Role of phospholipase Cγ1 in cell spreading requires association with a β-Pix/GIT1-containing complex, leading to activation of Cdc42 and Rac1

The significance of multiprotein signaling complexes in cell motility is becoming increasingly important. We have previously shown that phospholipase C gamma 1 (PLC gamma 1) is critical for integrin-mediated cell spreading and motility (N. Jones et al., J. Cell Sci. 118:2695-2706, 2005). In the current study we show that, on a basement membrane-type matrix, PLC gamma 1 associates with the adaptor protein GIT1 and the Rac1/Cdc42 guanine exchange factor beta-Pix; GIT1 and beta-Pix form tight complexes independently of PLC gamma 1. The association of PLC gamma 1 with the complex requires both GITI and beta-Pix and the specific array region (gamma SA) of PLC gamma 1. Mutations of PLC gamma 1 within the gamma SA region reveal that association with this complex is essential for the phosphorylation of PLC gamma 1 and the progression to an elongated morphology after integrin engagement. Short interfering RNA (siRNA) depletion of either beta-Pix or GIT1 inhibited cell spreading in a fashion similar to that seen with siRNA against PLC gamma l. Furthermore, siRNA depletion of PLC gamma 1, beta-Pix, or GIT1 inhibited Cdc42 and Rac1 activation, while constitutively active forms of Cdc42 or Rac1, but not RboA, were able to rescue the elongation of these cells. Signaling of the PLC gamma 1/GIT1/beta-Pix complex to Cdc42/Rac1 was found to involve the activation of calpains, calcium-dependent proteases. Therefore, we propose that the association of PLC gamma l with complexes containing GIT1 and beta-Pix is essential for its role in integrin-mediated cell spreading and motility. As a component of this complex, PLC gamma 1 is also involved in the activation of Cdc42 and Rac1.