Organization of the genetic locus for chicken myosin light chain kinase is complex: Multiple proteins are encoded and exhibit differential expression and localization

Abstract

We report that the genetic locus that encodes vertebrate smooth muscle and nonmuscle myosin light chain kinase (MLCK) and kinase-related protein (KRP) has a complex arrangement and a complex pattern of expression. Three proteins are encoded by 31 exons that have only one variation, that of the first exon of KRP, and the genomic locus spans approximately 100 kb of DNA. The three proteins can differ in their relative abundance and localization among tissues and with development. MLCK is a calmodulin (CaM) regulated protein kinase that phosphorylates the light chain of myosin II. The chicken has two MLCK isoforms encoded by the MLCK/KRP locus. KRP does not bind CaM and is not a protein kinase. However, KRP binds to and regulates the structure of myosin II. Thus, KRP and MLCK have the same subcellular target, the myosin II molecular motor system. We examined the tissue and cellular localization of KRP and MLCK in the chicken embryo and in adult chicken tissues. We report on the selective localization of KRP and MLCK among and within tissues and on a differential distribution of the proteins between embryonic and adult tissues. The results fill a void in our knowledge about the organization of the MLCK/KRP genetic locus, which appears to be a late evolving regulatory paradigm, and suggest an independent and complex regulation of expression of the gene products from the MLCK/KRP genetic locus that may reflect a basic principle found in other eukaryotic gene clusters that encode functionally linked proteins.

LFA-1-induced T cell migration on ICAM-1 involves regulation of MLCK-mediated attachment and ROCK-dependent detachment

“…In comparison with murine lung cells and other cell lines (data not shown), MLCK appears to be expressed in low abundance in human T cells. Four other variants of the MLCK protein have been described previously (Lazar and Garcia, 1999) and, from the chicken genomic sequence, the identification of canonical splice consensus sites in each of the 31 exons raises the possibility that more forms exist (Birukov et al, 1998).…”

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

“…Molecular cloning of MLCK from human endothelial cells (20) revealed a high molecular weight MLCK variant containing a unique 922-residue N-terminal domain not expressed in the low molecular weight MLCK isoform, which is abundantly expressed in smooth muscle. Comparison of the cDNA encoding human high and low molecular weight MLCKs, when combined with results of chromosome mapping of human MLCK to single locus with chromosomal localization to 3qcen-q21 (6), suggests that mammalian MLCK genomic organization is highly similar to the "gene within a gene" organization of the avian smooth muscle/nonmuscle MLCK gene expressing two size class MLCK variants and one nonkinase protein (KRP), which are encoded by exons 1-31, 15-31, and 29A-31, respectively (17,43). The complexity of the human MLCK genomic organization was recently further emphasized by the detection of five splice variants of high molecular weight MLCK in nonmuscle and smooth muscle tissues using RT-PCR approaches (28).…”

“…3 and 11). However, Western blot screening of a variety of embryonic and adult smooth muscle and nonmuscle tissues revealed expression of a high molecular weight MLCK variant with electrophoretic mobility in the range of 208 -214 kDa (17)(18)(19). Garcia and colleagues (20) subsequently sequenced the high molecular weight MLCK isoform cloned from a human endothelial cell cDNA library, revealing an open reading frame, which encodes a protein of 1914 amino acids.…”

A Differentiation-dependent Splice Variant of Myosin Light Chain Kinase, MLCK1, Regulates Epithelial Tight Junction Permeability

“…In smooth muscle, the 130-kDa smooth muscle MLCK (short MLCK) and a carboxyl-terminal fragment of MLCK, telokin, are produced as separate mRNA transcripts. In contrast, a 220-kDa MLCK (long MLCK) containing a 922-amino acid amino-terminal extension is expressed in many nonmuscle cells, including embryonic tissues and endothelium (13,14). Long MLCK is expressed as at least five different splice variants that all contain the amino-terminal extension; these variants are distinguished by the presence or absence of short (Ͻ70 amino acids) regions as a result of alternative mRNA splicing (15).…”