Gary M. Bokoch

NADPH oxidases: not just for leukocytes anymore!

In addition to their role in bacterial killing by leukocytes, reactive oxygen species (ROS) have been increasingly recognized as important components of signaling and host defense in other cell types. The formation of ROS in both phagocytic- and non-phagocytic cells involves membrane-localized NADPH oxidases (Noxs). Nox proteins show structural homology to the cytochrome b(558) of leukocytes but, until recently, their regulation has been poorly understood. Here, we describe our current understanding of Nox function, and discuss emerging paradigms for regulation of Nox activity by Rac GTPase and/or other cytosolic components.

Regulation of microtubule destabilizing activity of Op18/stathmin downstream of Rac1.

In the leading edge of migrating cells, a subset of microtubules exhibits net growth in a Rac1- and p21-activated kinase-dependent manner. Here, we explore the possibility of whether phosphorylation and inactivation of the microtubule-destabilizing protein Op18/stathmin could be a mechanism regulating microtubule dynamics downstream of Rac1 and p21-activated kinases. We find that, in vitro, Pak1 phosphorylates Op18/stathmin specifically at serine 16 and inactivates its catastrophe promoting activity in biochemical and time lapse microscopy microtubule assembly assays. Furthermore, phosphorylation of either serine 16 or 63 is sufficient to inhibit Op18/stathmin in vitro. In cells, the microtubule-destabilizing effect of an excess of Op18/stathmin can be partially overcome by expression of constitutively active Rac1(Q61L), which is dependent on Pak activity, suggesting that the microtubule cytoskeleton can be regulated through inactivation of Op18/stathmin downstream of Rac1 and Pak in vivo. However, in vivo, Pak1 activity alone is not sufficient to phosphorylate Op18, indicating that additional pathways downstream of Rac1 are required for Op18 regulation.

Akt Phosphorylation of Serine 21 on Pak1 Modulates Nck Binding and Cell Migration

ABSTRACT The p21-activated protein kinases (Paks) regulate cellular proliferation, differentiation, transformation, and survival through multiple downstream signals. Paks are activated directly by the small GTPases Rac and Cdc42 and several protein kinases including Akt and PDK-1. We found that Akt phosphorylated and modestly activated Pak1 in vitro. The major site phosphorylated by Akt on Pak1 mapped to serine 21, a site originally shown to be weakly autophosphorylated on Pak1 when Cdc42 or Rac activates it. A peptide derived from the region surrounding serine 21 was a substrate for Akt but not Pak1 in vitro, and Akt stimulated serine 21 phosphorylation on the full-length Pak1 much better than Rac did. The adaptor protein Nck binds Pak near serine 21, and its association is regulated by phosphorylation of this site. We found that either treatment of Pak1 in vitro with Akt or coexpression of constitutively active Akt with Pak1 reduced Nck binding to Pak1. In HeLa cells, green fluorescent protein-tagged Pak1 was concentrated at focal adhesions and was released when Akt was cotransfected. A peptide containing the Nck binding site of Pak1 fused to a portion of human immunodeficiency virus Tat to allow it to enter cells was used to test the functional importance of Nck/Pak binding in Akt-stimulated cell migration. This Tat-Nck peptide reduced Akt-stimulated cell migration. Together, these data suggest that Akt modulates the association of Pak with Nck to regulate cell migration.

Antagonistic Cross-talk between Rac and Cdc42 GTPases Regulates Generation of Reactive Oxygen Species

Cross-talk between Rho GTPase family members (Rho, Rac, and Cdc42) plays important roles in modulating and coordinating downstream cellular responses resulting from Rho GTPase signaling. The NADPH oxidase of phagocytes and nonphagocytic cells is a Rac GTPase-regulated system that generates reactive oxygen species (ROS) for the purposes of innate immunity and intracellular signaling. We recently demonstrated that NADPH oxidase activation involves sequential interactions between Rac and the flavocytochrome b558 and p67phox oxidase components to regulate electron transfer from NADPH to molecular oxygen. Here we identify an antagonistic interaction between Rac and the closely related GTPase Cdc42 at the level of flavocytochrome b558 that regulates the formation of ROS. Cdc42 is unable to stimulate ROS formation by NADPH oxidase, but Cdc42, like Rac1 and Rac2, was able to specifically bind to flavocytochrome b558 in vitro. Cdc42 acted as a competitive inhibitor of Rac1- and Rac2-mediated ROS formation in a recombinant cell-free oxidase system. Inhibition was dependent on the Cdc42 insert domain but not the Switch I region. Transient expression of Cdc42Q61L inhibited ROS formation induced by constitutively active Rac1 in an NADPH oxidase-expressing Cos7 cell line. Inhibition of Cdc42 activity by transduction of the Cdc42-binding domain of Wiscott-Aldrich syndrome protein into human neutrophils resulted in an enhanced fMetLeuPhe-induced oxidative response, consistent with inhibitory cross-talk between Rac and Cdc42 in activated neutrophils. We propose here a novel antagonism between Rac and Cdc42 GTPases at the level of the Nox proteins that modulates the generation of ROS used for host defense, cell signaling, and transformation.

Mechanism of p21-activated Kinase 6-mediated Inhibition of Androgen Receptor Signaling

PAK6 was first identified as an androgen receptor (AR)-interacting protein able to inhibit AR-mediated transcriptional responses. PAK6 is a serine/threonine kinase belonging to the p21-activated kinase (PAK) family implicated in actin reorganization and cell motility, gene transcription, apoptosis, and cell transformation. We investigated the biochemical basis for inhibition of AR signaling by PAK6. We compared the kinase activity of PAK6 with two other well characterized members of the PAK family, PAK1 and PAK4. Like PAK4, PAK6 possesses a constitutive basal kinase activity that, unlike PAK1, is not modulated by the binding of active Rac or Cdc42 GTPases. In order to test the involvement of PAK6 kinase activity in suppression of AR-mediated transcription, we generated kinase-dead (K436A) and kinase-active (S531N) mutants of PAK6. We show that PAK6 kinase activity is required for effective PAK6-induced repression of AR signaling. Suppression does not depend upon GTPase binding to PAK6 and is not mimicked by the closely related PAK1 and PAK4 isoforms. Kinase-dependent inhibition by PAK6 extended to the enhanced AR-mediated transcription seen in the presence of coactivating molecules and to the action of AR coinhibitors. Active PAK6 inhibited nuclear translocation of the stimulated AR, suggesting a possible mechanism for inhibition of AR responsiveness. Finally, we observe that autophosphorylated, active PAK6 protein is differently expressed among prostate cancer cell lines. Modulation of PAK6 activity may be responsible for regulation of AR signaling in various forms of prostate cancer.

The molecular basis for adhesion-mediated suppression of reactive oxygen species generation by human neutrophils

Human neutrophil adherence to ECMs induces an initial inhibition of stimulated reactive oxygen species (ROS) formation, followed by an enhanced phase of oxidant production. The initial integrin-mediated suppression of ROS constitutes a mechanism to prevent inappropriate tissue damage as leukocytes migrate to inflammatory sites. The Rac2 guanosine 5-triphosphatase (GTPase) is a critical regulatory component of the phagocyte NADPH oxidase. We show that activation of Rac2 is inhibited in adherent neutrophils, correlating with inhibition of ROS formation. Conversely, NADPH oxidase components p47 and p67 assemble normally, suggesting a specific action of adhesion on the Rac2 molecular switch. Reconstitution with activated Rac2 restored rapid NADPH oxidase activation kinetics to adherent neutrophils, establishing that inhibition was due to defective Rac2 activity. We provide evidence that integrins inhibit Rac2 activation via a membrane-associated guanine nucleotide exchange factor, likely to be Vav1. Activation of Vav1, but not its upstream activator, Syk, is suppressed by cell adhesion. Vav1 activity is inhibited due to dephosphorylation of the regulatory Tyr174 via enhanced tyrosine phosphatase activity in adherent cells. These studies identify an integrin-mediated pathway in which Vav1 is as a strong candidate for the critical regulatory point in suppression of Rac2 activation and ROS generation during inflammatory responses.