binding; D012689:Sequence Homology, Nucleic Acid; D013329:Structure-Activity Relationship; D013379:Substrate Specificity; D013489:Suppression, Genetic
D002147:Calmodulin; D016373:DNA, Antisense; D004247:DNA; D009219:Myosin-Light-Chain Kinase