Subcellular localization and function of alternatively spliced Noxo1 isoforms

Abstract

Nox organizer 1 (Noxo1), a p47 phox homolog, is produced as four isoforms with unique N-terminal PX domains derived by alternative mRNA splicing. We compared the subcellular distribution of these isoforms or their isolated PX domains produced as GFP fusion proteins, as well as their ability to support Nox1 activity in several transfected models. Noxo1α, β, γ, and δ show different subcellular localization patterns, determined by their PX domains. In HEK293 cells, Noxo1β exhibits prominent plasma membrane binding, Noxo1γ shows plasma membrane and nuclear associations, Noxo1α and δ localize primarily on intracellular vesicles or cytoplasmic aggregates, but not the plasma membrane. Nox1 activity correlates with Noxo1 plasma membrane binding in HEK293 cells, since Noxo1β supports the highest activity and Noxo1γ and Noxo1α support moderate or low activities, respectively. In COS-7 cells, where Noxo1α localizes on the plasma membrane, the activities supported by the three isoforms (α, β, and γ) do not differ significantly. The PX domains of β and γ bind the same phospholipids, including phosphatidic acid. These results indicate the variant PX domains are unique determinants of Noxo1 localization and Nox1 function. Finally, the overexpressed Noxo1 isoforms do not affect p22 phox localization, although Nox1 is needed to transport p22 phox to the plasma membrane.

Phosphatidylinositol (4,5)-bisphosphate Modulates Nox5 Localization via an N-Terminal Polybasic Region

“…The PX domain of p47phox binds with highest affinity to PtdIns(3,4)P 2 , and with lower, but significant, affinity to PtdIns(4,5)P 2 , PtdIns(3,4,5)P 3 , PtdIns(3,5)P 2 , and PtdIns(3)P (Kanai et al, 2001;Karathanassis et al, 2002). The PX domains of human NOXO1 and p40phox show a different lipid-binding specificity, with NOXO1 having highest affinity for PtdIns(3,5)P 2 , PtdIns(5)P, PtdIns(4)P, and p40phox exhibiting a preference for PtdIns(3)P Ellson et al, 2006;Takeya et al, 2006;Ueyama et al, 2007). Interaction between PtdIns phosphates and the PX domain is essential for membrane-targeting of these regulatory subunits, resulting in the formation of the active complex with Nox1 (through the PX-domain of NOXO1) or with Nox2 (through the PX-domains of p47phox and p40phox).…”

NADPH Oxidases as Novel Pharmacologic Targets against Influenza A Virus Infection

“…Also, like p47phox, the PX domain of NOXO1 associates with membrane phospholipids, and the identification of four alternative splice variants of NOXO1 Ueyama et al, 2007), which vary in the nature of their PX domain, influences the subcellular localization of NOX1 and ROS production (Opitz et al, 2007).…”

“…Similar to p67phox, NOXA1 (molecular size ∼51 kDa) contains four TPRs that associate with Rac and an activation domain for NOX1 binding, which are required for full NOX1 activity (Cheng et al, 2006;Miyano et al, 2006;Ueyama et al, 2007). Also, like p67phox, NOXA1 contains a PBI domain; however, it does not possess the conserved lysine residue and therefore fails to interact with p40phox (Ago et al, 2003).…”

The NOXO1β PX Domain Preferentially Targets PtdIns(4,5)P2 and PtdIns(3,4,5)P3

“…Its absence would alter the helical register, resulting in L52 being surface-exposed and K53 being buried, likely disrupting proper folding of the PX domain. Both NOXO1α and δ localize to intracellular vesicles and/or form large aggregates when overexpressed 11 , consistent with partial misfolding of the protein. NOXO1γ and δ differ from NOXO1β and α, respectively, in containing the five-residue insert GQASL between L74 and D75 (NOXO1β numbering).…”

“…Variations in the size of the pocket and in the identity of residues that contact the PtdIns head group appear to dictate specificity of a given PX domain for a given phosphorylated PtdIns. Protein-lipid overlay analyses of NOXO1β suggest a broad preference for anionic phosphatidylinositols, including PtdIns(3,5)P 2 , PtdIns(3)P, PtdIns(4)P, and PtdIns(5)P. Cell localization studies show that NOXO1β is constitutively membrane-bound and that this localization is dependent on the PX domain 9–11 . The constitutive membrane localization, and hence activation of Nox1, by NOXO1β is consistent with constitutive ROS production observed in HEK293 cells transfected with murine Nox1, NOXA1, and NOXO1 12 .…”