The myosin phosphatase targeting protein (MYPT) family: a regulated mechanism for achieving substrate specificity of the catalytic subunit of protein phosphatase type 1δ

The mammalian MYPT family consists of the products of five genes, denoted MYPT1, MYPT2, MBS85, MYPT3 and TIMAP, which function as targeting and regulatory subunits to confer substrate specificity and subcellular localization on the catalytic subunit of type 1δ protein serine/threonine phosphatase (PP1cδ). Family members share several conserved domains, including an RVxF motif for PP1c binding and several ankyrin repeats that mediate protein–protein interactions. MYPT1, MYPT2 and MBS85 contain C-terminal leucine zipper domains involved in dimerization and protein–protein interaction, whereas MYPT3 and TIMAP are targeted to membranes via a C-terminal prenylation site. All family members are regulated by phosphorylation at multiple sites by various protein kinases; for example, Rho-associated kinase phosphorylates MYPT1, MYPT2 and MBS85, resulting in inhibition of phosphatase activity and Ca²⁺ sensitization of smooth muscle contraction. A great deal is known about MYPT1, the myosin targeting subunit of myosin light chain phosphatase, in terms of its role in the regulation of smooth muscle contraction and, to a lesser extent, non-muscle motile processes. MYPT2 appears to be the key myosin targeting subunit of myosin light chain phosphatase in cardiac and skeletal muscles. MBS85 most closely resembles MYPT2, but little is known about its physiological function. Little is also known about the physiological role of MYPT3, although it is likely to target myosin light chain phosphatase to membranes and thereby achieve specificity for substrates involved in regulation of the actin cytoskeleton. MYPT3 is regulated by phosphorylation by cAMP-dependent protein kinase. TIMAP appears to target PP1cδ to the plasma membrane of endothelial cells where it serves to dephosphorylate proteins involved in regulation of the actin cytoskeleton and thereby control endothelial barrier function. With such a wide range of regulatory targets, MYPT family members have been implicated in diverse pathological events, including hypertension, Parkinson’s disease and cancer.     

Nuclear translocation of the N-terminal prodomain of interleukin-16

Interleukin-16 (IL-16) is a pleiotropic cytokine that functions as a chemoattractant factor, a modulator of T cell activation, and an inhibitor of human immunodeficiency virus (HIV) replication. These diverse functions are exclusively attributed to the secreted C-terminal peptide of 121 amino acids (mature IL-16), which is cleaved from the precursor protein (pro-IL-16) by caspase-3. Human pro-IL-16 is comprised of 631 amino acids with three PDZ domains, one of which is present in secreted mature IL-16. No cellular localization or biologic functions have been ascribed to the unusually large and highly conserved N-terminal prodomain formed as a result of proteolytic release of the third PDZ domain of pro-IL-16. Here we show that the N-terminal prodomain of pro-IL-16 translocates into the nucleus following cleavage of the C-terminal segment. The nuclear localization signal of pro-IL-16 consists of a classical bipartite nuclear targeting motif. We also show that the nuclear targeting of the IL-16 prodomain induces a G(0)/G(1) arrest in the cell cycle. Taken together, the high degree of conservation of the prodomain among species, the presence of two PDZ motifs, and the nuclear localization and subsequent inhibitory effect on cell cycle progression suggest that pro-IL-16 is cleaved into two functional proteins, a C-terminal-secreted cytokine and an N-terminal product, which affects the cell cycle.     

Involvement of rho kinase in the ouabain-induced contractions of the rat renal arteries

Agonist and depolarization-induced vascular smooth muscle contractions include the activation of rho/rho kinase pathway. However, there are no reports addressing the question whether this pathway is involved in ouabain-induced vascular smooth muscle contractions. Therefore, in this study, the possible participation of the rho/rho kinase pathway in ouabain-induced contractions was evaluated in rat renal arteries. Effects of rho kinase inhibitors (fasudil and Y-27632) on ouabain-induced contractions, and phosphorylation of myosin binding subunits (MYPT/MBS85) of myosin phosphatase were determined using isolated tissue and Western blot experiments, respectively. Fasudil and Y-27632 inhibited ouabain-induced contractions in a concentration-dependent manner. The phosphorylation of MYPT was not altered by ouabain. However, ouabain significantly increased MBS85 phosphorylation of myosin phosphatase. The phosphorylation of both subunits of myosin phosphatase was inhibited by Y-27632. These results indicate that activation of rho kinase and the subsequent phosphorylation of MBS85 are involved in ouabain-induced contraction of rat renal arteries. This mechanism may be important in essential hypertension with elevated endogenous ouabain levels.     

Cloning, characterization and expression analysis of two Tetraodon nigroviridis interleukin-16 isoform genes

Interleukin-16 (IL-16) is an important pro-inflammatory cytokine that functions as a chemoattractant factor and is well characterized in human and other mammals, but is largely unknown in fish. In the present study, two isoforms of pro-IL-16 homologues were cloned and characterized from pufferfish Tetraodon nigroviridis. The full-length T. nigroviridis pro-IL-16 isoform 1 cDNA exhibits 2453 bp in size including 291 bp 5'UTR (untranslated region), 1704 bp ORF (open reading frame) and 458 bp 3'UTR, while pro-IL-16 isoform 2 cDNA exhibits a 3801 bp ORF and a 458 bp 3'UTR. Bioinformatics analysis demonstrated that the pro-IL-16 isoform 1 with a predicted mass of 60.6 kDa contained two PDZ (postsynaptic density/disc large/zona occludens-1) domains, whereas the 138.2 kDa pro-IL-16 isoform 2 had two additional PDZ domains in its N-terminal extension. RT-PCR results revealed that ,almost in all examined organs and tissues, the mRNA of both pro-IL-16 isoforms can be detected, except in intestine and gill, where the isoform 2 mRNA is absent. The two putative precursor proteins showed 30.0-33.0% identity to various mammalian and avian homologues. This is the first report of such genes in teleostean fish and we hope the molecular characterization of these two pro-IL-16 isoforms will provide insights into the study of both evolution of IL-16 precursor proteins and the immune system as a whole.     

Myosin phosphatase: Structure,regulation and function

Phosphorylation of myosin II plays an important role in many cell functions, including smooth muscle contraction. The level of myosin II phosphorylation is determined by activities of myosin light chain kinase and myosin phosphatase (MP). MP is composed of 3 subunits: a catalytic subunit of type 1 phosphatase, PPlc; a targeting subunit, termed myosin phosphatase target subunit, MYPT; and a smaller subunit, M20, of unknown function. Most of the properties of MP are due to MYPT and include binding of PP1c and substrate. Other interactions are discussed. A recent discovery is the existence of an MYPT family and members include, MYPT1, MYPT2, MBS85, MYPT3 and TIMAP. Characteristics of each are outlined. An important discovery was that the activity of MP could be regulated and both activation and inhibition were reported. Activation occurs in response to elevated cyclic nucleotide levels and various mechanisms are presented. Inhibition of MP is a major component of Ca2+-sensitization in smooth muscle and various molecular mechanisms are discussed. Two mechanisms are cited frequently: (1) Phosphorylation of an inhibitory site on MYPT1, Thr696 (human isoform) and resulting inhibition of PP1c activity. Several kinases can phosphorylate Thr696, including Rho-kinase that serves an important role in smooth muscle function; and (2) Inhibition of MP by the protein kinase C-potentiated inhibitor protein of 17 kDa (CPI-17). Examples where these mechanisms are implicated in smooth muscle function are presented. The critical role of RhoA/Rho-kinase signaling in various systems is discussed, in particular those vascular smooth muscle disorders involving hypercontractility.     

Myosin phosphatase interacts with and dephosphorylates the retinoblastoma protein in THP-1 leukemic cells: its inhibition is involved in the attenuation of daunorubicin-induced cell death by calyculin-A

Reversible phosphorylation of the retinoblastoma protein (pRb) is an important regulatory mechanism in cell cycle progression. The role of protein phosphatases is less understood in this process, especially concerning the regulatory/targeting subunits involved. It is shown that pretreatment of THP-1 leukemic cells with calyculin-A (CL-A), a cell-permeable phosphatase inhibitor, attenuated daunorubicin (DNR)-induced cell death and resulted in increased pRb phosphorylation and protection against proteolytic degradation. Protein phosphatase-1 catalytic subunits (PP1c) dephosphorylated the phosphorylated C-terminal fragment of pRb (pRb-C) slightly, whereas when PP1c was complexed to myosin phosphatase target subunit-1 (MYPT1) in myosin phosphatase (MP) holoenzyme dephosphorylation was stimulated. The pRb-C phosphatase activity of MP was partially inhibited by anti-MYPT1(1-296) implicating MYPT1 in targeting PP1c to pRb. MYPT1 became phosphorylated on both inhibitory sites (Thr695 and Thr850) upon CL-A treatment of THP-1 cells resulting in the inhibition of MP activity. MYPT1 and pRb coprecipitated from cell lysates by immunoprecipitation with either anti-MYPT1 or anti-pRb antibodies implying that pRb-MYPT1 interaction occurred at cellular levels. Surface plasmon resonance-based experiments confirmed binding of pRb-C to both PP1c and MYPT1. In control and DNR-treated cells, MYPT1 and pRb were predominantly localized in the nucleus exhibiting partial colocalization as revealed by immunofluorescence using confocal microscopy. Upon CL-A treatment, nucleo-cytoplasmic shuttling of both MYPT1 and pRb, but not PP1c, was observed. The above data imply that MP, with the targeting role of MYPT1, may regulate the phosphorylation level of pRb, thereby it may be involved in the control of cell cycle progression and in the mediation of chemoresistance of leukemic cells.     

Myosin phosphatase is inactivated by caspase-3 cleavage and phosphorylation of myosin phosphatase targeting subunit 1 during apoptosis

In nonapoptotic cells, the phosphorylation level of myosin II is constantly maintained by myosin kinases and myosin phosphatase. During apoptosis, caspase-3–activated Rho-associated protein kinase I triggers hyperphosphorylation of myosin II, leading to membrane blebbing. Although inhibition of myosin phosphatase could also contribute to myosin II phosphorylation, little is known about the regulation of myosin phosphatase in apoptosis. In this study, we have demonstrated that, in apoptotic cells, the myosin-binding domain of myosin phosphatase targeting subunit 1 (MYPT1) is cleaved by caspase-3 at Asp-884, and the cleaved MYPT1 is strongly phosphorylated at Thr-696 and Thr-853, phosphorylation of which is known to inhibit myosin II binding. Expression of the caspase-3 cleaved form of MYPT1 that lacked the C-terminal end in HeLa cells caused the dissociation of MYPT1 from actin stress fibers. The dephosphorylation activity of myosin phosphatase immunoprecipitated from the apoptotic cells was lower than that from the nonapoptotic control cells. These results suggest that down-regulation of MYPT1 may play a role in promoting hyperphosphorylation of myosin II by inhibiting the dephosphorylation of myosin II during apoptosis.     

Myosin Phosphatase Target Subunit 1 (MYPT1) Regulates the Contraction and Relaxation of Vascular Smooth Muscle and Maintains Blood Pressure

Myosin light chain phosphatase with its regulatory subunit, myosin phosphatase target subunit 1 (MYPT1) modulates Ca²⁺-dependent phosphorylation of myosin light chain by myosin light chain kinase, which is essential for smooth muscle contraction. The role of MYPT1 in vascular smooth muscle was investigated in adult MYPT1 smooth muscle specific knock-out mice. MYPT1 deletion enhanced phosphorylation of myosin regulatory light chain and contractile force in isolated mesenteric arteries treated with KCl and various vascular agonists. The contractile responses of arteries from knock-out mice to norepinephrine were inhibited by Rho-associated kinase (ROCK) and protein kinase C inhibitors and were associated with inhibition of phosphorylation of the myosin light chain phosphatase inhibitor CPI-17. Additionally, stimulation of the NO/cGMP/protein kinase G (PKG) signaling pathway still resulted in relaxation of MYPT1-deficient mesenteric arteries, indicating phosphorylation of MYPT1 by PKG is not a major contributor to the relaxation response. Thus, MYPT1 enhances myosin light chain phosphatase activity sufficient for blood pressure maintenance. Rho-associated kinase phosphorylation of CPI-17 plays a significant role in enhancing vascular contractile responses, whereas phosphorylation of MYPT1 in the NO/cGMP/PKG signaling module is not necessary for relaxation.     

Proteolytic cleavage of PDZD2 generates a secreted peptide containing two PDZ domains

PDZD2 (PDZ-domain-containing 2; also known as PAPIN, AIPC and PIN1) is a ubiquitously expressed multi-PDZ-domain protein. We have shown that PDZD2, which shows extensive homology to pro-interleukin-16 (pro-IL-16), is localized mainly to the endoplasmic reticulum (ER). Pro-IL-16 is cleaved in a caspase-3-dependent mechanism to generate the secreted cytokine IL-16. The abundant expression of PDZD2 in the ER, and its sequence similarity to pro-IL-16, suggests that similar post-translational processing of PDZD2 may occur. Indeed, western blotting and mass spectrometry analysis of conditioned medium from cells transfected with epitope-tagged PDZD2 show that there is secretion of a PDZD2 peptide of approximately 37 kDa (sPDZD2, for secreted PDZD2) that contains two PDZ domains. Expression of PDZD2 was detected in several tissues. Furthermore, sPDZD2 secretion is suppressed by the mutation of a sequence that shows similarity to caspase recognition motifs or by treatment with a caspase inhibitor. In summary, PDZD2 is the first reported multi-PDZ protein that is processed by proteolytic cleavage to generate a secreted peptide containing two PDZ domains.     

Assembly of MYPT1 with protein phosphatase-1 in fibroblasts redirects localization and reorganizes the actin cytoskeleton

Dephosphorylation of actin-binding proteins by a specialized form of protein Ser/Thr phosphatase type-1 (PP1) regulates smooth muscle contraction and morphology and motility of nonmuscle cells. This myosin and ezrin/radixin/moesin (ERM)-targeted phosphatase comprises the delta isoform PP1 catalytic subunit plus a primary regulatory subunit called myosin phosphatase targeting (MYPT1). We reconstructed myosin/ERM phosphatase in living rat embryo fibroblasts (REF52 cells) by transient expression of epitope-tagged MYPT1 (myc-MYPT1) plus HA-tagged PP1. Unexpectedly, wild-type myc-MYPT1 expressed alone accumulated predominantly in the nucleus, as visualized by immunofluorescent microscopy, whereas if coexpressed with HA-PP1, it was localized in the cytosol and deposited on cytoskeleton myofilaments. The F38A mutation of MYPT1 that eliminates PP1 binding gave nuclear localization of myc-MYPT1, even when coexpressed with HA-PP1. Thus, expression of both subunits was necessary to form myosin/ERM phosphatase in situ and mediate myofilament localization. The results indicate there is little endogenous PP1 available for interaction or interchange with ectopic regulatory subunits in living cells. We concluded that myosin binding by the C-terminal domain of MYPT1 is not sufficient to override nuclear import in fibroblasts, but the binding of PP1 to myc-MYPT1 neutralizes nuclear import. Full-length myc-MYPT1 plus HA-PP1 induced only subtle changes in organization of the actin cytoskeleton, however coexpression of myc-MYPT1(1-300) with HA-PP1 dispersed stress fibers without major alteration in morphology and myc-MYPT1(1-498) disrupted the cytoskeleton and produced radically extended cells that appeared like neurons. Based on these responses, we conclude that the MYPT1 C-terminus functions as an auto-inhibitory domain, and a central domain in MYPT1 can mediate extensive reorganization of the actin cytoskeleton.     

Basis for the Isoform-specific Interaction of Myosin Phosphatase Subunits Protein Phosphatase 1c �� and Myosin Phosphatase Targeting Subunit 1

Myosin II association with actin, which triggers contraction, is regulated by orchestrated waves of phosphorylation/dephosphorylation of the myosin regulatory light chain. Blocking myosin regulatory light chain phosphorylation with small molecule inhibitors alters the shape, adhesion, and migration of many types of smooth muscle and cancer cells. Dephosphorylation is mediated by myosin phosphatase (MP), a complex that consists of a catalytic subunit (protein phosphatase 1c, PP1c), a large subunit (myosin phosphatase targeting subunit, MYPT), and a small subunit of unknown function. MYPT functions by targeting PP1c onto its substrate, phosphorylated myosin II. Using RNA interference, we show here that stability of PP1c β and MYPT1 is interdependent; knocking down one of the subunits decreases the expression level of the other. Associated changes in cell shape also occur, characterized by flattening and spreading accompanied by increased cortical actin, and cell numbers decrease secondary to apoptosis. Of the three highly conserved isoforms of PP1c, we show that MYPT1 binding is restricted to PP1c β, and, using chimeric analysis and site-directed mutations, that the central region of PP1c β confers the isoform-specific binding. This finding was unexpected because the MP crystal structure has been solved and was reported to identify the variable, C-terminal domain of PP1c β as being the region key for isoform-specific interaction with MYPT1. These findings suggest a potential screening strategy for cardiovascular and cancer therapeutic agents based on destabilizing MP complex formation and function.     

p116Rip targets myosin phosphatase to the actin cytoskeleton and is essential for RhoA/ROCK-regulated neuritogenesis

Activation of the RhoA-Rho kinase (ROCK) pathway stimulates actomyosin-driven contractility in many cell systems, largely through ROCK-mediated inhibition of myosin II light chain phosphatase. In neuronal cells, the RhoA-ROCK-actomyosin pathway signals cell rounding, growth cone collapse, and neurite retraction; conversely, inhibition of RhoA/ROCK promotes cell spreading and neurite outgrowth. The actin-binding protein p116(Rip), whose N-terminal region bundles F-actin in vitro, has been implicated in Rho-dependent neurite remodeling; however, its function is largely unknown. Here, we show that p116(Rip), through its C-terminal coiled-coil domain, interacts directly with the C-terminal leucine zipper of the regulatory myosin-binding subunits of myosin II phosphatase, MBS85 and MBS130. RNA interference-induced knockdown of p116(Rip) inhibits cell spreading and neurite outgrowth in response to extracellular cues, without interfering with the regulation of myosin light chain phosphorylation. We conclude that p116(Rip) is essential for neurite outgrowth and may act as a scaffold to target the myosin phosphatase complex to the actin cytoskeleton.     

Myosin phosphatase targeting subunit 1 affects cell migration by regulating myosin phosphorylation and actin assembly

Myosin II plays important roles in many contractile-like cell functions, including cell migration, adhesion, and retraction. Myosin II is activated by regulatory light chain (RLC) phosphorylation whereas RLC dephosphorylation by myosin light chain phosphatase containing a myosin phosphatase targeting subunit (MYPT1) leads to myosin inactivation. HeLa cells contain MYPT1 in addition to a newly identified human variant 2 containing an internal deletion. RLC dephosphorylation, cell migration, and adhesion were inhibited when either or both MYPT1 isoforms were knocked down by RNA interference. RLC was highly phosphorylated (60%) when both isoforms were suppressed by siRNA treatment relative to control cells (10%) with serum-starvation and ROCK inhibition. Prominent stress fibers and focal adhesions were associated with the enhanced RLC phosphorylation. The reintroduction of MYPT1 or variant 2 in siRNA-treated cells decreased stress fibers and focal adhesions. MYPT1 knockdown also led to an increase of F-actin relative to G-actin in HeLa cells. The myosin inhibitor blebbistatin did not inhibit this effect, indicating MYPT1 likely affects actin assembly independent of RLC phosphorylation. Proper expression of MYPT1 or variant 2 is critical for RLC phosphorylation and actin assembly, thus maintaining normal cellular functions by simultaneously controlling cytoskeletal architecture and actomyosin activation.     

Structural basis for spinophilin-neurabin receptor interaction

Neurabin and spinophilin are neuronal scaffolding proteins that play important roles in the regulation of synaptic transmission through their ability to target protein phosphatase 1 (PP1) to dendritic spines where PP1 dephosphorylates and inactivates glutamate receptors. However, thus far, it is still unknown how neurabin and spinophilin themselves are targeted to these membrane receptors. Spinophilin and neurabin contain a single PDZ domain, a common protein-protein interaction recognition motif, which are 86% identical in sequence. We report the structures of both the neurabin and spinophilin PDZ domains determined using biomolecular NMR spectroscopy. These proteins form the canonical PDZ domain fold. However, despite their high degree of sequence identity, there are distinct and significant structural differences between them, especially between the peptide binding pockets. Using two-dimensional 1H-15N HSQC NMR analysis, we demonstrate that C-terminal peptide ligands derived from glutamatergic AMPA and NMDA receptors and cytosolic proteins directly and differentially bind spinophilin and neurabin PDZ domains. This peptide binding data also allowed us to classify the neurabin and spinophilin PDZ domains as the first identified neuronal hybrid class V PDZ domains, which are capable of binding both class I and II peptides. Finally, the ability to bind to glutamate receptor subunits suggests that the PDZ domains of neurabin and spinophilin are important for targeting PP1 to C-terminal phosphorylation sites in AMPA and NMDA receptor subunits.     

Myosin phosphatase target subunit: Many roles in cell function

Phosphorylation of myosin II is important in many aspects of cell function and involves a myosin kinase, e.g. myosin light chain kinase, and a myosin phosphatase (MP). MP is regulated by the myosin phosphatase target subunit (MYPT1). The domain structure, properties, and genetic analyses of MYPT1 and its isoforms are outlined. MYPT1 binds the catalytic subunit of type 1 phosphatase, delta isoform, and also acts as an interactive platform for many other proteins. A key reaction for MP is with phosphorylated myosin II and the first process shown to be regulated by MP was contractile activity of smooth muscle. In cell division and cell migration myosin II phosphorylation also plays a critical role and these are discussed. However, based on the wide range of partners for MYPT1 it is likely that MP is implicated with substrates other than myosin II. Open questions are whether the diverse functions of MP reflect different cellular locations and/or specific roles for the MYPT1 isoforms.     

The essential role of PP1beta in Drosophila is to regulate nonmuscle myosin

Reversible phosphorylation of myosin regulatory light chain (MRLC) is a key regulatory mechanism controlling myosin activity and thus regulating the actin/myosin cytoskeleton. We show that Drosophila PP1beta, a specific isoform of serine/threonine protein phosphatase 1 (PP1), regulates nonmuscle myosin and that this is the essential role of PP1beta. Loss of PP1beta leads to increased levels of phosphorylated nonmuscle MRLC (Sqh) and actin disorganisation; these phenotypes can be suppressed by reducing the amount of active myosin. Drosophila has two nonmuscle myosin targeting subunits, one of which (MYPT-75D) resembles MYPT3, binds specifically to PP1beta, and activates PP1beta's Sqh phosphatase activity. Expression of a mutant form of MYPT-75D that is unable to bind PP1 results in elevation of Sqh phosphorylation in vivo and leads to phenotypes that can also be suppressed by reducing the amount of active myosin. The similarity between fly and human PP1beta and MYPT genes suggests this role may be conserved.     

A novel regulatory mechanism of myosin light chain phosphorylation via binding of 14-3-3 to myosin phosphatase

Myosin II phosphorylation-dependent cell motile events are regulated by myosin light-chain (MLC) kinase and MLC phosphatase (MLCP). Recent studies have revealed myosin phosphatase targeting subunit (MYPT1), a myosin-binding subunit of MLCP, plays a critical role in MLCP regulation. Here we report the new regulatory mechanism of MLCP via the interaction between 14-3-3 and MYPT1. The binding of 14-3-3beta to MYPT1 diminished the direct binding between MYPT1 and myosin II, and 14-3-3beta overexpression abolished MYPT1 localization at stress fiber. Furthermore, 14-3-3beta inhibited MLCP holoenzyme activity via the interaction with MYPT1. Consistently, 14-3-3beta overexpression increased myosin II phosphorylation in cells. We found that MYPT1 phosphorylation at Ser472 was critical for the binding to 14-3-3. Epidermal growth factor (EGF) stimulation increased both Ser472 phosphorylation and the binding of MYPT1-14-3-3. Rho-kinase inhibitor inhibited the EGF-induced Ser472 phosphorylation and the binding of MYPT1-14-3-3. Rho-kinase specific siRNA also decreased EGF-induced Ser472 phosphorylation correlated with the decrease in MLC phosphorylation. The present study revealed a new RhoA/Rho-kinase-dependent regulatory mechanism of myosin II phosphorylation by 14-3-3 that dissociates MLCP from myosin II and attenuates MLCP activity.     

Involvement of 67-kDa laminin receptor-mediated myosin phosphatase activation in antiproliferative effect of epigallocatechin-3-O-gallate at a physiological concentration on Caco-2 colon cancer cells

Previously we reported that 67-kDa laminin receptor (67LR) mediates epigallocatechin-3-O-gallate (EGCG)-induced cell growth inhibition and reduction of myosin regulatory light chain (MRLC) phosphorylation at Thr-18/Ser-19, which is important for cytokinesis. Here, we found that human colon adenocarcinoma Caco-2 cells exhibited higher expression level of 67LR and EGCG at a physiologically achievable concentration (1 microM) significantly accumulated the cells in G(2)/M phase without affecting expression of Wnt-signaling components. We also found that myosin phosphatase targeting subunit 1 (MYPT1) phosphorylation at Thr-696, which inhibits myosin phosphatase and promotes MRLC phosphorylation, was reduced in response to 1 microM EGCG. 67LR knockdown by RNA interference abolished the inhibitory effects of 1 microM EGCG on cell cycle progression and the phosphorylation of MRLC and MYPT1. These results suggest that through 67LR, EGCG at a physiological concentration can activate myosin phosphatase by reducing MYPT1 phosphorylation and that may be involved in EGCG-induced cell growth inhibition.