The small GTPase Rho is implicated in cytoskeletal rearrangements including stress fiber and focal adhesion formation and in the transcriptional activation of c-fos serum response element. In vitro, Rho-kinase, which is activated by Rho, phosphorylates not only myosin light chain (MLC) (thereby activating myosin ATPase) but also myosin phosphatase, thus inactivating myosin phosphatase. Rho-kinase is involved in the formation of stress fibers and focal adhesions in fibroblasts. Here we show that the expression of constitutively active Rho-kinase increased the level of MLC phosphorylation. The activity of Rho-kinase was necessary for maintaining the vinculin-containing focal adhesions, whereas organized actin stress fibers were not necessary for this. The microinjection of constitutively active Rho-kinase into fibroblasts induced the formation of focal adhesions to some extent under the conditions where organized actin stress fibers were disrupted. The expression of constitutively active Rhokinase also stimulated the transcriptional activity of c-fos serum response element. These results suggest that Rho-kinase has distinct roles in divergent pathways downstream of Rho, which include MLC phosphorylation leading to stress fiber formation, focal adhesion formation, and gene expression.

SummarySite-specific phosphorylation of intermediate filament (IF) proteins on serine and threonine residues leads to alteration of the filament structure, in vitro and in vivo. Protein kinases involved in cell signaling and those activated in mitosis dynamically control spatial and temporal organization of intracellular IF phosphorylation. Thus, IF phosphorylation appears to be one of the most predominant strategies in coordinating intracellular organization of the IF Accepted 6 March 1996 network.

Keratins, constituent proteins of intermediate filaments of epithelial cells, are phosphoproteins containing phosphoserine and phosphothreonine. We examined the in vitro phosphorylation of keratin filaments by CAMPdependent protein kinase, protein kinase C and Ca2 +/calmodulin-dependent protein kinase 11. When rat liver keratin filaments reconstituted by type I keratin 18 (molecular mass 47 kDa; acidic type) and type I1 keratin 8 (molecular mass 55 kDa; basic type) in a 1 : 1 ratio were used as substrates, all the protein kinases phosphorylated both of the constituent proteins to a significant rate and extent, and disassembly of the keratin filament structure occurred. Kinetic analysis suggested that all these protein kinases preferentially phosphorylate keratin 8, compared to keratin 18. The amino acid residues of keratins 8 and 18 phosphorylated by CAMP-dependent protein kinase or protein kinase C were almost exclusively serine, while those phosphorylated by Ca2 c calmoddin-dependent protein kinase I1 were serine and threonine. Peptide mapping analysis indicated that these protein kinases phosphorylate keratins 8 and 18 in a different manner. These observations gave the way for in vivo studies of the role of phosphorylation in the reorganization of keratin filaments.

VASCULAR AND INTERVENTIONAL RADIOLOGYC olorectal cancer (CRC) is one of the most common malignancies in the world, with metastases to the lungs in approximately 10%-20% of patients (1). Surgery is performed when both the primary tumor and the lung metastases are completely resectable, with a 3-year overall survival (OS) rate of 53%-82% after lung metastasectomy (2-6). Systemic chemotherapy is administered for patients who are not candidates for surgery, but its 3-year OS rate is less than 50% (7,8). Stereotactic radiation therapy is another treatment option for CRC lung metastases but is usually performed in patients who are not candidates for surgery, with a reported 3-year OS rate of 43%-51% (9,10).Radiofrequency ablation (RFA) is another minimally invasive local-regional treatment for malignant lung neoplasms. It has been considered a reasonable alternative to stereotactic radiation therapy, with the advantages of requiring only one treatment procedure, with less cost, and the feasibility of repeat RFA for residual or recurrent tumor (11). The OS rate of RFA for the treatment of CRC lung metastases in patients who are not candidates for surgery is similar to that of stereotactic radiation therapy. In fact, the 3-year OS rates after RFA for colorectal pulmonary metastasis are 46%-57% in prospective studies (12,13) and 46%-76% in retrospective studies (14-16). From Results: Seventy participants with CRC (mean age, 66 years 6 10; 49 men) were evaluated. The 3-year OS rate was 84% (59 of 70 participants; 95% confidence interval [CI]: 76%, 93%). In multivariable analysis, factors associated with worse OS included rectal rather than colon location (hazard ratio [

Smooth muscle contraction is primarily activated by an increase in cytosolic Ca 2ϩ , which activates myosin light chain kinase (MLCK), 1 a Ca 2ϩ -calmodulin-dependent kinase that specifically phosphorylates myosin regulatory light chain (MLC 20 ), thus activating myosin motor function and muscle contraction. However, it has been realized that the smooth muscle contractile response is modulated by factors other than Ca 2ϩ . One such factor is the small G-protein Rho (1, 2). Takai and co-workers (3) originally reported that GTP␥S enhanced contraction of saponin-permeabilized smooth muscle at submaximal Ca 2ϩ concentrations, but this effect diminished in the presence of exoenzyme C3, a Rho-specific inhibitor. Subsequently it was reported that Rho can decrease myosin phosphatase activity, which anticipates the increase in the level of myosin phosphorylation (4). The target proteins of Rho have been identified recently by several laboratories. Among these Rho targets, the Rho kinases and the myosin-binding subunit of myosin phosphatase (5) have been suggested to play an important role in the regulation of smooth muscle contraction. It was found in an in vitro system that Rho kinase can phosphorylate myosin phosphatase (specifically the myosin-binding subunit), which had the effect of decreasing myosin phosphatase activity (5). It was also suggested that Rho kinase can directly phosphorylate MLC 20 at serine 19 in vitro, thus activating actomyosin ATPase activity (6, 7).The question addressed in the present study is how external stimuli initiated at the plasma membrane can activate the Rho-dependent pathway to increase the phosphorylation of myosin in the contractile domain of the cells. In epithelial cells it was demonstrated by immunocytochemistry (8) and by electron microscopy (9) that the active form of RhoA in transfected cells localizes at the plasma membrane or in submembranous cortical actin networks. Consistent with these findings, a translocation of RhoA to the particulate fraction during agonist stimulation was reported using cell fractionation methods in smooth muscle (10) and fibroblasts (11). Although these previous results suggest the recruitment of Rho to the plasma membrane by external stimuli, it remains obscure whether or not the translocation of Rho is kinetically coupled with changes in myosin phosphorylation and thus contraction. This issue is directly relevant to the question of how the Ca 2ϩ pathway and Rho pathway differentially contribute to changes in myosin phosphorylation, because the change in cytosolic Ca 2ϩ in smooth muscle cells is achieved within a few seconds after

No abstract

Autophosphorylation of smooth muscle myosin light chain kinase was initially reported by Foyt et al. [Foyt, H. L., & Means, A. R. (1985) J. Cyclic Nucleotide Protein Phosphorylation Res. 260, 8978-8983], however, the effects of autophosphorylation on the kinase activity as well as the location of the sites have not been elucidated. Here we demonstrate that MLCK is autophosphorylated at three sites, Thr 803, Ser 815, and Ser 823, and this phosphorylation alters MLCK activity. Two phosphorylation sites are located in the regulatory domain of the kinase, the threonine site toward the autoinhibitory region and the serine site (Ser 815) in close proximity to the calmodulin anchoring site. The autophosphorylation was significantly inhibited by the binding of calmodulin. The autophosphorylation at Thr 803 is an intramolecular process, and the alignment of the basic amino acid residues nearby Thr 803 was highly homologous to the phosphorylation site of myosin light chain, suggesting that the regulatory site is in close proximity to the catalytic site in the three-dimensional structure. The phosphorylation at the threonine site activated the calmodulin-independent activity while the phosphorylation at the serine site inhibited the calmodulin-dependent activity due to a decrease in the affinity for calmodulin. This finding shows another example of the activation of calmodulin-dependent kinases by autophosphorylation at its autoinhibitory region and provides a new clue for understanding the calmodulin/MLCK signalling pathway.

Fast MR blood flow measurement at baseline is highly useful for predicting significant stenosis in internal mammary arterial grafts.