Molecular characterization of a mammalian smooth muscle myosin light chain kinase

Abstract

A 5.6-kilobase cDNA clone has been isolated which includes the entire coding region for the myosin light chain kinase from rabbit uterine tissue. This cDNA, expressed in COS cells, encodes a Ca2+/calmodulin-dependent protein kinase with catalytic properties similar to other purified smooth muscle myosin light chain kinases. A module (TLKPVGNIKPAE), repeated sequentially 15 times, has been identified near the N terminus of this smooth muscle kinase. It is not present in chicken gizzard or rabbit skeletal muscle myosin light chain kinases. This repeat module and a subrepeat (K P A/V) are similar in amino acid content to repeated motifs present in other proteins, some of which have been shown to associate with chromatin structures. Immunoblot analysis after sodium dodecyl sulfate-polyacrylamide gel electrophoresis, used to compare myosin light chain kinase present in rabbit, bovine, and chicken smooth and nonmuscle tissues, showed that within each species both tissue types have myosin light chain kinases with indistinguishable molecular masses. These data suggest that myosin light chain kinases present in smooth and nonmuscle tissues are the same protein.

Fig. 1. Construction of a full-length rabbit uterine smooth muscle MLCK

A, schematic representation of the full-length cDNA showing the relative locations of some of the restriction enzyme sites that were used to assemble the full-length molecule. The deduced coding region of the cDNA occurs between nucleotides 306 and 3746. B, Schematic representation of the three overlapping cDNA fragments obtained from a λ-gt11, oligo(dT) (top), SMPE-I (middle) and SMPE-II (bottom) primed libraries. C, schematic representation of the N- (upper, filled line) and C-terminal (middle, open line) fragments which were ligated at the XmnI site common to both constructs. The KpnI and XbaI sites originated from a pGEM polylinker and were used to ligate the 3998-bp cDNA fragment to pCMV5 generating the pCMV5-SMMLCK COS cell expression vector.

Fig. 2. Nucleotide and deduced amino acid sequence of the cDNA encoding the rabbit uterine smooth muscle MLCK

The nucleotide sequence of the full-length cDNA and the deduced amino acid sequence of coding region (uppercase letters) are shown. The 5′ noncoding region has been translated and is shown in lowercase letters. Residues which are underlined are those which were determined by sequencing of peptide fragments derived from purified bovine tracheal MLCK. The nucleotides for translational initiation and termination as well as the polyadenylation signal are shown in bold letters. Nucleotides 1-14 and 5601-5608 are sequences of an adaptor and linker, respectively, which were used in construction of the cDNA libraries.

Fig. 3. Nucleotide sequence of a portion of the rabbit smooth muscle MLCK gene

The nucleotide and deduced amino acid sequence of the 5′ portion of the rabbit smooth muscle MLCK gene are shown. Amino acids in uppercase letters are those which are also present in the rabbit uterine smooth muscle MLCK cDNA. Amino acids which are in bold and underlined are those beginning with and overlapping the translational start site predicted for the cDNA. The first bp of overlap of the genomic sequence with the cDNA sequence has been indicated by a *. Nucleotides which are underlined are those which are proposed as a potential transcriptional start site for the rabbit smooth muscle MLCK mRNA. Nucleotides in bold are those corresponding to a primer used in the primer extension analysis.

Fig. 4. Northern analysis of RNA isolated from smooth muscle tissues

Total cellular RNA (20 μg each lane) from rabbit uterine tissue (lane 1), rat uterine tissue (lane 2), bovine tracheal tissue (lane 3) were fractionated through a 1.2% agarose gel as described in “Experimental Procedures”. The blot was probed with a ³²P-labeled cDNA probe corresponding to nucleotides 574–1405 (N-terminal probe) of the full-length cDNA. Lane 4 is 5 μg of poly(A⁺) mRNA from rabbit uterine tissue which was probed with a ³²P-labeled cDNA probe corresponding to nucleotides 3350–3998 (C-terminal probe) of the full-length cDNA. The positions of the RNA molecular weight standards are shown in kilobases on the left and an arrowhead on the right side corresponds of the position of the 5.8-kb mRNAs. The lanes representing the rabbit uterine tissue (lane 1 and lane 4) were exposed for 24 h at −70 °C, all other lanes were exposed for 72 h at −70 °C.

Fig. 5. Immunoblot analysis of MLCK

A, immunoblot analysis of recombinant smooth muscle MLCK expressed in COS cells. Lysates from COS cells transfected with either pCMV5 (mock), pCMV5-SMMLCK or homogenates from bovine tracheal or rabbit uterine smooth muscle tissue were prepared as described under “Experimental Procedures.” Lane 1, 2 ng of purified bovine tracheal muscle MLCK; lane 2, 4 μg of bovine tracheal tissue extract; lane 3, 39 μg of total protein rabbit uterine tissue extract; lane 4, 30 μl of COS cell lysate from cells transfected with pCMV5-SMMLCK; lane 5, 30 μl of COS cell lysate from cells transfected with pCMV5(mock). B, immunoblot analysis of smooth and nonmuscle rabbit, chicken and bovine tissues. Tissue extracts and immunoblots were prepared as described in “Experimental Procedures.” The amount of total protein loaded per lane varies as indicated and is not representative of relative amounts of immunoreactive protein present in these tissues. Rabbit uterus (39 μg, lane 1), rabbit trachea (178 μg, lane 2), rabbit aorta (86 μg, lane 3), rabbit ileum (83 μg, lane 4), rabbit kidney (168 μg, lane 5), rabbit adrenal (220 μg, lane 6), and rabbit uterus (39 μg, lane 7); chicken gizzard (12 μg, lane 8), chicken liver (38 μg, lane 9); bovine trachea (4 μg, lane 10), bovine adrenal (13 μg, lane 11) and purified bovine tracheal MLCK, 2 ng (lane 12). C, immunoblot analysis of cultured cells. Lane 1, chicken gizzard tissue (12 μg of total protein); lane 2, 60 μl of cell lysate from chicken embryo fibroblast cells. The smaller molecular weight immunoreactive bands appearing in lane 1 are degradative fragments of the chicken gizzard MLCK. The positions of the molecular mass standards are indicated on the right side of the figures and apparent molecular masses for MLCKs from these tissues are summarized in Table II.

Fig. 6. Amino acid alignment of chicken nonmuscle, chicken smooth muscle, and rabbit smooth muscle MLCKs

Residues 239–972 of the chicken embryo fibroblast nonmuscle (CKNM, top), 1–972 of the chicken gizzard smooth muscle (CKSM, middle), and 1–1147 of the rabbit uterine smooth muscle MLCK (RBSM, bottom) have been aligned for maximum homology. Residues which are identical and similar in all three sequences have been indicated by a * and ., respectively, below the alignment. Residues contained within the catalytic core and regulatory/calmodulin-binding domains of the enzymes are in bold letters (Olson et al., 1990).

Fig. 7. Alignment of the repeated amino acid sequence within the N terminus of the rabbit smooth muscle MLCK

The K P (A/V) amino acids are represented in bold letters.