Dataset Information

Regulation of a Coupled MARCKS-PI3K Lipid Kinase Circuit by Calmodulin: Single-Molecule Analysis of a Membrane-Bound Signaling Module.

ABSTRACT: Amoeboid cells that employ chemotaxis to travel up an attractant gradient possess a signaling network assembled on the leading edge of the plasma membrane that senses the gradient and remodels the actin mesh and cell membrane to drive movement in the appropriate direction. In leukocytes such as macrophages and neutrophils, and perhaps in other amoeboid cells as well, the leading edge network includes a positive feedback loop in which the signaling of multiple pathway components is cooperatively coupled. Cytoplasmic Ca2+ is a recently recognized component of the feedback loop at the leading edge where it stimulates phosphoinositide-3-kinase (PI3K) and the production of its product signaling lipid phosphatidylinositol 3,4,5-trisphosphate (PIP3). A previous study implicated Ca2+-activated protein kinase C (PKC) and the phosphatidylinositol 4,5-bisphosphate (PIP2) binding protein MARCKS as two important players in this signaling, because PKC phosphorylation of MARCKS releases free PIP2 that serves as the membrane binding target and substrate for PI3K. This study asks whether calmodulin (CaM), which is known to directly bind MARCKS, also stimulates PIP3 production by releasing free PIP2. Single-molecule fluorescence microscopy is used to quantify the surface density and enzyme activity of key protein components of the hypothesized Ca2+-CaM-MARCKS-PIP2-PI3K-PIP3 circuit. The findings show that CaM does stimulate PI3K lipid kinase activity by binding MARCKS and displacing it from PIP2 headgroups, thereby releasing free PIP2 that recruits active PI3K to the membrane and serves as the substrate for the generation of PIP3. The resulting CaM-triggered activation of PI3K is complete in seconds and is much faster than PKC-triggered activation, which takes minutes. Overall, the available evidence implicates both PKC and CaM in the coupling of Ca2+ and PIP3 signals and suggests these two different pathways have slow and fast activation kinetics, respectively.

SUBMITTER: Ziemba BP

PROVIDER: S-EPMC5632315 | biostudies-literature | 2016 Nov

REPOSITORIES: biostudies-literature

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Regulation of a Coupled MARCKS-PI3K Lipid Kinase Circuit by Calmodulin: Single-Molecule Analysis of a Membrane-Bound Signaling Module.

Ziemba Brian P BP Swisher G Hayden GH Masson Glenn G Burke John E JE Williams Roger L RL Falke Joseph J JJ

Biochemistry 20161110 46

Amoeboid cells that employ chemotaxis to travel up an attractant gradient possess a signaling network assembled on the leading edge of the plasma membrane that senses the gradient and remodels the actin mesh and cell membrane to drive movement in the appropriate direction. In leukocytes such as macrophages and neutrophils, and perhaps in other amoeboid cells as well, the leading edge network includes a positive feedback loop in which the signaling of multiple pathway components is cooperatively ...[more]

PMID: 27933776

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Project description:In chemotaxing ameboid cells, a complex leading-edge signaling circuit forms on the cytoplasmic leaflet of the plasma membrane and directs both actin and membrane remodeling to propel the leading edge up an attractant gradient. This leading-edge circuit includes a putative amplification module in which Ca(2+)-protein kinase C (Ca(2+)-PKC) is hypothesized to phosphorylate myristoylated alanine-rich C kinase substrate (MARCKS) and release phosphatidylinositol-4,5-bisphosphate (PIP2), thereby stimulating production of the signaling lipid phosphatidylinositol-3,4,5-trisphosphate (PIP3) by the lipid kinase phosphoinositide-3-kinase (PI3K). We investigated this hypothesized Ca(2+)-PKC-MARCKS-PIP2-PI3K-PIP3 amplification module and tested its key predictions using single-molecule fluorescence to measure the surface densities and activities of its protein components. Our findings demonstrate that together Ca(2+)-PKC and the PIP2-binding peptide of MARCKS modulate the level of free PIP2, which serves as both a docking target and substrate lipid for PI3K. In the off state of the amplification module, the MARCKS peptide sequesters PIP2 and thereby inhibits PI3K binding to the membrane. In the on state, Ca(2+)-PKC phosphorylation of the MARCKS peptide reverses the PIP2 sequestration, thereby releasing multiple PIP2 molecules that recruit multiple active PI3K molecules to the membrane surface. These findings 1) show that the Ca(2+)-PKC-MARCKS-PIP2-PI3K-PIP3 system functions as an activation module in vitro, 2) reveal the molecular mechanism of activation, 3) are consistent with available in vivo data, and 4) yield additional predictions that are testable in live cells. More broadly, the Ca(2+)-PKC-stimulated release of free PIP2 may well regulate the membrane association of other PIP2-binding proteins, and the findings illustrate the power of single-molecule analysis to elucidate key dynamic and mechanistic features of multiprotein signaling pathways on membrane surfaces.

Project description:The leukocyte chemosensory pathway detects attractant gradients and directs cell migration to sites of inflammation, infection, tissue damage, and carcinogenesis. Previous studies have revealed that local Ca2+ and PIP3 signals at the leading edge of polarized leukocytes play central roles in positive feedback loop essential to cell polarization and chemotaxis. These prior studies showed that stimulation of the leading edge Ca2+ signal can strongly activate PI3K, thereby triggering a larger PIP3 signal, but did not elucidate the mechanistic link between Ca2+ and PIP3 signaling. A hypothesis explaining this link emerged, postulating that Ca2+-activated PKC displaces the MARCKS protein from plasma membrane PIP2, thereby releasing sequestered PIP2 to serve as the target and substrate lipid of PI3K in PIP3 production. In vitro single molecule studies of the reconstituted pathway on lipid bilayers demonstrated the feasibility of this PKC-MARCKS-PI3K regulatory module linking Ca2+ and PIP3 signals in the reconstituted system. The present study tests the model predictions in live macrophages by quantifying the effects of: (a) two pathway activators-PDGF and ATP that stimulate chemoreceptors and Ca2+ influx, respectively; and (b) three pathway inhibitors-wortmannin, EGTA, and Go6976 that inhibit PI3K, Ca2+ influx, and PKC, respectively; on (c) four leading edge activity sensors-AKT-PH-mRFP, CKAR, MARCKSp-mRFP, and leading edge area that report on PIP3 density, PKC activity, MARCKS membrane binding, and leading edge expansion/contraction, respectively. The results provide additional evidence that PKC and PI3K are both essential elements of the leading edge positive feedback loop, and strongly support the existence of a PKC-MARCKS-PI3K regulatory module linking the leading edge Ca2+ and PIP3 signals. As predicted, activators stimulate leading edge PKC activity, displacement of MARCKS from the leading edge membrane and increased leading edge PIP3 levels, while inhibitors trigger the opposite effects. Comparison of the findings for the ameboid chemotaxis of leukocytes with recently published findings for the mesenchymal chemotaxis of fibroblasts suggests that some features of the emerging leukocyte leading edge core pathway (PLC-DAG-Ca2+-PKC-MARCKS-PIP2-PI3K-PIP3) may well be shared by all chemotaxing eukaryotic cells, while other elements of the leukocyte pathway may be specialized features of these highly optimized, professional gradient-seeking cells. More broadly, the findings suggest a molecular mechanism for the strong links between phospho-MARCKS and many human cancers.

Project description:Targeting activated fibroblasts, including myofibroblast differentiation, has emerged as a key therapeutic strategy in patients with idiopathic pulmonary fibrosis (IPF). However, there is no available therapy capable of selectively eradicating myofibroblasts or limiting their genesis. Through an integrative analysis of the regulator genes that are responsible for the activation of IPF fibroblasts, we noticed the phosphatidylinositol 4,5-bisphosphate (PIP2)-binding protein, myristoylated alanine-rich C-kinase substrate (MARCKS), as a potential target molecule for IPF. Herein, we have employed a 25-mer novel peptide, MARCKS phosphorylation site domain sequence (MPS), to determine if MARCKS inhibition reduces pulmonary fibrosis through the inactivation of PI3K/protein kinase B (AKT) signaling in fibroblast cells. We first observed that higher levels of MARCKS phosphorylation and the myofibroblast marker α-smooth muscle actin (α-SMA) were notably overexpressed in all tested IPF lung tissues and fibroblast cells. Treatment with the MPS peptide suppressed levels of MARCKS phosphorylation in primary IPF fibroblasts. A kinetic assay confirmed that this peptide binds to phospholipids, particularly PIP2, with a dissociation constant of 17.64 nM. As expected, a decrease of phosphatidylinositol (3,4,5)-trisphosphate pools and AKT activity occurred in MPS-treated IPF fibroblast cells. MPS peptide was demonstrated to impair cell proliferation, invasion, and migration in multiple IPF fibroblast cells in vitro as well as to reduce pulmonary fibrosis in bleomycin-treated mice in vivo. Surprisingly, we found that MPS peptide decreases α-SMA expression and synergistically interacts with nintedanib treatment in IPF fibroblasts. Our data suggest MARCKS as a druggable target in pulmonary fibrosis and also provide a promising antifibrotic agent that may lead to effective IPF treatments.-Yang, D. C., Li, J.-M., Xu, J., Oldham, J., Phan, S. H., Last, J. A., Wu, R., Chen, C.-H. Tackling MARCKS-PIP3 circuit attenuates fibroblast activation and fibrosis progression.

Project description:Lipid rafts are thought to be key organizers of membrane-protein complexes in cells. Many proteins that interact with rafts have bulky polymeric components such as intrinsically disordered protein domains and polysaccharide chains. Therefore, understanding the interaction between membrane domains and membrane-bound polymers provides insights into the roles rafts play in cells. Multiple studies have demonstrated that high concentrations of membrane-bound polymeric domains create significant lateral steric pressure at membrane surfaces. Furthermore, our recent work has shown that lateral steric pressure at membrane surfaces opposes the assembly of membrane domains. Building on these findings, here we report that membrane-bound polymers are potent suppressors of membrane phase separation, which can destabilize lipid domains with substantially greater efficiency than globular domains such as membrane-bound proteins. Specifically, we created giant vesicles with a ternary lipid composition, which separated into coexisting liquid ordered and disordered phases. Lipids with saturated tails and poly(ethylene glycol) (PEG) chains conjugated to their head groups were included at increasing molar concentrations. When these lipids were sparse on the membrane surface they partitioned to the liquid ordered phase. However, as they became more concentrated, the fraction of GUVs that were phase-separated decreased dramatically, ultimately yielding a population of homogeneous membrane vesicles. Experiments and physical modeling using compositions of increasing PEG molecular weight and lipid miscibility phase transition temperature demonstrate that longer polymers are the most efficient suppressors of membrane phase separation when the energetic barrier to lipid mixing is low. In contrast, as the miscibility transition temperature increases, longer polymers are more readily driven out of domains by the increased steric pressure. Therefore, the concentration of shorter polymers required to suppress phase separation decreases relative to longer polymers. Collectively, our results demonstrate that crowded, membrane-bound polymers are highly efficient suppressors of phase separation and suggest that the ability of lipid domains to resist steric pressure depends on both their lipid composition and the size and concentration of the membrane-bound polymers they incorporate.

Dataset Information

Regulation of a Coupled MARCKS-PI3K Lipid Kinase Circuit by Calmodulin: Single-Molecule Analysis of a Membrane-Bound Signaling Module.

Publications

Regulation of a Coupled MARCKS-PI3K Lipid Kinase Circuit by Calmodulin: Single-Molecule Analysis of a Membrane-Bound Signaling Module.

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