Mutations in the PX-SH3A linker of p47phox decouple PI(3,4)P2 binding from NADPH oxidase activation.
NADPH oxidase is essential in the human innate immune response. p47 (phox), a cytosolic NADPH oxidase component, plays a regulatory role in the activation of NADPH oxidase. Our manipulation of p47 (phox) by mutation and amino acid deletion shows that the linker region between the PX and N-terminal SH3 domain plays a role in blocking the binding of the phosphoinositide 3,4-bisphosphate [PI(3,4)P2], a lipid second messenger generated upon neutrophil activation. Replacement of linker residues 151-158 with glycine alters NMR-measured spin lattice relaxation rates and sedimentation velocity compared to those of the wild-type protein, suggesting that the PX domain is released from its autoinhibited conformation. Liposome binding and surface plasmon resonance experiments confirm this result, showing that this mutant has a similar binding affinity for the isolated PX domain toward PI(3,4)P2. However, an in vitro NADPH oxidase activity assay reveals that this glycine mutant of the full-length protein greatly reduced NADPH oxidase activity upon activation even though it displayed PI(3,4)P2 binding activity comparable to that of the isolated PX domain. Our results highlight an active role of the PX-SH3 linker region in maintaining p47 (phox) in its fully autoinhibited form and demonstrate that binding of p47 (phox) to membrane phospholipids is mechanistically distinct from NADPH oxidase activation.
Shen
Kai
K
Department of Biochemistry, Wake Forest University School of Medicine, Winston-Salem, North Carolina 27157-1016, USA.
Sergeant
Susan
S
Hantgan
Roy R
RR
McPhail
Linda C
LC
Horita
David A
DA
D000595:Amino Acid Sequence; D002942:Circular Dichroism; D005998:Glycine / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D006801:Humans; D008081:Liposomes / Q000378:metabolism; D009682:Magnetic Resonance Spectroscopy; D008969:Molecular Sequence Data; D009154:Mutation; D019255:NADPH Oxidases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D010716:Phosphatidylinositols / Q000378:metabolism*; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D017386:Sequence Homology, Amino Acid; D020349:Surface Plasmon Resonance; D018909:src Homology Domains / Q000235:genetics
D008081:Liposomes; D010716:Phosphatidylinositols; C118301:phosphoinositide-3,4-bisphosphate; D019255:NADPH Oxidases; C060505:neutrophil cytosolic factor 1; D005998:Glycine