Abstract 1364: Direct phosphorylation and attenuation of the DLC1 tumor suppressor by SRC kinase: a new mechanism of SRC-dependent activation of Rho with translational implications

Many tumors have high SRC activity and constitutive up-regulation of Rho-GTP, which is frequently implicated in the neoplastic process and tumor progression to metastasis, but the mechanistic link is unclear. Here we identify the DLC1 tumor suppressor, which encodes a RhoGAP, as a previously unrecognized direct target of the SRC kinase, in untransformed cells and in tumor-derived cell lines, which upregulates Rho-GTP. SRC kinase directly phosphorylates two Tyrosines in DLC1: Y451 and Y701. SRC phosphorylation of DLC1-Y701, which is located in the DLC1 RhoGAP domain, attenuates its RhoGAP activity and tumor suppressor function by inhibiting the binding of active Rho-GTP to the RhoGAP domain, reducing the hydrolysis of active Rho-GTP to inactive Rho-GDP and resulting in an increase in Rho-GTP. The role of SRC phosphorylation of DLC1-Y451 is to diminish DLC1 binding to tensin, which further reduces the tumor suppressor functions, as measured by cell migration rate, anchorage-independent growth, and tumor formation in nude mice, independently of Rho-GTP. We also found that ERK1 cooperates with SRC by phosphorylating DLC1-S129, which enhances the binding of the SRC SH3 domain to this region of DLC1, and increases SRC-dependent phosphorylation of Y451 and Y701. The cooperative effects reduce RhoGAP activity, increase Rho-GTP and Rho/ROCK/MRLC signaling, and attenuate DLC1 tumor suppressor functions. DLC1 preferentially binds active SRC, which reduces overall SRC activity. Therefore, SRC activity tends to be lower in cells that express high levels of endogenous DLC1, compared with cells in which DLC1 expression has been down-regulated. Although SRC targets two DLC1 functions that contribute to its tumor suppressor activity, DLC1 binds several ligands in addition to tensin that also contribute to its tumor suppressor functions. Therefore, it would not be surprising if high SRC activity were associated with selective pressure for down-regulation of DLC1 expression. Consistent with this hypothesis, the combination high SRC expression and low DLC1 expression was associated with a poor prognosis in lung adenocarcinomas (p=0.005) in TCGA. The potentially reversible nature of the SRC-induced attenuation of DLC1 function can be exploited therapeutically. In two systems that have high SRC activity and express DLC1 - the mouse MMTV-PyMT cancer model and a human lung cancer cell line - the Src inhibitor PP1 has potent antitumor activity, as it reduces DLC1 Tyrosine phosphor