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.

Abstract

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.

Death associated proteins (DAPs): from gene identification to the analysis of their apoptotic and tumor suppressive functions

“…The removal of the calmodulin regulatory domain (DCaM mutant) generated a constitutively active kinase (i.e., a gain of function mutation). This is consistent with previous information on other well studied calmodulin-dependent kinases, in which the calmodulin-regulatory domain has inhibitory e ects on the kinase activity, relieved by binding to Ca 2+ / calmodulin (Shoemaker et al, 1990). DAP-kinase activity was abolished by the substitution of a conserved lysine by alanine residue within the kinase domain (K42A), thus generating an inactive kinase mutant with potential dominant-negative activity .…”

Role of the N‐terminal region of the skeletal muscle myosin light chain kinase target sequence in its interaction with calmodulin

“…A double charge cluster reversal mutation where D l 18-El20 was also replaced by KKK yielded almost no activation of sk-MLCK (Weber et al, 1989). Shoemaker et al (1990) showed that for fibroblast MLCK (CaM binding sequence identical to sm-MLCK), the K, for the substrate peptide was higher in the presence of E84K or E120K mutant CaMs, but decreased to the same value as the wild type if a charge reversa1 RRK-EEE was introduced at the beginning of the CaM binding region. In the crystal structure of CaM with the sm-MLCK peptide, both E120 and El 14 in the C-domain interact with basic residues in the N-terminal region of the peptide (Meador et al, 1992).…”

Differential Regulation of Alternatively Spliced Endothelial Cell Myosin Light Chain Kinase Isoforms by p60Src

“…EC MLCK-1 exhibited substantial time-dependent p60 src -catalyzed incorporation of radioactive phosphate (Fig. 2), whereas p60 src -mediated 32 P incorporation did not occur in either MLCK-2, the EC MLCK splice variant, lacking the 69-amino acid stretch containing the p60 src consensus site, nor in SM MLCK, which completely lacks the novel N terminus (Fig. 2).…”

“…Phosphorylation of MLCK diluted in reaction buffer was started by adding 0.2 mM ATP, 10 Ci/ml [␥- 32 P]ATP and 75 units/ml recombinant p60 src kinase (Upstate Biotechnology, Inc., Lake Placid, NY; final concentrations). Synthetic MLCK-1 peptides were used for p60 src phosphorylation assays at 0.1 mg/ml final concentration.…”