Dissecting Activation of the PAK1 Kinase at Protrusions in Living Cells

Institut Curie, Centre de Recherche, Paris, France.
Journal of Biological Chemistry (Impact Factor: 4.57). 08/2009; 284(36):24133-43. DOI: 10.1074/jbc.M109.015271
Source: PubMed


The p21-activated kinase (PAK) 1 kinase, an effector of the Cdc42 and Rac1 GTPases, regulates cell protrusions and motility by controlling actin and adhesion dynamics. Its deregulation has been linked to human cancer. We show here that activation of PAK1 is necessary for protrusive activity during cell spreading. To investigate PAK1 activation dynamics at live protrusions, we developed a conformational biosensor, based on fluorescence resonance energy transfer. This novel PAK1 biosensor allowed the spatiotemporal visualization of PAK1 activation during spreading of COS-7 cells and during motility of normal rat kidney cells. By using this imaging approach in COS-7 cells, the following new insights on PAK1 regulation were unveiled. First, PAK1 acquires an intermediate semi-open conformational state upon recruitment to the plasma membrane. This semi-open PAK1 species is selectively autophosphorylated on serines in the N-terminal regulatory region but not on the critical threonine 423 in the catalytic site. Second, this intermediate PAK1 state is hypersensitive to stimulation by Cdc42 and Rac1. Third, interaction with PIX proteins contributes to PAK1 stimulation at membrane protrusions, in a GTPase-independent way. Finally, trans-phosphorylation events occur between PAK1 molecules at the membrane possibly playing a relevant role for its activation. This study leads to a model for the complex and accurate regulation of PAK1 kinase in vivo at cell protrusions.

  • Source
    • "GFP-Akt-YFP Calleja et al., 2003 B-Raf Prin-BRaf Terai and Matsuda, 2006 C-Raf Prin-CRaf Terai and Matsuda, 2005 Death associated protein kinase 1 (DAPK1) DAPK1 sensor Pilji c et al., 2011 CaMKII Camui Takao et al., 2005; Kwok et al., 2008 Erk Miu2 Fujioka et al., 2006 MAPK-activated protein kinase 2 (MK2) GMB Neininger et al., 2001 Myosin light-chain kinase (MLCK) exMLCK Geguchadze et al., 2004 P21-activated kinase 1 (PAK1) Pakabi Parrini et al., 2009 PDK1 PARE Gao et al., 2011 Activity Probes Protein kinase A AKAR Zhang et al., 2001; Komatsu et al., 2011 Abl kinase Abl indicator Ting et al., 2001 Akt AktAR Gao and Zhang, 2008; Komatsu et al. 2011 Aktus Sasaki et al., 2003 BAR Zhang et al., 2007 BKAR Kunkel et al., 2005 AMPK AMPKAR Tsou et al., 2011 Aurora B kinase Aurora B sensor Chu et al., 2011 ATM kinase Atomic Johnson et al., 2007 Protein kinase C CKAR Violin et al., 2003; Komatsu et al., 2011; Wu-Zhang et al., 2012 Cyclin-dependent kinase 1 Cdk1 FRET sensor Gavet and Pines, 2010 Protein kinase D DKAR Fuchs et al., 2009; Eisler et al., 2012 EGFR EGFR indicator Ting et al., 2001 Erk Erkus Sato et al., 2007 EKAR Harvey et al. 2008; Komatsu et al., 2011 REV Xu et al., 2013 Focal adhesion kinase (FAK) FAK sensor Seong et al., 2011 Insulin receptor Phocus Sato and Umezawa, 2004 c-Jun N-terminal kinase (JNK) JNKAR Fosbrink et al., 2010; Komatsu et al., 2011 Microtubule affinity regulating kinase (MARK) MARK sensor Timm et al., 2011 Polo-like kinase 1 Plk sensor Mac urek et al., 2008 RSK Eevee-RSK Komatsu et al., 2011 S6K Eevee-S6K Komatsu et al., 2011 Stress-activated protein kinase kinase kinase SAP3K activity reporter Tomida et al., 2009 Src Src biosensor Ting et al., 2001; Wang et al., 2005; Ouyang et al., 2008 188 Chemistry & Biology 21, February 20, 2014 ª2014 Elsevier Ltd All rights reserved "
    [Show abstract] [Hide abstract]
    ABSTRACT: Fluorescence-based, genetically encodable biosensors are widely used tools for real-time analysis of biological processes. Over the last few decades, the number of available genetically encodable biosensors and the types of processes they can monitor have increased rapidly. Here, we aim to introduce the reader to general principles and practices in biosensor development and highlight ways in which biosensors can be used to illuminate outstanding questions of biological function. Specifically, we focus on sensors developed for monitoring kinase activity and use them to illustrate some common considerations for biosensor design. We describe several uses to which kinase and second-messenger biosensors have been put, and conclude with considerations for the use of biosensors once they are developed. Overall, as fluorescence-based biosensors continue to diversify and improve, we expect them to continue to be widely used as reliable and fruitful tools for gaining deeper insights into cellular and organismal function.
    Preview · Article · Jan 2014 · Chemistry & biology
  • Source
    • "Membrane targeted C-terminus CAAX Pak can result in up to an order of magnitude increase in activity relative to the wild type kinase [83] [101]. In a recent FRET-based analysis, expression of Pak-CAAX resulted in ser192/197 phosphorylation, but lacked phosphorylation at thr402 [102], the status of the Pak studied here. Furthermore, Nef mutations that mediate loss of Pak association also display a decrease in viral growth in primary T cells [12] [82], and a loss of enhanced T cell activity [12]. "
    [Show abstract] [Hide abstract]
    ABSTRACT: The HIV-1 Nef protein brings about increased T cell activity and viral titers through mechanisms that are poorly understood. Nef activity has been described as an enhancer, but not an inducer, of certain signaling pathways that lead to T cell activation and viral production, particularly from resting T cells. The protein has also been found to associate with and promote autophosphorylation of a serine kinase, Pak2, but the Nef-associated kinase level is very low and difficult to study. Here we demonstrate that Nef expression mediates phosphorylation of Mek1 serine298 in T cell lines as well as primary human T cells, thus directly affecting the Erk cascade. This phosphorylation is through a Pak and Rac activity. We also find that Pak2 in Nef expressing cells is phosphorylated on serine192/197, the first biochemical description of the Nef-mediated activation state for this kinase.
    Full-text · Article · Jun 2013 · Current HIV research
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: Cell motility requires the spatial and temporal coordination of forces in the actomyosin cytoskeleton with extracellular adhesion. The biochemical mechanism that coordinates filamentous actin (F-actin) assembly, myosin contractility, adhesion dynamics, and motility to maintain the balance between adhesion and contraction remains unknown. In this paper, we show that p21-activated kinases (Paks), downstream effectors of the small guanosine triphosphatases Rac and Cdc42, biochemically couple leading-edge actin dynamics to focal adhesion (FA) dynamics. Quantitative live cell microscopy assays revealed that the inhibition of Paks abolished F-actin flow in the lamella, displaced myosin IIA from the cell edge, and decreased FA turnover. We show that, by controlling the dynamics of these three systems, Paks regulate the protrusive activity and migration of epithelial cells. Furthermore, we found that expressing Pak1 was sufficient to overcome the inhibitory effects of excess adhesion strength on cell motility. These findings establish Paks as critical molecules coordinating cytoskeletal systems for efficient cell migration.
    Full-text · Article · Jun 2011 · The Journal of Cell Biology
Show more