The integral membrane protein p22phox is an indispensable component of the 
superoxide-generating phagocyte NADPH oxidase, whose catalytic core is the 
membrane-associated gp91phox (also known as Nox2). p22phox associates with 
gp91phox and, through its proline-rich C terminus, provides a binding site for 
the tandem Src homology 3 domains of the activating subunit p47phox. Whereas 
p22phox is expressed ubiquitously, its participation in regulating the activity 
of other Nox enzymes is less clear. This study investigates the requirement of 
p22phox for Nox enzyme activity and explores the role of its proline-rich region 
(PRR) for regulating activity. Coexpression of specific Nox catalytic subunits 
(Nox1, Nox2, Nox3, Nox4, or Nox5) along with their corresponding regulatory 
subunits (NOXO1/NOXA1 for Nox1; p47phox/p67phox/Rac for Nox2; NOXO1 for Nox3; no 
subunits for Nox4 or Nox5) resulted in marked production of reactive oxygen. 
Small interfering RNAs decreased endogenous p22phox expression and inhibited 
reactive oxygen generation from Nox1, Nox2, Nox3, and Nox4 but not Nox5. 
Truncated forms of p22phox that disrupted the PRR-inhibited reactive oxygen 
generation from Nox1, Nox2, and Nox3 but not from Nox4 and Nox5. Similarly, 
p22phox (P156Q), a mutation that disrupts Src homology 3 binding by the PRR, 
potently inhibited reactive oxygen production from Nox1 and Nox2 but not from 
Nox4 and Nox5. Expression of p22phox (P156Q) inhibited NOXO1-stimulated Nox3 
activity, but co-expression of NOXA1 overcame the inhibitory effect. The P157Q 
and P160Q mutations of p22phox showed selective inhibition of 
Nox2/p47phox/p67phox, and selectivity was specific for the organizing subunit 
(p47phox or NOXO1) rather than the Nox catalytic subunit. These studies stress 
the importance of p22phox for the function of Nox1, Nox2, Nox3, and Nox4, and 
emphasize the key role of the PRR for regulating Nox proteins whose activity is 
dependent upon p47phox or NOXO1.