p21-activated Kinase (PAK1) Is Phosphorylated and Activated by 3-Phosphoinositide-dependent Kinase-1 (PDK1)

Departments of Immunology and Cell Biology, Scripps Research Institute, La Jolla, California 92037, USA.
Journal of Biological Chemistry (Impact Factor: 4.57). 12/2000; 275(52):41201-41209. DOI: 10.1074/jbc.M006553200


In this study, we show that phosphorylated 3-phosphoinositide-dependent kinase 1 (PDK1) phosphorylates p21-activated kinase
1 (PAK1) in the presence of sphingosine. We identify threonine 423, a conserved threonine in the activation loop of kinase
subdomain VIII, as the PDK1 phosphorylation site on PAK1. Threonine 423 is a previously identified PAK1 autophosphorylation
site that lies within a PAK consensus phosphorylation sequence. After pretreatment with phosphatases, autophosphorylation
of PAK1 occurred at all major sites except threonine 423. A phosphothreonine 423-specific antibody detected phosphorylation
of recombinant, catalytically inactive PAK1 after incubation with wild-type PAK1, indicating phosphorylation of threonine
423 occurs by an intermolecular mechanism. The biological significance of PDK1 phosphorylation of PAK1 at threonine 423 in vitro is supported by the observation that these two proteins interact in vivo and that PDK1-phosphorylated PAK1 has an increased activity toward substrate. An increase of phosphorylation of catalytically
inactive PAK1 was observed in COS-7 cells expressing wild-type, but not catalytically inactive, PDK1 upon elevation of intracellular
sphingosine levels. PDK1 phosphorylation of PAK1 was not blocked by pretreatment with wortmannin or when PDK1 was mutated
to prevent phosphatidylinositol binding, indicating this process is independent of phosphatidylinositol 3-kinase activity.
The data presented here provide evidence for a novel mechanism for PAK1 regulation and activation.

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    • "PAK1 has been most extensively characterized among the group I PAKs and the signaling pathways of PAK1 are shown in Figure 2. The activity of PAK1 is regulated by a wide spectrum of upstream signaling molecules, including the small Rho GTPases Cdc42 and Rac, phosphoinositide-dependent kinase 1 (PDK1) [28], protein kinase A (PKA) [29], Phosphoinositide 3-Kinase (PI3K) [30] and AKT via phosphorylation and protein-protein interaction [31]. Activation of PAK1 by releasing it from the AID and the maintenance of kinase activity are resulted from the phosphorylations of Ser144, Ser199 and Ser204 and Thr423 [32,33]. "
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    ABSTRACT: The p21-activated kinases (PAKs) are downstream effectors of the Rho family small GTPases as well as a wide variety of mitogenic factors and have been implicated in cancer formation, development and metastasis. PAKs phosphorylate a wide spectrum of substrates to mediate extracellular signals and regulate cytoskeletal remodeling, cell motility and survival. In this review, we aim to summarize the findings regarding the oncogenic role and the underlying mechanisms of PAKs signaling in various cancers, and in particular highlight the prime importance of PAKs in hepatocellular carcinoma (HCC) progression and metastasis. Recent studies exploring the potential therapeutic application of PAK inhibitors will also be discussed.
    Full-text · Article · Aug 2014 · Journal of Molecular Signaling
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    • "Currently, most evidence indicates that Pak1 autophosphorylation occurs through an inter-molecular mechanism consisting of auto-phosphorylation between two Pak1 monomers (King et al., 2000; Parrini et al., 2002). Therefore, Pak1 must have intrinsic capacity to maintain a dynamic dimer structure FIGURE 1 | (A) p21 activated kinase-1 is divided into N-termial and C-terminal halves. "
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    ABSTRACT: P21 activated kinase-1 (Pak1) has diverse functions in mammalian cells. Although a large number of phosphoproteins have been designated as Pak1 substrates from in vitro studies, emerging evidence has indicated that Pak1 may function as a signaling molecule through a unique molecular mechanism - scaffolding. By scaffolding, Pak1 delivers signals through an auto-phosphorylation-induced conformational change without transfer of a phosphate group to its immediate downstream effector(s). Here we review evidence for this regulatory mechanism based on structural and functional studies of Pak1 in different cell types and research models as well as in vitro biochemical assays. We also discuss the implications of Pak1 scaffolding in disease-related signaling processes and the potential in cardiovascular drug development.
    Full-text · Article · Sep 2013 · Frontiers in Pharmacology
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    • "It is a downstream effector of the small Rho GTPase, including Rac1 and Cdc42, which regulates diverse cellular processes, including cell cycle progression, cell motility, cell polarity and apoptosis [5]. Activated Rho GTPase binds to PAKs on the Cdc42/Rac interactive binding (CRIB) domain, causing the relief of autoinhibitory domain (AID), subsequent autophosphorylation of the catalytic domain and kinase activation [6]. Among the multiple autophosphorylation sites, threonine-423 (T423) is particularly important for counteracting autoinhibition and maintaining the complete activated state [7]. "
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    ABSTRACT: Hepatocellular carcinoma (HCC) is one of the major malignancies worldwide and is associated with poor prognosis due to the high incidences of metastasis and tumor recurrence. Our previous study showed that overexpression of p21-activated protein kinase 1 (PAK1) is frequently observed in HCC and is associated with a more aggressive tumor behavior, suggesting that PAK1 is a potential therapeutic target in HCC. In the current study, an allosteric small molecule PAK1 inhibitor, IPA-3, was evaluated for the potential in suppressing hepatocarcinogenesis. Consistent with other reports, inhibition of PAK1 activity was observed in several human HCC cell lines treated with various dosages of IPA-3. Using cell proliferation, colony formation and BrdU incorporation assays, we demonstrated that IPA-3 treatment significantly inhibited the growth of HCC cells. The mechanisms through which IPA-3 treatment suppresses HCC cell growth are enhancement of apoptosis and blockage of activation of NF-κB. Furthermore, our data suggested that IPA-3 not only inhibits the HCC cell growth, but also suppresses the metastatic potential of HCC cells. Nude mouse xenograft assay demonstrated that IPA-3 treatment significantly reduced the tumor growth rate and decreased tumor volume, indicating that IPA-3 can suppress the in vivo tumor growth of HCC cells. Taken together, our demonstration of the potential preclinical efficacy of IPA-3 in HCC provides the rationale for cancer therapy.
    Full-text · Article · Jul 2013 · PLoS ONE
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