Myosin light chain kinase (MLCK) is a dedicated Ca2+/calmodulin-dependent protein kinase that phosphorylates a specific site (Ser19) on the regulatory light chain (RLC) of myosin II. There are two MLCK genes in vertebrates: one for the short skeletal/cardiac MLCK consisting of a catalytic domain and a regulatory domain with a Ca2+/CaM binding region, the other MLCK gene has three alternative promoters giving rise to three proteins: a short (130 kDa) and a long (220 kDa) smooth muscle MLCK and a C-terminal Ig module that gives rise to telokin, also known as kinase-related protein (Kamm and stull, 2001). Telokin lacks the kinase and regulatory domains and is expressed selectively in smooth muscle, while short and long MLCK are ubiquitously expressed. Skeletal/cardiac MLCK are expressed in the adult heart (Herring et al., 2000). Differential expression of MLCK isoforms in different types of muscles and non-muscle cells in embryonic and adult tissues, suggest the possibility that they serve different functions in regulation of the cytoskeletal and in cell motility. Apart from the wellknown role of MLCK in switching on smooth muscle contractility through phosphorylation of the RLCs, critical roles for MLCK have been proposed in nonmuscle cells, such as the regulation of endothelial cells permeability, stress fiber formation, cell migration, fibroblasts contractile activity and proliferation, cytokinesis, secretion, neurite growth cone extension, modulation of ion channel currents, signaling pathways leading to fibroblast apoptosis, and maintenance of normal cardiac function. The short smooth muscle MLCK is expressed in the embryonic and adult heart, where it may regulate non-muscle myosin II motor activity and is thought to play a critical role in cardiac development. Therefore, it was extremely surprising to find that mice showing complete lack of expression of vertebrate short and long MLCK smooth muscle isoforms and telokin developed to full size (Figure 1) although they died within 1–5h after birth. The phenotype is not due to the loss of telokin as telokinnull mice survive and breed normally. To generate these mice, we designed a targeted construct to allow cre recombinase dependent disruption of required telokin promoter sequences (including a CArG element) located within intron 27 of the MLCK gene that is located between exons encoding the calmodulin-binding domain in the kinase. In generating the telokin-null mouse by homologous recombination, we found that insertion of the full-length targeted construct into this locus (including a loxP flanked neomycin selection cassette) resulted in complete disruption of the MLCK gene expression in the absence of cre recombination. Thus, these mice containing the neomycin cassette were null for the short and long MLCK isoforms as well as telokin. Telokin selective knockout (KO) mice was generated by crossing heterozygous mice with the Prm-cre mouse and then breeding the resulting heterozygous telokin KO mice to homozygosity. Western blot analysis of the stomach and intestines of MLCK)/) animals show an absence of both long and short MLCK and telokin. Skeletal MLCK was not detected and expression was limited to adult skeletal muscle.

The MLCK KO mice showed a ratio of)/):+/):+/+ close to that expected based on Mendelian inheritance with a normal litter size of nine. However, at E14. 5, 20% of the)/) embryos had deformed heads, which have also been observed in some null animals at birth. The inconsistent deformed heads of the)/) embryos likely represents variable penetrance, as the mice are not on a congenic background.