Abstract. The ERM family members, ezrin, radixin, and moesin, localizing just beneath the plasma membranes, are thought to be involved in the actin iliamerit/plasma membrane association. To identify the integral membrane protein directly associated with ERM family members, we performed immunoprecipitation studies using antimoesin mAb and cultured baby hamster kidney (BHK) cells metabolically labeled with [35S]methionine or surface-labeled with biotin. The results indicated that moesin is directly associated with a 140-kD integral membrane protein. Using BHK cells as antigens, we obtained a mAb that recognized the 140-kD membrane protein. We next cloned a cDNA encoding the 140-kD membrane protein and identified it as CD44, a broadly distributed cell surface glycoprotein. Immunoprecipitation with various anti-CIM4 mAbs showed that ezrin and radixin, as well as moesin, are associated with CIM4, not only in BHK cells, but also in mouse L fibroblasts. Furthermore, immunofluorescence microscopy revealed that in both BHK and L cells, the Triton X-100-insoluble CIM4 is precisely colocalized with ERM family members. We concluded that ERM family members work as molecular linkers between the cytoplasmic domain of CIM4 and actin-based cytoskeletons.

Abstract-Two myosin light chain (MLC) kinase (MLCK) proteins, smooth muscle (encoded by mylk1 gene) and skeletal (encoded by mylk2 gene) MLCK, have been shown to be expressed in mammals. Even though phosphorylation of its putative substrate, MLC2, is recognized as a key regulator of cardiac contraction, a MLCK that is preferentially expressed in cardiac muscle has not yet been identified. In this study, we characterized a new kinase encoded by a gene homologous to mylk1 and -2, named cardiac MLCK, which is specifically expressed in the heart in both atrium and ventricle. In fact, expression of cardiac MLCK is highly regulated by the cardiac homeobox protein Nkx2-5 in neonatal cardiomyocytes. The overall structure of cardiac MLCK protein is conserved with skeletal and smooth muscle MLCK; however, the amino terminus is quite unique, without significant homology to other known proteins, and its catalytic activity does not appear to be regulated by Ca 2ϩ /calmodulin in vitro. Cardiac MLCK is phosphorylated and the level of phosphorylation is increased by phenylephrine stimulation accompanied by increased level of MLC2v phosphorylation. Both overexpression and knockdown of cardiac MLCK in cultured cardiomyocytes revealed that cardiac MLCK is likely a new regulator of MLC2 phosphorylation, sarcomere organization, and cardiomyocyte contraction. (Circ Res. 2008;102:571-580.)Key Words: kinase Ⅲ transcription Ⅲ contraction P hosphorylation of both myosin heavy chain and myosin light chain (MLC) affects motor activity and thick filament assembly. 1 In smooth muscle cells, phosphorylation of MLC2 by smooth muscle MLCK is thought to be responsible for the initiation of contraction. 2 In skeletal and cardiac muscles, however, initiation of muscle contraction depends on voltage-gated L-type Ca 2ϩ channels in the plasma membrane and T-tubules. Increased local Ca 2ϩ concentrations allow the sarcoplasmic reticulum to release large amounts of Ca 2ϩ , which bind to troponin C followed by myosin-actin cross-bridge formation. During this process, MLCK potentiates peak tension in skeletal muscle 1,3 and the force and rate of cross-bridge recruitment in cardiac myocytes. 4,5 To date, smooth muscle (encoded by mylk1 gene) and skeletal (encoded by mylk2 gene) MLCKs have been characterized. 3 Mouse skeletal muscle MLCK is predominantly expressed in skeletal muscle, and mouse smooth muscle MLCK is expressed in several tissues but predominantly in smooth muscle. 6,7 Mutations in human skeletal MLCK on human chromosome 20 have been mapped to a disease locus for familial cardiac hypertrophy (Online Mendelian Inheritance in Man no. 606566), suggesting that abnormal function of skeletal MLCK stimulates cardiac hypertrophy. 8 However, the abundance of skeletal MLCK expression in the heart is controversial, 8 -10 and gene-targeted mice for skeletal MLCK appear to have normal cardiac function. 10 Short-form (130-kDa) smooth muscle MLCK is expressed in the heart at lower levels than those detected in smooth muscle-rich organs such as gut, ...