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.

Single-Molecule Study Reveals How Receptor and Ras Synergistically Activate PI3Kα and PIP3 Signaling

“…Except where noted otherwise, fluors, synthetic lipids, synthetic phospho-Tyr peptide containing the activation loop sequence of platelet-derived growth factor receptor (an RTK), and other reagents and materials were obtained as previously described from the same suppliers ( 18 , 19 ). In addition, the maleimide headgroup phospholipid dioleolyl-phosphoenthanolamine-N-[4-(p-maleimidomethyl)cyclohexane-carboxamide] (DOPE-MCC or Mal-PE) was obtained from Avanti Polar Lipids (Alabaster, AL).…”

“…To address these mechanistic questions, this study employs single-molecule total internal reflection fluorescence microscopy (TIRFM) to investigate a representative receptor-Ras-PI3K-PIP 3 signaling module reconstituted from four protein/peptide components and two lipid components on a supported lipid bilayer: 1) A widely employed soluble phospho-Tyr peptide (pYp) is used to mimic the flexible, regulatory phospho-Tyr loop of an RTK receptor ( 7 , 8 , 9 , 10 , 18 , 19 ). This peptide possesses phospho-Tyr at one or both conserved phosphorylation sites and is derived from the platelet-derived growth factor receptor, an RTK central to chemotaxis and inflammation.…”

Myristoylated alanine-rich C kinase substrate (MARCKS): a multirole signaling protein in cancers

“…This interaction allows PKC (and possibly calmodulin) [24] to control levels of PIP 2 and by extension the secondary messengers and products of PIP 2 hydrolysis, inositol triphosphate (IP 3 ) and diacylglycerol (DAG). This control of PIP 2 in turn allows MARCKS to regulate cell-signaling pathway effectors including phospholipase D (PLD) [25,26] and phosphoinositide 3-kinase (PI3K) [27,28], the latter of which activates AKT signaling via the phosphorylation of PIP 2 to PIP 3.…”

“…When localized to the plasma membrane, MARCKS crosslinks F-actin in a PIP 2 -dependent manner [26,28]; when phosphorylated and released into the cytosol, no such interactions occur [29,30], suggesting a role in control of the cytoskeleton. Although MARCKS is known to interact directly with F-actin via its effector domain, Laux et al's investigation of the mechanism, currently the most thorough, points to indirect regulation of cortical actin via sequestration of PIP 2 , thereby interfering with its ability to sequester downstream effector proteins that do interact directly with cytoskeletal proteins [31].…”

PIRT the TRP Channel Regulating Protein Binds Calmodulin and Cholesterol-Like Ligands

“…It has been reported to interact with P2 × 3 channels and that it could impact overactive bladder [26], and also co-localizes with P2X2 within the enteric nervous system [27]. Given that PIRT interacts with PIP 2 , and that a number of PIP 2 -dependent channels are also calmodulin-regulated [11,16,[28][29][30], we sought to investigate if PIRT interacts with calmodulin and other common modalities that impact ion channel function.…”