Rsu1 contributes to cell adhesion and spreading in MCF10A cells via effects on P38 map kinase signaling

Abstract

The ILK, PINCH, Parvin (IPP) complex regulates adhesion and migration via binding of ILK to β1 integrin and α-parvin thus linking focal adhesions to actin cytoskeleton. ILK also binds the adaptor protein PINCH which connects signaling proteins including Rsu1 to the complex. A recent study of Rsu1 and PINCH1 in non-transformed MCF10A human mammary epithelial cells revealed that the siRNA-mediated depletion of either Rsu1 or PINCH1 decreased the number of focal adhesions (FAs) and altered the distribution and localization of FA proteins. This correlated with reduced adhesion, failure to spread or migrate in response to EGF and a loss of actin stress fibers and caveolae. The depletion of Rsu1 caused significant reduction in PINCH1 implying that Rsu1 may function in part by regulating levels of PINCH1. However, Rsu1, but not PINCH1, was required for EGF-induced activation of p38 Map kinase and ATF2 phosphorylation, suggesting a Rsu1 function independent from the IPP complex. Reconstitution of Rsu1-depleted cells with a Rsu1 mutant (N92D) that does not bind to PINCH1 failed to restore FAs or migration but did promote IPP-independent spreading and constitutive as well as EGF-induced p38 activation. In this commentary we discuss p38 activity in adhesion and how Rsu1 expression may be linked to Map kinase kinase (MKK) activation and detachment-induced stress kinase signaling.

Keywords: FA; focal adhesion; FAK; focal adhesion kinase; ILK; Integrin linked kinase; IPP; Intergrin linked kinase-PINCH-Parvin; LRR; leucine rich repeat; MCF10A cells; MKK; Map kinase kinase; NLS; nuclear localization sequence; PINCH1; RALGEF; Ral Guanine nucleotide exchange factor; ROS; reactive oxygen species; Rsu1; Rsu1; Ras suppressor protein 1.; adhesion; migration; p38 Map kinase.

Figure 1.

siRNA-mediated depletion of Rsu1 blocks activation of MKK4 in MCF10A cells. MCF10A cells transfected with a Rsu1 specific or a negative control siRNA were stimulated with EGF (10ng/ml) at 96 hours post transfection and lysates were harvested and examined by western blotting as described previously. Antibodies for the expression of proteins include: phospho-MKK4 Ser257 (Cell Signaling Technology #4514), MKK4 (Santa Cruz Biotechnology #166168), phospho-MKK3 Ser189/MKK6 Ser207 (Cell Signaling Technology #9231) Tubulin (Santa Cruz Biotechnology #8035) was used as loading control.

Figure 2.

The absence of Rsu1-PINCH1 binding alters MKK4 and p38 Map kinase activation in MCF10A cells. (Left) Expression of either endogenous Rsu1 protein or wt-Rsu1 in the absence of endogenous protein allows activation of MKK4 and phosphorylation of p38 Map kinase in MCF10A cells. (Middle) In cells that are detached from substrate, or depleted of Rsu1, integrin engagement and focal adhesion formation are reduced or absent. This results in a block in the activation of MKK4, but MCF10A cell detachment leads to the activation of p38 by MKK6. (Right) The reconstitution of Rsu1-depleted cells with N92D-Rsu1 does not completely restore FA formation but it does promote MKK4 activation.