title: 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 PIP2that 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 PIP3production 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-PIP3circuit. The findings show that CaM does stimulate PI3K lipid kinase activity by binding MARCKS and displacing it from PIP2headgroups, thereby releasing free PIP2that 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 PIP3signals and suggests these two different pathways have slow and fast activation kinetics, respectively.
authors.affiliation: Molecular Biophysics Program and Department of Chemistry and Biochemistry, University of Colorado , Boulder, Colorado 80309-0215, United States.
authors.forename: Brian P
authors.initials: BP
authors.lastname: Ziemba
authors.affiliation: Molecular Biophysics Program and Department of Chemistry and Biochemistry, University of Colorado , Boulder, Colorado 80309-0215, United States.
authors.forename: G Hayden
authors.initials: GH
authors.lastname: Swisher
authors.affiliation: Laboratory of Molecular Biology, Medical Research Council , Cambridge CB2 0QH, U.K.
authors.forename: Glenn
authors.initials: G
authors.lastname: Masson
authors.affiliation: Laboratory of Molecular Biology, Medical Research Council , Cambridge CB2 0QH, U.K.
authors.forename: John E
authors.initials: JE
authors.lastname: Burke
authors.affiliation: Laboratory of Molecular Biology, Medical Research Council , Cambridge CB2 0QH, U.K.
authors.forename: Roger L
authors.initials: RL
authors.lastname: Williams
authors.affiliation: Molecular Biophysics Program and Department of Chemistry and Biochemistry, University of Colorado , Boulder, Colorado 80309-0215, United States.
authors.forename: Joseph J
authors.initials: JJ
authors.lastname: Falke
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D002118:Calcium / Q000378:metabolism; D002147:Calmodulin / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D002462:Cell Membrane / Q000378:metabolism*; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008565:Membrane Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008856:Microscopy, Fluorescence; D008958:Models, Molecular; D000076250:Myristoylated Alanine-Rich C Kinase Substrate; D058539:Phosphatidylinositol 3-Kinase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D019269:Phosphatidylinositol 4,5-Diphosphate / Q000378:metabolism; D018129:Phosphatidylinositol Phosphates / Q000378:metabolism; D011485:Protein Binding; D000072417:Protein Domains; D061987:Sf9 Cells; D015398:Signal Transduction; D018411:Spodoptera; D013997:Time Factors
chemical_list: D002147:Calmodulin; D047908:Intracellular Signaling Peptides and Proteins; C000619710:MARCKS protein, human; D008565:Membrane Proteins; D019269:Phosphatidylinositol 4,5-Diphosphate; D018129:Phosphatidylinositol Phosphates; C060974:phosphatidylinositol 3,4,5-triphosphate; D000076250:Myristoylated Alanine-Rich C Kinase Substrate; D058539:Phosphatidylinositol 3-Kinase; D002118:Calcium