Mitosis-specific activation of LIM motif-containing protein kinase and roles of cofilin phosphorylation and dephosphorylation in mitosis

Actin filament dynamics play a critical role in mitosis and cytokinesis. LIM motif-containing protein kinase 1 (LIMK1) regulates actin reorganization by phosphorylating and inactivating cofilin, an actin-depolymerizing and -severing protein. To examine the role of LIMK1 and cofilin during the cell cycle, we measured cell cycle-associated changes in the kinase activity of LIMK1 and in the level of cofilin phosphorylation. Using synchronized HeLa cells, we found that LIMK1 became hyperphosphorylated and activated in prometaphase and metaphase, then gradually returned to the basal level as cells entered into telophase and cytokinesis. Although Rho-associated kinase and p21-activated protein kinase phosphorylate and activate LIMK1, they are not likely to be involved in mitosis-specific activation and phosphorylation of LIMK1. Immunoblot and immunofluorescence analyses using an anti-phosphocofilin-specific antibody revealed that the level of cofilin phosphorylation, similar to levels of LIMK1 activity, increased during prometaphase and metaphase then gradually declined in telophase and cytokinesis. Ectopic expression of LIMK1 increased the level of cofilin phosphorylation throughout the cell cycle and induced the formation of multinucleate cells. These results suggest that LIMK1 is involved principally in control of mitosis-specific cofilin phosphorylation and that dephosphorylation and reactivation of cofilin at later stages of mitosis play a critical role in cytokinesis of mammalian cells.     

Signaling oscillations: Molecular mechanisms and functional roles

Mounting evidence shows that oscillatory activity is widespread in cell signaling. Here, we review some of this recent evidence, focusing on both the molecular mechanisms that potentially underlie such dynamical behavior, and the potential advantages that signaling oscillations might have in cell function. The biological processes considered include cellular differentiation and tissue maintenance, intermittent responses in pluripotent stem cells, and collective cell migration during wound healing. With the aid of mathematical modeling, we review recent examples in which delayed negative feedback has been seen to act as a unifying principle that underpins this wide variety of phenomena.     

Molecular mechanism of cofilin dephosphorylation by ouabain

We previously reported that phosphorylated cofilin-triosephosphate isomerase (TPI) complex interacts with Na,K-ATPase and enhances the pump activity through the phosphorylation of cofilin via Rho-mediated signaling pathway. In this study, we tested the hypothesis that the dephosphorylation of cofilin may be induced through Na,K-ATPase inhibition by ouabain. The phosphorylation level of cofilin by ouabain which decreases in a time- and dose-dependent manner in various human cell lines, remains unchanged by pretreatment with Src inhibitor, PP2; epidermal growth factor receptor (EGFR) inhibitor, AG1478; Raf-1 kinase (Raf) inhibitor, GW5074; and ERK kinase (MEK) inhibitor, PD98059, and by transfection of Ras dominant negative mutant (RasN17). This suggests that ouabain dephosphorylates cofilin through the Src/EGFR/Ras/Raf/MEK pathway. Ouabain activates Ras/Raf/MEK pathway, but down-regulates Rho kinase (ROCK)/LIM kinase (LIMK)/cofilin pathway, implying that there may be a cross-talk by ouabain between the Ras/Raf/MEK and the ROCK/LIMK/cofilin pathways. Immunofluorescence and flow cytometry suggest that ouabain-induced active form of cofilin may be involved in cytoskeletal reorganization and cell volume regulation. Thus, these findings demonstrate a new molecular mechanism for the dephosphorylation of cofilin through the inhibition of Na,K-ATPase by ouabain.     

Calcium signal-induced cofilin dephosphorylation is mediated by Slingshot via calcineurin

Cofilin, an essential regulator of actin filament dynamics, is inactivated by phosphorylation at Ser-3 and reactivated by dephosphorylation. Although cofilin undergoes dephosphorylation in response to extracellular stimuli that elevate intracellular Ca2+ concentrations, signaling mechanisms mediating Ca2+-induced cofilin dephosphorylation have remained unknown. We investigated the role of Slingshot (SSH) 1L, a member of a SSH family of protein phosphatases, in mediating Ca2+-induced cofilin dephosphorylation. The Ca2+ ionophore A23187 and Ca2+-mobilizing agonists, ATP and histamine, induced SSH1L activation and cofilin dephosphorylation in cultured cells. A23187- or histamine-induced SSH1L activation and cofilin dephosphorylation were blocked by calcineurin inhibitors or a dominant-negative form of calcineurin, indicating that calcineurin mediates Ca2+-induced SSH1L activation and cofilin dephosphorylation. Importantly, knockdown of SSH1L expression by RNA interference abolished A23187- or calcineurin-induced cofilin dephosphorylation. Furthermore, calcineurin dephosphorylated SSH1L and increased the cofilin-phosphatase activity of SSH1L in cell-free assays. Based on these findings, we suggest that Ca2+-induced cofilin dephosphorylation is mediated by calcineurin-dependent activation of SSH1L.     

Capsaicin induces cofilin dephosphorylation in human intestinal cells: the triggering role of cofilin in tight-junction signaling

Previously, we demonstrated that capsaicin induces tight-junction (TJ) opening in human intestinal Caco-2 cells. In order to clarify the mechanism underlying the TJ opening action of capsaicin, we performed a proteomics study on capsaicin-treated Caco-2 cells. Phosphorylated cofilin was decreased significantly by capsaicin treatment. In addition, capsaicin induced Ca2+ influx in Caco-2 cells and there was a clear correlation between Ca2+) influx and cofilin dephosphorylation (activation). The Ca2+-chelating reagent EGTA blocked the cofilin dephosphorylation induced by both capsaicin and ionomycin, suggesting that the dephosphorylation was mediated by Ca2+ influx. Finally, transepithelial electrical resistance measurements showed that TJ opening accompanied cofilin dephosphorylation. Our data suggest that TJ opening is mediated by cofilin dephosphorylation, which is caused by capsaicin stimuli, including Ca2+ influx. This is the first report of capsaicin action via the dephosphorylation of cofilin in human intestinal cells.     

Porphyromonas gingivalis SerB-mediated dephosphorylation of host cell cofilin modulates invasion efficiency

Porphyromonas gingivalis, a host-adapted opportunistic pathogen, produces a serine phosphatase, SerB, known to affect virulence, invasion and persistence within the host cell. SerB induces actin filament rearrangement in epithelial cells, but the mechanistic basis of this is not fully understood. Here we investigated the effects of SerB on the actin depolymerizing host protein cofilin. P. gingivalis infection resulted in the dephosphorylation of cofilin in gingival epithelial cells. In contrast, a SerB-deficient mutant of P. gingivalis was unable to cause cofilin dephosphorylation. The involvement of cofilin in P. gingivalis invasion was determined by quantitative image analysis of epithelial cells in which cofilin had been knocked down or knocked in with various cofilin constructs. siRNA-silencing of cofilin led to a significant decrease in numbers of intracellular P. gingivalis marked by an absence of actin colocalization. Transfection with wild-type cofilin or constitutively active cofilin both increased numbers of intracellular bacteria, while constitutively inactive cofilin abrogated invasion. Expression of LIM kinase resulted in reduced P. gingivalis invasion, an effect that was reversed by expression of constitutively active cofilin. These results show that P. gingivalis SerB activity induces dephosphorylation of cofilin, and that active cofilin is required for optimal invasion into gingival epithelial cells.     

Src kinase regulation by phosphorylation and dephosphorylation

Src and Src-family protein-tyrosine kinases are regulatory proteins that play key roles in cell differentiation, motility, proliferation, and survival. The initially described phosphorylation sites of Src include an activating phosphotyrosine 416 that results from autophosphorylation, and an inhibiting phosphotyrosine 527 that results from phosphorylation by C-terminal Src kinase (Csk) and Csk homologous kinase. Dephosphorylation of phosphotyrosine 527 increases Src kinase activity. Candidate phosphotyrosine 527 phosphatases include cytoplasmic PTP1B, Shp1 and Shp2, and transmembrane enzymes include CD45, PTPalpha, PTPepsilon, and PTPlambda. Dephosphorylation of phosphotyrosine 416 decreases Src kinase activity. Thus far PTP-BL, the mouse homologue of human PTP-BAS, has been shown to dephosphorylate phosphotyrosine 416 in a regulatory fashion. The platelet-derived growth factor receptor protein-tyrosine kinase mediates the phosphorylation of Src Tyr138; this phosphorylation has no direct effect on Src kinase activity. The platelet-derived growth factor receptor and the ErbB2/HER2 growth factor receptor protein-tyrosine kinases mediate the phosphorylation of Src Tyr213 and activation of Src kinase activity. Src kinase is also a substrate for protein-serine/threonine kinases including protein kinase C (Ser12), protein kinase A (Ser17), and CDK1/cdc2 (Thr34, Thr46, and Ser72). Of the three protein-serine/threonine kinases, only phosphorylation by CDK1/cdc2 has been demonstrated to increase Src kinase activity. Although considerable information on the phosphoprotein phosphatases that catalyze the hydrolysis of Src phosphotyrosine 527 is at hand, the nature of the phosphatases that mediate the hydrolysis of phosphotyrosine 138 and 213, and phosphoserine and phosphothreonine residues has not been determined.     

Glucose phosphorylation and dephosphorylation in chicken liver.

1. Glucokinase was absent from chicken liver and only the low Km hexokinases, inhibited by AMP, ADP but not ATP, were present. 2. The Km of chicken liver glucose-6-phosphatase for glucose-6-phosphate was reduced from 5.65 to 3.75 mM following starvation, and the enzyme was inhibited by glucose. 3. Starvation of chickens for 24 hr slightly lowered the hexokinase activity and doubled glucose-6-phosphatase activity; it did not change subcellular distribution of the enzymes. Oral glucose rapidly restored the activities to fed values. 4. It was concluded that glucose uptake into, and efflux from, chicken hepatocytes, was regulated by the activity and kinetic characteristics of glucose-6-phosphatase and by the glucose-6-phosphate concentration, and that the hexokinases had little regulatory function.     

Direct Rho-associated kinase inhibiton induces cofilin dephosphorylation and neurite outgrowth in PC-12 cells

Axons fail to regenerate in the adult central nervous system (CNS) following injury. Developing strategies to promote axonal regeneration is therapeutically attractive for various CNS pathologies such as traumatic brain injury, stroke and Alzheimer’s disease. Because the RhoA pathway is involved in neurite outgrowth, Rho-associated kinases (ROCKs), downstream effectors of GTP-bound Rho, are potentially important targets for axonal repair strategies in CNS injuries. We investigated the effects and downstream mechanisms of ROCK inhibition in promoting neurite outgrowth in a PC-12 cell model. Robust neurite outgrowth (NOG) was induced by ROCK inhibitors Y-27632 and H-1152 in a time-and dose-dependent manner. Dramatic cytoskeletal reorganization was noticed upon ROCK inhibition. NOG initiated within 5 to 30 minutes followed by neurite extension between 6 and 10 hours. Neurite processes were then sustained for over 24 hours. Rapid cofilin dephosphorylation was observed within 5 minutes of Y-27632 and H-1152 treatment. Re-phosphorylation was observed by 6 hours after Y-27632 treatment, while H-1152 treatment produced sustained cofilin dephosphorylation for over 24 hours. The results suggest that ROCK-mediated dephosphorylation of cofilin plays a role in the initiation of NOG in PC-12 cells.     

Regulation of spermidine acetyltransferase activity by phosphorylation and dephosphorylation

Spermidine acetyltransferase activity is more than 10-fold higher in the pancreas of a 20-hr-fasted than in that of a fed chicken. The preparation of the fed bird inactivates the other. The effect is due to a thermolabile component of microsomes, and is also obtained with alkaline phosphatase. The inactivated preparation partially recovers its activity through phosphorylation catalyzed by a cAMP-dependent protein kinase. The results presented strongly suggest that spermidine acetyltransferase activity is regulated by phosphorylation and dephosphorylation.     

Regulation through phosphorylation/dephosphorylation cascade systems

The cyclic interconversion of enzymes between phosphorylated and unphosphorylated forms comprises a major mechanism of cellular regulation. A theoretical analysis of reversible covalent modification systems (Stadtman, E.R., and Chock, P.B. (1977) Proc. Natl. Acad. Sci. U.S.A. 74, 2761-2765) revealed that they are endowed with extraordinary regulatory capacities; they may exhibit smooth, flexible responses to changes in single and multiple metabolite levels, signal amplification, and apparent positive cooperativity. To test qualitatively and quantitatively the theories and equations involved in this analysis, a model in vitro phosphorylation/dephosphorylation cyclic cascade was developed in which the converter enzymes catalyzing the covalent modifications were cAMP-dependent protein kinase (EC 2.7.1.37; type II) and phosphoprotein phosphatase (EC 3.1.3.16; Mr = 38,000), both purified to near homogeneity from bovine heart. The kinetic constants for both enzymes were fully characterized using the nanopeptide Leu-Arg-Arg-Ala-Ser-Val-Ala-Gln-Leu as the interconvertible substrate, cAMP as an activator for the kinase, and Pi as an inhibitor for the phosphatase. In the presence of a nearly constant concentration of ATP, a steady-state level of phosphorylation of the peptide was attained which was determined by the relative concentrations of the kinase, phosphatase, and effectors. As predicted by the cyclic cascade model, this monocyclic cascade exhibited both signal amplification and an increase in sensitivity to variations in multiple effector concentrations. In addition, the data show that the steady-state level of phosphorylation obtained in the presence of an activator of the kinase (e.g. cAMP) and an inhibitor of the phosphatase (e.g. Pi) is a function of the product of the relative effector concentrations. Finally, the results reveal that when the concentration of enzyme-substrate complex is not negligible, cyclic cascades are potentially more sensitive to variations in effector concentrations and can achieve even greater signal amplification than predicted previously.     

Concerted regulation of protein phosphorylation and dephosphorylation by calmodulin

The multiple functions of calmodulin in brain bring to light an apparent paradox in the mechanism of action of this multifunctional regulatory protein: How can the simultaneous calmodulin stimulation of enzymes with opposing functions such as cyclic nucleotide phosphodiesterases and adenylate cyclase, which are responsible for the degradation and synthesis of cAMP, respectively, be physiologically significant? The same question applies to the simultaneous activation of protein kinases (in particular calmodulin kinase II) and a protein phosphatase (calcineurin). One could propose that the protein kinase(s) and the phosphatase may be located in different cells or in different cellular compartments, and are therefore not antagonizing each other. The same result could be achieved if the specific substrates of these enzymes have different cellular localizations. This does not seem to be the case. In many areas of the brain the two enzymes and their substrates coexist in the same cell. For example, the hippocampus is rich in calmodulin kinase II, calcineruin and substrates for the two enzymes. A more general scheme is presented here, based on different mechanisms of the calmodulin regulation of the two classes of enzyme, which helps to solve this apparent inconsistency in the mechanism of action of calmodulin.     

Involvement of slingshot in the Rho-mediated dephosphorylation of ADF/cofilin during Xenopus cleavage

ADF/cofilin is a key regulator for actin dynamics during cytokinesis. Its activity is suppressed by phosphorylation and reactivated by dephosphorylation. Little is known, however, about regulatory mechanisms of ADF/cofilin function during formation of contractile ring actin filaments. Using Xenopus cycling extracts, we found that ADF/cofilin was dephosphorylated at prophase and telophase. In addition, constitutively active Rho GTPase induced dephosphorylation of ADF/cofilin in the egg extracts. This dephosphorylation was inhibited by Na(3)VO (4) but not by other conventional phosphatase-inhibitors. We cloned a Xenopus homologue of Slingshot phosphatase (XSSH), originally identified in Drosophila and human as an ADF/cofilin phosphatase, and raised antibody specific for the catalytic domain of XSSH. This inhibitory antibody significantly suppressed the Rho-induced dephosphorylation of ADF/cofilin in extracts, suggesting that the dephosphorylation at telophase is dependent on XSSH. XSSH bound to actin filaments with a dissociation constant of 0.4 microM, and the ADF/cofilin phosphatase activity was increased in the presence of F-actin. When latrunculin A, a G-actin-sequestering drug, was added to extracts, both Rho-induced actin polymerization and dephosphorylation of ADF/cofilin were markedly inhibited. Jasplakinolide, an actin-stabilizing drug, alone induced actin polymerization in the extracts and lead to dephosphorylation of ADF/cofilin. These results suggest that Rho-induced dephosphorylation of ADF/cofilin is dependent on the XSSH activation that is caused by increase in the amount of F-actin induced by Rho signaling. XSSH colocalized with both actin filaments and ADF/cofilin in the actin patches formed on the surface of the early cleavage furrow. Injection of inhibitory antibody blocked cleavage of blastomeres. Thus, XSSH may reorganize actin filaments through dephosphorylation and reactivation of ADF/cofilin at early stage of contractile ring formation.     

U73122 inhibits the dephosphorylation and translocation of cofilin in activated macrophage-like U937 cells

Cofilin, an actin-binding protein, plays an important role in the migration, phagocytosis, and superoxide production of activated phagocytes through cytoskeletal reorganization. In unstimulated phagocytes, cofilin is a major phosphoprotein. However, upon activation, the phosphoprotein is dephosphorylated and translocated from cytosol to plasma membranes. Only the unphosphorylated form of cofilin is an active form that binds actin, whereas the regulatory mechanisms of cofilin have not been elucidated. We found that 1-[6-[[17beta-3-methoxyestra-1,3,5(10)-trien-17-yl]amino]hexyl]-1H-pyrrole-2,5-dione (U73122), an inhibitor of phospholipase C (PLC), suppressed both opsonized zymosan (OZ)-induced dephosphorylation and translocation of cofilin in macrophage-like U937 cells at 4 microM concentration. OZ triggered an increase in inositol 1,4,5-trisphosphate (IP3), and U73122 inhibited it. 1-[6-[[17beta-3-Methoxyestra-1,3,5(10)-trien-17-yl]amino]hexyl]-1H-pyrrole-2,5-pyrrodione-dione (U73343), which was employed as an inactive analogue, had no such inhibitory activities as did U73122. Furthermore, herbimycin A, an inhibitor of src-type tyrosine kinase, also inhibited OZ-triggered IP3 formation. These results suggest that the activity and localization of cofilin are regulated by PLC at the downstream of src-family tyrosine kinase.