An Adaptor Role for Cytoplasmic Sam68 in Modulating Src Activity during Cell Polarization

Abstract

The Src-associated substrate during mitosis with a molecular mass of 68 kDa (Sam68) is predominantly nuclear and is known to associate with proteins containing the Src homology 3 (SH3) and SH2 domains. Although Sam68 is a Src substrate, little is known about the signaling pathway that link them. Src is known to be activated transiently after cell spreading, where it modulates the activity of small Rho GTPases. Herein we report that Sam68-deficient cells exhibit loss of cell polarity and cell migration. Interestingly, Sam68-deficient cells exhibited sustained Src activity after cell attachment, resulting in the constitutive tyrosine phosphorylation and activation of p190RhoGAP and its association with p120rasGAP. Consistently, we observed that Sam68-deficient cells exhibited deregulated RhoA and Rac1 activity. By using total internal reflection fluorescence microscopy, we observed Sam68 near the plasma membrane after cell attachment coinciding with phosphorylation of its C-terminal tyrosines and association with Csk. These findings show that Sam68 localizes near the plasma membrane during cell attachment and serves as an adaptor protein to modulate Src activity for proper signaling to small Rho GTPases.

Regulatory Mechanisms that Mediate Tenascin C-Dependent Inhibition of Oligodendrocyte Precursor Differentiation

“…7A). This cytosolic localization of Sam68 has previously been reported in other cells (Grange et al, 2004;Huot et al, 2009). The expression of Sam68 within all stages of oligodendrocyte development was confirmed by immunoblotting, which also Figure 6.…”

“…Moreover, Fyn activity regulates expression and alternative splicing of MBP, involving the RNA-binding molecule quaking I (Lu et al, 2005). The quaking/ STAR family (Lukong and Richard, 2003) also includes Sam68 that functions as a signaling transducer for Fyn-mediated migration in fibroblasts (Huot et al, 2009) and as a phosphorylationdependent splicing regulator for Bcl-X and CD44 in different cell lines (Matter et al, 2002;Paronetto et al, 2007). As yet, Sam68 has not been implicated in oligodendrocyte development but is specifically downregulated by Tnc in neural stem cells (Moritz et al, 2008) and hence represents a plausible Tnc target in oligodendrocytes.…”

The RNA-binding protein Sam68 is a multifunctional player in human cancer

“…Cell migration and polarized movements are promoted by Sam68 in an RNA-binding-independent manner (Huot et al 2009a). Sam68 seems to be required for negative feedback inhibition of Src, as the activity of the kinase is constitutively high in Sam68 K/K murine embryonic fibroblasts (Huot et al 2009a). Thus, deregulated Src activity might underlie the defects in actin cytoskeleton and cell migration observed in Sam68-deficient fibroblasts.…”

“…Notably, both Sam68 and BRK are upregulated in breast cancer and support cell proliferation and invasiveness (Barker et al 1997, Ostrander et al 2010, Song et al 2010. Cell migration and polarized movements are promoted by Sam68 in an RNA-binding-independent manner (Huot et al 2009a). Sam68 seems to be required for negative feedback inhibition of Src, as the activity of the kinase is constitutively high in Sam68 K/K murine embryonic fibroblasts (Huot et al 2009a).…”

Emerging roles for Sam68 in adipogenesis and neuronal development

“…Studies have also identified Sam68 as an adaptor protein during cell spreading where it functions to recruit Csk via C-terminal tyrosines to modulate Src kinase activity during cell attachment. 21 Transgenic animal models have helped to uncover the critical functional consequences of dysregulated alternative splicing and have facilitated further understanding of the underlying molecular mechanisms that coordinate distinct cellular differentiation programs involved in tissue development. The generation of Sam68-deficient mice has revealed numerous unexpected physiological roles for this RNA binding protein.…”