APKCζ affects directed cell migration through the regulation of myosin light chain phosphorylation.
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ABSTRACT:
SUBMITTER: Petrov D
PROVIDER: S-EPMC5569965 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Myosin light chain kinase (MLCK) has long been implicated in the myosin phosphorylation and force generation required for cell migration. Here, we surprisingly found that the deletion of MLCK resulted in fast cell migration, enhanced protrusion formation, and no alteration of myosin light chain phosphorylation. The mutant cells showed reduced membrane tether force and fewer membrane F-actin filaments. This phenotype was rescued by either kinase-dead MLCK or five-DFRXXL motif, a MLCK fragment with potent F-actin-binding activity. Pull-down and co-immunoprecipitation assays showed that the absence of MLCK led to attenuated formation of transmembrane complexes, including myosin II, integrins and fibronectin. We suggest that MLCK is not required for myosin phosphorylation in a migrating cell. A critical role of MLCK in cell migration involves regulating the cell membrane tension and protrusion necessary for migration, thereby stabilizing the membrane skeleton through F-actin-binding activity. This finding sheds light on a novel regulatory mechanism of protrusion during cell migration.
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Project description:Myosin light chain (MLC) phosphorylation plays important roles in various cellular functions such as cellular morphogenesis, motility, and smooth muscle contraction. MLC phosphorylation is determined by the balance between activities of Rho-associated kinase (Rho-kinase) and myosin phosphatase. An impaired balance between Rho-kinase and myosin phosphatase activities induces the abnormal sustained phosphorylation of MLC, which contributes to the pathogenesis of certain vascular diseases, such as vasospasm and hypertension. However, the dynamic principle of the system underlying the regulation of MLC phosphorylation remains to be clarified. Here, to elucidate this dynamic principle whereby Rho-kinase regulates MLC phosphorylation, we developed a mathematical model based on the behavior of thrombin-dependent MLC phosphorylation, which is regulated by the Rho-kinase signaling network. Through analyzing our mathematical model, we predict that MLC phosphorylation and myosin phosphatase activity exhibit bistability, and that a novel signaling pathway leading to the auto-activation of myosin phosphatase is required for the regulatory system of MLC phosphorylation. In addition, on the basis of experimental data, we propose that the auto-activation pathway of myosin phosphatase occurs in vivo. These results indicate that bistability of myosin phosphatase activity is responsible for the bistability of MLC phosphorylation, and the sustained phosphorylation of MLC is attributed to this feature of bistability.
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Project description:Perturbed Nicotinamide adenine dinucleotide (NAD+) homeostasis is involved in cancer progression and metastasis. Quinolinate phosphoribosyltransferase (QPRT) is the rate-limiting enzyme in the kynurenine pathway participating in NAD+ generation. In this study, we demonstrated that QPRT expression was upregulated in invasive breast cancer and spontaneous mammary tumors from MMTV-PyVT transgenic mice. Knockdown of QPRT expression inhibited breast cancer cell migration and invasion. Consistently, ectopic expression of QPRT promoted cell migration and invasion in breast cancer cells. Treatment with QPRT inhibitor (phthalic acid) or P2Y11 antagonist (NF340) could reverse the QPRT-induced invasiveness and phosphorylation of myosin light chain. Similar reversibility could be observed following treatment with Rho inhibitor (Y16), ROCK inhibitor (Y27632), PLC inhibitor (U73122), or MLCK inhibitor (ML7). Altogether, these results indicate that QPRT enhanced breast cancer invasiveness probably through purinergic signaling and might be a potential prognostic indicator and therapeutic target in breast cancer.
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Project description:The proper orientation of the mitotic spindle is essential for mitosis; however, how these events unfold at the molecular level is not well understood. AMP-activated protein kinase (AMPK) regulates energy homeostasis in eukaryotes, and AMPK-null Drosophila mutants have spindle defects. We show that threonine(172) phosphorylated AMPK localizes to the mitotic spindle poles and increases when cells enter mitosis. AMPK depletion causes a mitotic delay with misoriented spindles relative to the normal division plane and a reduced number and length of astral microtubules. AMPK-depleted cells contain mitotic actin bundles, which prevent astral microtubule-actin cortex attachments. Since myosin regulatory light chain (MRLC) is an AMPK downstream target and mediates actin function, we investigated whether AMPK signals through MRLC to control spindle orientation. Mitotic levels of serine(19) phosphorylated MRLC (pMRLC(ser19)) and spindle pole-associated pMRLC(ser19) are abolished when AMPK function is compromised, indicating that AMPK is essential for pMRLC(ser19) spindle pole activity. Phosphorylation of AMPK and MRLC in the mitotic spindle is dependent upon calcium/calmodulin-dependent protein kinase kinase (CamKK) activity in LKB1-deficient cells, suggesting that CamKK regulates this pathway when LKB1 function is compromised. Taken together, these data indicate that AMPK mediates spindle pole-associated pMRLC(ser19) to control spindle orientation via regulation of actin cortex-astral microtubule attachments.
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Project description:The enhanced migration found in tumor cells is often caused by external stimuli and the sequential participation of cytoskeleton-related signaling molecules. However, until now, the molecular connection between the lysophosphatidic acid (LPA) receptor and nonmuscle myosin II (NM II) has not been analyzed in detail for LPA-induced migration. Here, we demonstrate that LPA induces migration by activating the LPA(1) receptor which promotes phosphorylation of the 20 kDa NM II light chain through activation of Rho kinase (ROCK). We show that LPA-induced migration is insensitive to pertussis toxin (PTX) but does require the LPA(1) receptor as determined by siRNA and receptor antagonists. LPA activates ROCK and also increases GTP-bound RhoA activity, concomitant with the enhanced membrane recruitment of RhoA. LPA-induced migration and invasion are attenuated by specific inhibitors including C3 cell-permeable transferase and Y-27632. We demonstrate that NM II plays an important role in LPA-induced migration and invasion by inhibiting its cellular function with blebbistatin and shRNA lentivirus directed against NM II-A or II-B. Inhibition or loss of either NM II-A or NM II-B in 4T1 cells results in a decrease in migration and invasion. Restoration of the expression of NM II-A or NM II-B also rescued LPA-induced migration. Taken together, these results suggest defined pathways for signaling through the LPA(1) receptor to promote LPA-mediated NM II activation and subsequent cell migration in 4T1 breast cancer cells.
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Project description:Hyperphosphorylation of myosin regulatory light chain (RLC) in cardiac muscle is proposed to cause compensatory hypertrophy. We therefore investigated potential mechanisms in genetically modified mice. Transgenic (TG) mice were generated to overexpress Ca2+/calmodulin-dependent myosin light chain kinase specifically in cardiomyocytes. Phosphorylation of sarcomeric cardiac RLC and cytoplasmic nonmuscle RLC increased markedly in hearts from TG mice compared with hearts from wild-type (WT) mice. Quantitative measures of RLC phosphorylation revealed no spatial gradients. No significant hypertrophy or structural abnormalities were observed up to 6 months of age in hearts of TG mice compared with WT animals. Hearts and cardiomyocytes from WT animals subjected to voluntary running exercise and isoproterenol treatment showed hypertrophic cardiac responses, but the responses for TG mice were attenuated. Additional biochemical measurements indicated that overexpression of the Ca2+/calmodulin-binding kinase did not perturb other Ca2+/calmodulin-dependent processes involving Ca2+/calmodulin-dependent protein kinase II or the protein phosphatase calcineurin. Thus, increased myosin RLC phosphorylation per se does not cause cardiac hypertrophy and probably inhibits physiological and pathophysiological hypertrophy by contributing to enhanced contractile performance and efficiency.
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Project description:The paracellular gap formed by endothelial cell (EC) contraction is fundamental for endothelium permeability, but the mechanism underlying EC contraction has yet to be determined. Here, we identified the zipper-interacting protein kinase (ZIPK) as the kinase for EC contraction and myosin light chain (MLC) phosphorylation. Inhibition of ZIPK activity by pharmacological inhibitors and small interfering RNAs led to a significant decrease in the mono- and diphosphorylation of MLCs along with a contractile response to thrombin, suggesting an essential role of ZIPK in EC paracellular permeability. To assess the role of ZIPK in vivo, we established mouse lines with conditional deletion of Zipk gene. The endothelium-specific deletion of Zipk led to embryonic lethality, whereas the UBC-CreERT2-mediated deletion of Zipk by tamoxifen induction at adulthood caused no apparent phenotype. The induced deletion of Zipk significantly inhibited ischemia-reperfusion-induced blood-brain barrier dysfunction and neuronal injuries from middle cerebral artery occlusion and reperfusion, as evidenced by reduced infarct and edema volume, attenuated Evans blue dye leakage, and improved neuronal behavior. We thus concluded that ZIPK and its phosphorylation of MLC were required for EC contraction and ischemic neuronal injuries. ZIPK may be a prospective therapeutic target for stroke.-Zhang, Y., Zhang, C., Zhang, H., Zeng, W., Li, S., Chen, C., Song, X., Sun, J., Sun, Z., Cui, C., Cao, X., Zheng, L., Wang, P., Zhao, W., Zhang, Z., Xu, Y., Zhu, M., Chen, H. ZIPK mediates endothelial cell contraction through myosin light chain phosphorylation and is required for ischemic-reperfusion injury.
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Project description:Familial hypertrophic cardiomyopathy (HCM) is characterized by left ventricular hypertrophy and myofibrillar disarray, and often results in sudden cardiac death. Two HCM mutations, N47K and R58Q, are located in the myosin regulatory light chain (RLC). The RLC mechanically stabilizes the myosin lever arm, which is crucial to myosin's ability to transmit contractile force. The N47K and R58Q mutations have previously been shown to reduce actin filament velocity under load, stemming from a more compliant lever arm (Greenberg, 2010). In contrast, RLC phosphorylation was shown to impart stiffness to the myosin lever arm (Greenberg, 2009). We hypothesized that phosphorylation of the mutant HCM-RLC may mitigate distinct mutation-induced structural and functional abnormalities. In vitro motility assays were utilized to investigate the effects of RLC phosphorylation on the HCM-RLC mutant phenotype in the presence of an ?-actinin frictional load. Porcine cardiac ?-myosin was depleted of its native RLC and reconstituted with mutant or wild-type human RLC in phosphorylated or non-phosphorylated form. Consistent with previous findings, in the presence of load, myosin bearing the HCM mutations reduced actin sliding velocity compared to WT resulting in 31-41% reductions in force production. Myosin containing phosphorylated RLC (WT or mutant) increased sliding velocity and also restored mutant myosin force production to near WT unphosphorylated values. These results point to RLC phosphorylation as a general mechanism to increase force production of the individual myosin motor and as a potential target to ameliorate the HCM-induced phenotype at the molecular level.
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Project description:The effect of phosphorylation on the conformation of the regulatory light chain (cRLC) region of myosin in ventricular trabeculae from rat heart was determined by polarized fluorescence from thiophosphorylated cRLCs labelled with bifunctional sulforhodamine (BSR). Less than 5% of cRLCs were endogenously phosphorylated in this preparation, and similarly low values of basal cRLC phosphorylation were measured in fresh intact ventricle from both rat and mouse hearts. BSR-labelled cRLCs were thiophosphorylated by a recombinant fragment of human cardiac myosin light chain kinase, which was shown to phosphorylate cRLCs specifically at serine 15 in a calcium- and calmodulin-dependent manner, both in vitro and in situ. The BSR-cRLCs were exchanged into demembranated trabeculae, and polarized fluorescence intensities measured for each BSR-cRLC in relaxation, active isometric contraction and rigor were combined with RLC crystal structures to calculate the orientation distribution of the C-lobe of the cRLC in each state. Only two of the four C-lobe orientation populations seen during relaxation and active isometric contraction in the unphosphorylated state were present after cRLC phosphorylation. Thus cRLC phosphorylation alters the equilibrium between defined conformations of the cRLC regions of the myosin heads, rather than simply disordering the heads as assumed previously. cRLC phosphorylation also changes the orientation of the cRLC C-lobe in rigor conditions, showing that the orientation of this part of the myosin head is determined by its interaction with the thick filament even when the head is strongly bound to actin. These results suggest that cRLC phosphorylation controls the contractility of the heart by modulating the interaction of the cRLC region of the myosin heads with the thick filament backbone.
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Project description:Praziquantel (PZQ) is the drug of choice for schistosomiasis and probably is the only highly effective drug currently available for treating schistosomiasis-infected individuals. The mode of action of PZQ involves increasing the calcium uptake of the parasite, resulting in tegumental damage and death of the parasite. Despite its remarkable function, the target of PZQ has not been identified yet. To begin to understand where PZQ acts, in this study we expressed the cDNA library of Schistosoma mansoni on the surface of T7 bacteriophages and screened this library with labeled PZQ. This procedure identified a clone that strongly bound to PZQ. Subsequent DNA analysis of inserts showed that the clone coded for regulatory myosin light chain protein. The gene was then cloned, and recombinant S. mansoni myosin light chain (SmMLC) was expressed. Immunoblot analysis using antibodies raised to recombinant SmMLC (rSmMLC) showed that SmMLC is abundantly expressed in schistosomula and adult stages compared to the amount in cercarial stages. In vitro analyses also confirmed that PZQ strongly binds to rSmMLC. Further, peptide mapping studies showed that PZQ binds to amino acids 46 to 76 of SmMLC. Immunoprecipitation analysis confirmed that SmMLC is phosphorylated in vivo upon exposure to PZQ. Interestingly, significant levels of anti-SmMLC antibodies were present in vaccinated mice compared to the amount in infected mice, suggesting that SmMLC may be a potential target for protective immunity in schistosomiasis. These findings suggest that PZQ affects SmMLC function, and this may have a role in PZQ action.
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