© 2000 Macmillan Magazines Ltd articles NATURE CELL BIOLOGY VOL 2 SEPTEMBER 2000 http://cellbio.nature.com 574 Pyk2 and FAK regulate neurite out- growth induced by growth factors and integrins Inga Ivankovic-Dikic*, Eva Grönroos*, Andree Blaukat*, Bernd-Uwe Barth* and Ivan Dikic* *Ludwig Institute for Cancer Research, Box 595, Husargatan 3, Uppsala, S- 75124 , Sweden e-mail: Ivan.Dikic@licr.uu.se Integration of signalling pathways initiated by receptor tyrosine kinases and integrins is essential for growth-factor- mediated biological responses. Here we show that co-stimulation of growth-factor receptors and integrins activates the focal-adhesion kinase (FAK) family to promote outgrowth of neurites in PC12 and SH-SY5Y cells. Pyk2 and FAK associate with adhesion-based complexes that contain epidermal growth factor (EGF) receptors, through their car- boxy- and amino-terminal domains. Expression of the C-terminal domain of Pyk2 or of FAK is sufficient to block neu- rite outgrowth, but not activation of extracellular-signal-regulated kinase (ERK). Moreover, activation and autophos- phorylation of Pyk2/FAK, as well as of effectors of their adhesion-targeting domains, such as paxillin, are important for propagation of signals that control neurite formation. Thus, Pyk2/FAK have important functions in signal integra- tion proximal to integrin/growth-factor receptor complexes in neurons. R eceptor tyrosine kinases (RTKs) regulate a variety of the cel- lular responses elicited by growth factors, including prolifer- ation, differentiation and migration 1 . Binding of growth fac- tors to RTKs promotes the dimerization and subsequent activation of receptors 1,2 . This in turn promotes autophosphorylation of RTKs and tyrosine phosphorylation of cellular proteins, which are critical steps in post-receptor signal transduction 1 . Recent evi- dence has shown that further extracellular signals are needed for efficient and potent cellular responses to growth-factor stimula- tion. Growth-factor-induced proliferation, cell-cycle progression and angiogenesis have been shown to require adhesion of cells to the extracellular matrix (ECM) 3–6 , a process that is mediated by the integrin family of cell-surface receptors 7 . Integrins are aggregated in transmembrane complexes known as focal contacts, which are highly enriched in cytoskeletal proteins 8 ; clustered integrins also activate several signalling cascades to control transcription and gene expression 7,8 . Moreover, integrins have been shown to physi- cally associate with RTKs and promote growth-factor-independ- ent activation of RTKs 5,9–11 . It has been proposed that proteins that act as converging points in signalling by these receptor systems may have important functions in the control of cellular responses induced by growth factors and cell adhesion. Previous studies have identified the focal-adhesion kinases FAK and Pyk2 as potential common mediators of signalling by growth factors and integrins 8,12,13 . FAK is rapidly tyrosine phosphorylated upon cell adhesion, leading to activation and phosphorylation of cytoskeleton-linked proteins that are responsible for signal propaga- tion downstream of integrins 14 . Pyk2 is readily activated by several soluble factors and has also been shown to participate in signalling by integrins with cell-type-specific differences 13,15–17 . Pyk2 is highly a b EGF IGF-1 – – PC12 SH-SY5Y Plastic Plastic Laminin Laminin Figure 1 Effect of integrin engagement on neurite outgrowth in PC12 or SH- SY5Y cells treated with EGF or IGF-1. a, PC12 cells or b, SH-SY5Y cells were plated on plastic or laminin-coated dishes and incubated in serum-free medium (–) or serum-free medium supplemented with 100 ng ml –1 EGF or 50 ng ml –1 IGF-1 respectively. The cells were photographed after 3 days. Magnification 200×.