Role of Phospholipase Cγ1 in Cell Spreading Requires Association with a β-Pix/GIT1-Containing Complex, Leading to Activation of Cdc42 and Rac1

Abstract

The significance of multiprotein signaling complexes in cell motility is becoming increasingly important. We have previously shown that phospholipase Cγ1 (PLCγ1) 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, PLCγ1 associates with the adaptor protein GIT1 and the Rac1/Cdc42 guanine exchange factor β-Pix; GIT1 and β-Pix form tight complexes independently of PLCγ1. The association of PLCγ1 with the complex requires both GIT1 and β-Pix and the specific array region (γSA) of PLCγ1. Mutations of PLCγ1 within the γSA region reveal that association with this complex is essential for the phosphorylation of PLCγ1 and the progression to an elongated morphology after integrin engagement. Short interfering RNA (siRNA) depletion of either β-Pix or GIT1 inhibited cell spreading in a fashion similar to that seen with siRNA against PLCγ1. Furthermore, siRNA depletion of PLCγ1, β-Pix, or GIT1 inhibited Cdc42 and Rac1 activation, while constitutively active forms of Cdc42 or Rac1, but not RhoA, were able to rescue the elongation of these cells. Signaling of the PLCγ1/GIT1/β-Pix complex to Cdc42/Rac1 was found to involve the activation of calpains, calcium-dependent proteases. Therefore, we propose that the association of PLCγ1 with complexes containing GIT1 and β-Pix is essential for its role in integrin-mediated cell spreading and motility. As a component of this complex, PLCγ1 is also involved in the activation of Cdc42 and Rac1.

We thank Graham Carpenter, Eunjoon Kim, Peter Hordijk, Richard Premont, Chris Marshall, and David Sacks for their kind gift of PLCγ1, GIT1, β-Pix, Cdc42, rac PAK-RBD, and WASP_CRIB constructs.

This work was funded by a grant from Cancer Research UK.