TY - JOUR
T1 - Regulation of a coupled MARCKS-PI3K lipid kinase circuit by calmodulin
T2 - Single-molecule analysis of a membrane-bound signaling module
AU - Ziemba, Brian P.
AU - Swisher, G. Hayden
AU - Masson, Glenn
AU - Burke, John E.
AU - Williams, Roger L.
AU - Falke, Joseph J.
N1 - Funding Information:
Work was funded by National Institutes of Health (NIH) Grant R01 GM063235 (to J.J.F.), by MC Grant U10518430 (to R.L.W.), and NIH Grant T32 GM065103 (to G.H.S.).
Publisher Copyright:
© 2016 American Chemical Society.
PY - 2016/11/22
Y1 - 2016/11/22
N2 - 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.
AB - 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.
UR - http://www.scopus.com/inward/record.url?scp=84997795089&partnerID=8YFLogxK
U2 - 10.1021/acs.biochem.6b00908
DO - 10.1021/acs.biochem.6b00908
M3 - Article
C2 - 27933776
AN - SCOPUS:84997795089
SN - 0006-2960
VL - 55
SP - 6395
EP - 6405
JO - Biochemistry
JF - Biochemistry
IS - 46
ER -