Identification of Focal Adhesion Kinase (FAK) and Phosphatidylinositol 3-kinase (PI3-kinase) as Par3 Partners by Proteomic Analysis Norimichi Itoh, 1 Masanori Nakayama, 1 Takashi Nishimura, 2 Shin Fujisue, 1 Tomoki Nishioka, 1 Takashi Watanabe, 1,3 and Kozo Kaibuchi 1,3,4 * 1 Department of Cell Pharmacology, Graduate School of Medicine, Nagoya University, 65 Tsurumai, Showa-ku, Nagoya, Japan 2 Laboratory for Cell Asymmetry, RIKEN Center for Developmental Biology, 2-2-3 Minatojima-Minamimachi, Chuo-ku, Kobe, Japan 3 Institute for Advanced Research, Nagoya University, Furo, Chikusa-ku, Nagoya, Japan 4 CREST Japan Science and Technology Agency, 4-1-8, Honcho, Kawaguchi, Japan Received 24 December 2009; Revised 8 February 2010; Accepted 11 February 2010 Monitoring Editor: Makoto Kinoshita Partition defective 3 (Par3) is involved in a variety of po- larity events including establishment of apico-basal polar- ity of epithelial cell, axon/dendrite specification of neurons and directional migration of cells with front-rear polarity. Par3 is thought to regulate cell polarity as a scaffold protein by interacting with various partner pro- teins such as Par6, aPKC, Tiam1/2 and Numb. However, the mode of actions of Par3 in polarized migration remains largely unknown. To explore Par3 functions, we screened Par3-interacting proteins by combining Par3 af- finity column chromatography and shotgun analysis using liquid chromatography-tandem mass spectrometry (LC-MS/MS). We obtained about two hundred Par3- interacting proteins from the rat brain cytosol fraction. Among them, we focused on FAK and PI3-kinase, as both of them participate in directional cell migration. FAK associated with the PDZ domain and the coiled-coil region of Par3 and p110 of PI3-kinase associated with the coiled-coil region of Par3. Par3 was partially colocal- ized with FAK in spreading cells. Depletion of Par3 by RNA interference inhibited adhesion-induced activation of FAK and PI3-kinase, and RNA interference-resistant Par3 restored the inhibitory effects. In addition, Par3 was required for the adhesion-induced cell spreading as well as for directional cell migration toward collagen. These results suggest that Par3 directly interacts with FAK and PI3-kinase, enhancing their activities for polarized cell migration. V C 2010 Wiley-Liss, Inc. Key Words: proteomics, shotgun analysis, LC-MS/MS, cell-ECM adhesion, polarized cell migration Introduction D irectional cell migration is required for a variety of cellular processes including tissue development, chemo- taxis and wound healing [Ridley et al., 2003]. To achieve directional migration, cells have to establish and maintain polarity towards a direction, so called ‘‘front-rear polarity’’ and are characterized with a front leading edge and rear tail [Ridley et al., 2003]. Accumulating evidence suggests that several key proteins such as Rho family small GTPases, phos- phatidylinositol 3-kinase (PI3-kinase) and the Par (partition defective) protein complex play a pivotal role in the polar- ized cell migration [Dow and Humbert, 2007; Goldstein and Macara, 2007; Cain and Ridley, 2009]. In the front area of migrating cells, Rac1, a member of the Rho family small GTPases, and PI3-kinase are highly active [Haugh et al., 2000; Kraynov et al., 2000; Itoh et al., 2002; Merlot and Firtel, 2003]. PI3-kinase activity is required for Rac1 activa- tion, and activated Rac1 generates a vectorial protrusion for the polarized migration [Welch et al., 2003; Pankov et al., 2005]. The protrusion is stabilized by adhering to the extrac- ellular matrix including fibronectin and collagen through adhesion molecules such as integrin. Integrin engagement, in turn, activates Rac1 through PI3-kinase [Marcoux and Vuori, 2003]. This positive circuit between PI3-kinase/Rac1 and integrin is critical for the front-rear polarity of the migrating cells [Ridley et al., 2003]. Activated integrin, composed of a and b subunits, transmits the signal to the downstream sig- naling proteins such as focal adhesion kinase (FAK) and PI3- kinase through the direct interaction of integrin b to talin. Talin recruits FAK and PI3-kinase to the site of activated integrin [Mitra et al., 2005]. High activity of RhoA, another member of the Rho family small GTPases, is observed in the Additional Supporting Information may be found in the online version of this article. *Address correspondence to: Kozo Kaibuchi, Department of Cell Pharmacology, Graduate School of Medicine, Nagoya University, 65 Tsurumai, Showa-ku, Nagoya 466-8550, Japan. E-mail: kaibuchi@med.nagoya-u.ac.jp Published online 16 March 2010 in Wiley InterScience (www.interscience. wiley.com). RESEARCH ARTICLE Cytoskeleton, May 2010 67:297–308 (doi: 10.1002/cm.20444) V C 2010 Wiley-Liss, Inc. 297 n