Crystal Structures of the p21-Activated Kinases PAK4, PAK5, and PAK6 Reveal Catalytic Domain Plasticity of Active Group II PAKs

Eswaran, Jeyanthy; Lee, Wen‐Hwa; Debreczeni, J.; Filippakopoulos, P.; Turnbull, Andrew V.; Fedorov, O.; Deacon, Sean; Peterson, Jeffrey R.; Knapp, Stefan

doi:10.1016/j.str.2007.01.001

Cited by 107 publications

(110 citation statements)

References 61 publications

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“…Briefly, cells were lysed, and RNA was isolated according to the protocol of the manufacturer using the RNAeasy Plus mini kit (Qiagen, Valencia, CA). Next, 25 l of cDNA was produced by RT 2 First Strand kit (SABioscience, Frederick, MD), mixed with quantitative PCR SYBR Green master mix and loaded into the Human Cancer Pathway RT 2 Profiler PCR Array (catalog no. PAHS-033, SABiosciences), which detects 85 different cancer-related gene expression changes per array, and the data were normalized to eight different housekeeping genes.…”

Section: Methodsmentioning

confidence: 99%

P21 Activated Kinase-1 (Pak1) Promotes Prostate Tumor Growth and Microinvasion via Inhibition of Transforming Growth Factor β Expression and Enhanced Matrix Metalloproteinase 9 Secretion

Goc

Al-Azayzih

Abdalla

et al. 2013

Journal of Biological Chemistry

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Background:The significance of Pak1 in prostate cancer remains unclear. Results: Pak1 knockdown impaired prostate tumor growth via increased expression of TGF␤ and reduced secretion of MMP9. Conclusions:We demonstrated that Pak1 is a more potent mediator of prostate cancer cell migration and tumor growth than Pak6, the predominant isoform in the prostate. Significance: A novel role of Pak1 in prostate cancer is identified.

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Section: Methodsmentioning

confidence: 99%

P21 Activated Kinase-1 (Pak1) Promotes Prostate Tumor Growth and Microinvasion via Inhibition of Transforming Growth Factor β Expression and Enhanced Matrix Metalloproteinase 9 Secretion

Goc

Al-Azayzih

Abdalla

et al. 2013

Journal of Biological Chemistry

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“…Structural studies of the kinase domains of type II PAKs have suggested that the conformational plasticity of the glycine-rich loop acts as a molecular sensor for ATP binding, governing structural rearrangements important for maintenance of a competent active state (31). How these conformational rearrangements are controlled at the molecular level, however, remains to be elucidated.…”

mentioning

confidence: 99%

Type II p21-activated kinases (PAKs) are regulated by an autoinhibitory pseudosubstrate

Ha¹,

Davis²,

Chen³

et al. 2012

Proc. Natl. Acad. Sci. U.S.A.

101

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The type II p21-activated kinases (PAKs) are key effectors of RHOfamily GTPases involved in cell motility, survival, and proliferation. Using a structure-guided approach, we discovered that type II PAKs are regulated by an N-terminal autoinhibitory pseudosubstrate motif centered on a critical proline residue, and that this regulation occurs independently of activation loop phosphorylation. We determined six X-ray crystal structures of either fulllength PAK4 or its catalytic domain, that demonstrate the molecular basis for pseudosubstrate binding to the active state with phosphorylated activation loop. We show that full-length PAK4 is constitutively autoinhibited, but mutation of the pseudosubstrate releases this inhibition and causes increased phosphorylation of the apoptotic regulation protein Bcl-2/Bcl-X L antagonist causing cell death and cellular morphological changes. We also find that PAK6 is regulated by the pseudosubstrate region, indicating a common type II PAK autoregulatory mechanism. Finally, we find Src SH3, but not β-PIX SH3, can activate PAK4. We provide a unique understanding for type II PAK regulation.autoregulation | protein kinase | RHO GTPase effector | signaling T he RHO-family small GTPases RAC1 and CDC42 control many cellular functions, including cytoskeletal organization, morphological changes, cell motility, and cell-cycle progression (1). These enzymes are regulated by cycling between GDPbound and GTP-bound states, whereby the GTP-bound state binds to and activates multiple effector molecules, triggering distinct downstream events. Pathological mutations in these proteins can alter cellular outcomes, and recurrent activating mutations suffer high mutational burdens in cancer (2). There is consequently significant interest in understanding the intrinsic details of these molecules and their complex signaling pathways.An important group of proteins that directly interact with RAC1 and CDC42 are the p21-activated kinases (PAKs) (3). These Ste20 family serine-threonine kinases are regulators of the actin cytoskeleton, cell survival, cell adhesion, cytokine signaling, and transcription (3, 4). There are two subgroups of PAK kinase, denoted type I (PAK1, PAK2, and PAK3) and type II (PAK4, PAK5, and PAK6). PAK4 is the best-studied type II PAK family member, is widely expressed (5), and is essential for viability in mice (6). Downstream substrates of PAK4 include the RHO GTPase guanine nucleotide exchange factor H1 (7), the cytoskeletal regulator LIM domain kinase 1 (LIMK1) (8), the adhesion receptor integrin β5 (9), the focal adhesion scaffolding protein paxillin (10), and the apoptotic regulation protein Bcl-2/ Bcl-X L antagonist causing cell death (BAD) (11). This array of substrates are thought to mediate effects of PAK4 activation in cells, including loss of focal adhesions (12), cell rounding (12, 13), cytoskeleton changes (13), and protection from apoptosis (11). Together, these observations indicate that PAK4 plays roles both as a regulator of the actin cytoskeleton and as a promoter of ...

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“…Thus, inhibitors of Pak1 have been suggested as a novel oncologic therapy Nheu et al, 2002). Although no highly selective inhibitors of Pak1 have been reported, several compounds originally identified for their ability to target other kinases also inhibit Pak family members (Eswaran et al, 2007;Nheu et al, 2002;Porchia et al, 2007). Here we report the identification and characterization of a highly selective, non-ATP-competitive inhibitor that targets the autoregulatory mechanism of group I Paks.…”

Section: Introductionmentioning

confidence: 94%

“…The following proteins were purified as described: Cdc42-GTPgS (Peterson et al, 2001), Pak2 (Rennefahrt et al, 2007), Pak4 and Pak5 catalytic domains (Eswaran et al, 2007), and full-length Pak1 (Rennefahrt et al, 2007). Full-length Pak3 (Bagrodia et al, 1995) and Pak5 (Cotteret et al, 2003) were expressed in HEK293 cells and immunopurified (see below).…”

Section: Experimental Procedures Protein Purification and Characterizmentioning

confidence: 99%

Analysis of p21-Activated Kinase Function in Neurofibromatosis Type 2

Chernoff¹

2009

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Public reporting burden for this collection of information is estimated to average 1 hour per response, including the time for reviewing instructions, searching existing data sources, gathering and maintaining the data needed, and completing and reviewing this collection of information. Send comments regarding this burden estimate or any other aspect of this collection of information, including suggestions for reducing this burden to Department of Defense, Washington Headquarters Services, Directorate for Information Operations and Reports (0704-0188), 1215 Jefferson Davis Highway, Suite 1204, Arlington, VA 22202-4302. Respondents should be aware that notwithstanding any other provision of law, no person shall be subject to any penalty for failing to comply with a collection of information if it does not display a currently valid OMB control number. PLEASE DO NOT RETURN YOUR FORM TO THE ABOVE ADDRESS. REPORT DATE SPONSOR/MONITOR'S REPORT NUMBER(S) DISTRIBUTION / AVAILABILITY STATEMENTApproved for Public Release; Distribution Unlimited SUPPLEMENTARY NOTES ABSTRACTThe NF2 product, Merlin, has recently been shown to inhibit p21-activated kinases (Paks), enzymes known to activate cell cycle progression and to induce changes in the actin cytoskeleton. These findings suggest that loss of Pak function might inhibit the abnormal growth and/or movement of cells lacking Merlin. We had proposed two aims: to test if loss of all Pak function affects signaling in NF2-/-cells and to test if Paks are required for tumorigenesis in an NF2 mouse model system. In the third year of this project, we published a manuscript describing the first selective small molecule inhibitor of group A Paks; completed crossing NF2 and Pak1-/-mice into a C3H genetic background; used a retroviral vector to express an enhanced Pak inhibitor (the Pak2 inhibitor domain) in normal and NF2 BBA (dominant negative) mutant mouse fibroblasts, and showed that loss of Pak function impedes NF2-driven cell proliferation and abnormal morphology; and used the same approach in xenograft experiments to show that loss of Pak function reduces NF2-induced tumor formation. With these advances, we have matched and in some cases exceeded our timetable for year three. Chernoff, Jonathan 4 INTRODUCTION:The goal of this project is to determine if group A p21-activated kinases (Paks) are important elements in signaling in neurofibromatosis type II (NF2). Our hypothesis is that inactivation of the NF2 gene disrupts a signaling pathway emanating from the small GTPase Rac and its effector, p21-activated kinase (Pak). We propose that stimulation of the Rac/Pak signaling axis in cells lacking Merlin leads to changes in transcriptional activity and cytoskeletal dynamics, ultimately resulting in enhanced cell proliferation and motility, which are hallmarks of tumorigenesis. If this hypothesis is correct, then inhibition of Pak signaling should disable the growth advantages of cells lacking Merlin. We intend to test this theory using Pak loss-of-function cells and animals. BODY:We s...

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Crystal Structures of the p21-Activated Kinases PAK4, PAK5, and PAK6 Reveal Catalytic Domain Plasticity of Active Group II PAKs

Cited by 107 publications

References 61 publications

P21 Activated Kinase-1 (Pak1) Promotes Prostate Tumor Growth and Microinvasion via Inhibition of Transforming Growth Factor β Expression and Enhanced Matrix Metalloproteinase 9 Secretion

P21 Activated Kinase-1 (Pak1) Promotes Prostate Tumor Growth and Microinvasion via Inhibition of Transforming Growth Factor β Expression and Enhanced Matrix Metalloproteinase 9 Secretion

Type II p21-activated kinases (PAKs) are regulated by an autoinhibitory pseudosubstrate

Analysis of p21-Activated Kinase Function in Neurofibromatosis Type 2

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