c-Yes Tyrosine Kinase Is a Potent Suppressor of ES Cell Differentiation and Antagonizes the Actions of Its Closest Phylogenetic Relative, c-Src

Abstract

ES cells are derived from the inner cell mass of the blastocyst stage embryo and are characterized by self-renewal and pluripotency. Previous work has shown that Src-family tyrosine kinases display dynamic expression and activity changes during ES cell differentiation, suggesting distinct functions in the control of developmental fate. Here we used ES cells to test the hypothesis that c-Src and its closest phylogenetic relative, c-Yes, act in biological opposition despite their strong homology. Unlike c-Src, enforced expression of active c-Yes blocked ES cell differentiation to embryoid bodies by maintaining pluripotency gene expression. To explore the interplay of c-Src and c-Yes in ES cell differentiation, we engineered c-Src and c-Yes mutants that are resistant to A-419259, a potent pyrrolopyrimidine inhibitor of the Src kinase family. Previous studies have shown that A-419259 treatment blocks all Src-family kinase activity in ES cells, preventing differentiation while maintaining pluripotency. Expression of inhibitor-resistant c-Src but not c-Yes rescued the A-419259 differentiation block, resulting in a cell population with properties of both primitive ectoderm and endoderm. Remarkably, when inhibitor-resistant c-Src and c-Yes were expressed together in ES cells, c-Yes activity suppressed c-Src mediated differentiation. These studies show that even closely related kinases such as c-Src and c-Yes have unique and opposing functions in the same cell type. Selective agonists or inhibitors of c-Src vs. c-Yes activity may allow more precise pharmacological manipulation of ES cell fate and have broader applications in other biological systems which express multiple Src family members such as tumor cells.

Src-family tyrosine kinase activities are essential for differentiation of human embryonic stem cells

“…While the c-Yes protein is expressed in both pluripotent hES cells and differentiated EBs derived from them, c-Yes kinase activity is downregulated as hES cells differentiate to EBs (Anneren et al, 2004; Zhang et al, 2014). This observation provided the first evidence that c-Yes, and possibly other members of the Src kinase family, may regulate hES cell fate.…”

“…These results suggest that Lck and c-Yes activity may influence self-renewal, while c-Src and Fyn signaling may be related to differentiation. Previous results have shown c-Yes is expressed and active in both mouse and human ES cells under self-renewal conditions (Anneren et al, 2004), and that enforced expression of c-Yes interferes with EB formation from mES cells (Zhang et al, 2014). On the other hand, both c-Src and Fyn are present and active in self-renewing mES cells as well as EBs formed from them.…”

“…RNA isolation, RT-PCR, quantitative real-time RT-PCR, and data analysis were performed as described in detail elsewhere (Zhang et al, 2014). …”

“…SFK proteins were immunoprecipitated from equivalent amounts of clarified ES cell lysates with isoform-specific antibodies and protein G-Sepharose as described elsewhere (Zhang et al, 2014; Meyn, III and Smithgall, 2009; Meyn, III et al, 2005). Immunoprecipitated proteins as well as cell lysates were separated by SDS-PAGE, transferred to PVDF membranes and probed with antibodies to the SFK activation loop, each SFK protein, Oct4, and actin as a loading control.…”

Serum Inter-α-inhibitor Activates the Yes Tyrosine Kinase and YAP/TEAD Transcriptional Complex in Mouse Embryonic Stem Cells

“…Yes, YAP, and TEAD2 are highly expressed in self-renewing mES cells and are downregulated when cells are induced to differentiate. In addition, the Yes kinase has been shown to suppress differentiation and block embryoid body maturation when overexpressed in mES cells (15). Moreover, TEAD2 can directly associate with the Oct3/4 promoter and activation of the Yes pathway induces, whereas suppression inhibits, Oct3/4 and Nanog promoter activities.…”