Structure and Expression of a Calcium-Binding Protein Gene Contained within a Calmodulin-Regulated Protein Kinase Gene

Abstract

We have determined the first genomic structure and characterized the mRNA and protein products of a novel vertebrate gene that encodes a calcium-binding protein with amino acid sequence identity to a protein kinase domain. The elucidation of the complete DNA sequence of this transcription unit and adjacent genomic DNA, Southern blot and polymerase chain reaction analyses of cellular genomic DNA, and examination of mRNA and protein species revealed that the calcium-binding kinase-related protein (KRP)-encoding gene is contained within the gene for a calmodulin-regulated protein kinase, myosin light-chain kinase (MLCK). The KRP gene transcription unit is composed of three exons and a 5'-flanking sequence containing a canonical TATA box motif. The TATA box, the transcription initiation site, and the first 109 nucleotides of the 5' noncoding region of the KRP mRNA correspond to an MLCK gene intron sequence. Both KRP and MLCK are produced in the same adult chicken tissue in relatively high abundance from a single contiguous stretch of genomic DNA and utilize the same reading frame and common exons to produce distinct mRNAs (2.7 and 5.5 kb, respectively) that encode proteins with dissimilar biochemical functions. There appears to be no precedent in vertebrate molecular biology for such a relationship. This may represent a mechanism whereby functional diversity can be achieved within the same vertebrate tissue by use of common exons to produce shuffled domains with identical amino acid sequences in different molecular contexts.

Characterization of the Chicken Telokin Heterogeneity by Time-of-Flight Mass Spectrometry

“…Telokin concentration in gizzard has been estimated to 90 µM (Shirinsky et al, 1993). The telokin promoter most probably lies within an intron in the 3′ region of the MLCK gene (Guerriero et al, 1986;Collinge et al, 1992). Thus, the primary structure of chicken telokin was first inferred from the sequence of the C-terminal region of MLCK.…”

“…However, the actual translational start site on the telokin mRNA has not been defined, first because the N-terminus of telokin is blocked and cannot be sequenced, secondly because telokin mRNA contains three possible ATG codons in the same open reading frame. On the basis of cDNA sequence analysis and amino acid composition of telokin, Ito et al (1989) proposed, for the turkey gizzard telokin N-terminus, the Ile 4 residue contained within the N-terminal sequence 1 MAMISGM 7 , while others proposed the 2 AMISGM 7 N-terminal sequence (Gallagher & Herring, 1991;Collinge et al, 1992). The whole telokin primary structure has not been determined by direct amino acid sequencing yet, and partial sequencing experiments did not encompass the C-terminal tail of the protein (Ito et al, 1989).…”

Autophosphorylation of Smooth Muscle Myosin Light Chain Kinase at Its Regulatory Domain

“…Smooth muscle and nonmuscle MLCKs contain a catalytic core at the center of the molecule and a regulatory region residing toward the C-terminus of the catalytic core (Olson of the molecule which is not present in skeletal MLCK is characterized by its acidic property, and it is found that this portion of molecule is independently expressed in situ, termed telokin (Ito et al, 1989;Gallagher et al, 1991;Collinge et al, 1992; Yoshikai & Ikebe, 1992) although its physiological function is not known.…”

Modular Structure of Smooth Muscle Myosin Light Chain Kinase: Hydrodynamic Modeling and Functional Implications

“…Finally, at the C-terminus is one more Ig motif (Ig3) terminated with a stretch of Glu residues (polyE). The 154-residue C-terminal segment of smMLCK containing the Ig3 domain is also expressed in a manner independent of the full-length MLCK and is termed telokin (23) or kinase-related protein (KRP) (24). Telokin was found to promote filament formation of unphosphorylated myosin in the presence of ATP presumably through its binding to the head-tail junction of myosin (25).…”