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.

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    • "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 "
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