Identification of focal adhesion kinase (FAK) and phosphatidylinositol 3-kinase (PI3-kinase) as Par3 partners by proteomic analysis.
Partition defective 3 (Par3) is involved in a variety of polarity events including establishment of apico-basal polarity 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 proteins 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 affinity 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 colocalized 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.
Itoh
Norimichi
N
Department of Cell Pharmacology, Graduate School of Medicine, Nagoya University, 65 Tsurumai, Showa-ku, Nagoya, Japan.
Nakayama
Masanori
M
Nishimura
Takashi
T
Fujisue
Shin
S
Nishioka
Tomoki
T
Watanabe
Takashi
T
Kaibuchi
Kozo
K
D048868:Adaptor Proteins, Signal Transducing; D000818:Animals; D019556:COS Cells; D018797:Cell Cycle Proteins / Q000378:metabolism*; D002465:Cell Movement; D016764:Cell Polarity / Q000502:physiology; D002522:Chlorocebus aethiops; D002846:Chromatography, Affinity; D002853:Chromatography, Liquid; D051416:Focal Adhesion Protein-Tyrosine Kinases / Q000378:metabolism*; D006367:HeLa Cells; D006801:Humans; D008565:Membrane Proteins / Q000378:metabolism*; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism*; D040901:Proteomics; D053719:Tandem Mass Spectrometry
D048868:Adaptor Proteins, Signal Transducing; D018797:Cell Cycle Proteins; D051416:Focal Adhesion Protein-Tyrosine Kinases; D008565:Membrane Proteins; D019869:Phosphatidylinositol 3-Kinases; C509689:PARD3 protein, human