Small molecule inhibitors of Lck: the search for specificity within a kinase family.

The Src family of non-transmembrane protein kinases is comprised of eleven homologous members in mammals. Together, these kinases regulate a wide variety of cellular processes including cell survival, proliferation, differentiation, and motility. One member of this family, Lck, plays a pivotal role in T-cell signaling. Inhibition of Lck with small molecules has significant potential for therapeutic immunosuppression and treatment of diseases such as rheumatoid arthritis and asthma. Critical for successful clinical use of any Lck inhibitor is high specificity for Lck as inhibition of other members of the Src kinase family may result in unwanted side effects. In this review we provide an overview of the various synthetic compounds currently under investigation as Lck-specific inhibitors. In addition we provide an analysis of the properties of these compounds that account for the specificity required for the inhibition of one of eleven highly similar kinases.

c-Yes tyrosine kinase is a potent suppressor of ES cell differentiation and antagonizes the actions of its closest phylogenetic relative, c-Src.

Embryonic stem (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 versus c-Yes activity may allow more precise pharmacological manipulation of ES cell fate and have broader applications in other biological systems that express multiple Src family members such as tumor cells.

Chemical genetics identifies c-Src as an activator of primitive ectoderm formation in murine embryonic stem cells.

Multiple Src family kinases (SFKs) are present in murine embryonic stem (mES) cells. Whereas complete inhibition of SFK activity blocks mES cell differentiation, sole inhibition of the SFK member c-Yes induces differentiation. Thus, individual SFKs may have opposing roles in the regulation of mES cell fate. To test this possibility, we generated SFK mutants with engineered resistance to a nonselective SFK inhibitor. The presence of an inhibitor-resistant c-Src mutant, but not analogous mutants of Hck, Lck, c-Yes, or Fyn, reversed the differentiation block associated with inhibitor treatment, resulting in the formation of cells with properties of primitive ectoderm. These results show that distinct SFK signaling pathways regulate mES cell fate and demonstrate that the formation of primitive ectoderm is regulated by the activity of c-Src.

Src family kinases phosphorylate the Bcr-Abl SH3-SH2 region and modulate Bcr-Abl transforming activity.

Bcr-Abl is the oncogenic protein-tyrosine kinase responsible for chronic myelogenous leukemia. Recently, we observed that inhibition of myeloid Src family kinase activity (e.g. Hck, Lyn, and Fyn) induces growth arrest and apoptosis in Bcr-Abl-transformed cells, suggesting that cell transformation by Bcr-Abl involves Src family kinases (Wilson, M. B., Schreiner, S. J., Choi, H. J., Kamens, J., and Smithgall, T. E. (2002) Oncogene 21, 8075-8088). Here, we report the unexpected observation that Hck, Lyn, and Fyn strongly phosphorylate the SH3-SH2 region of Bcr-Abl. Seven phosphorylation sites were identified by matrix-assisted laser desorption ionization time-of-flight mass spectrometry: Tyr89 and Tyr134 in the Abl-derived SH3 domain; Tyr147 in the SH3-SH2 connector; and Tyr158, Tyr191, Tyr204, and Tyr234 in the SH2 domain. SH3 domain Tyr89, the most prominent phosphorylation site in vitro, was strongly phosphorylated in chronic myelogenous leukemia cells in a Src family kinase-dependent manner. Substitution of the SH3-SH2 tyrosine phosphorylation sites with phenylalanine substantially reduced Bcr-Abl-mediated transformation of TF-1 myeloid cells to cytokine independence. The positions of these tyrosines in the crystal structure of the c-Abl core and the transformation defect of the corresponding Bcr-Abl mutants together suggest that phosphorylation of the SH3-SH2 region by Src family kinases impacts Bcr-Abl protein conformation and signaling.

SRC family kinase activity is required for murine embryonic stem cell growth and differentiation.

Self-renewal and differentiation of embryonic stem (ES) cells are regulated by cytokines and growth factors through tyrosine kinase-dependent signaling pathways. In murine ES cells, signals for self-renewal are generated by the leukemia inhibitory factor (LIF). LIF and other growth factors are linked to the activation of the Src family of cytoplasmic protein-tyrosine kinases (SFKs), which consists of eight members having shared structural architecture. In this article, we show that murine ES cells express seven SFKs, three of which (Hck, Src, and Fyn) exhibit constitutive activity in self-renewing ES cells. Differentiation of ES cells to embryoid bodies was associated with rapid transcriptional silencing of Hck and Lck and with the loss of the corresponding kinase proteins. The expression of other family members remained relatively constant, although some loss of Fgr and Lyn proteins was observed during differentiation. Like ES cells, embryoid bodies maintained constitutive Src and Fyn kinase activity. Partial inhibition of endogenous SFK activity with the ATP-competitive inhibitors 4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine or Src kinase inhibitor-1 induced differentiation of ES cells in the presence of LIF. In contrast, suppression of all SFK activity with higher concentrations of these inhibitors, or with the more potent compound A-419259 (Bioorg Med Chem Lett 12:1683-1686, 2002) blocked differentiation in response to LIF withdrawal. It is surprising that these inhibitor-treated cells remained pluripotent despite the absence of LIF. Our results implicate individual members of the Src kinase family in distinct ES cell renewal and differentiation pathways and show that small-molecule SFK inhibitors can control ES cell fate.

The destruction box of the cyclin Clb2 binds the anaphase-promoting complex/cyclosome subunit Cdc23.

Properly regulated cyclin proteolysis is critical for normal cell cycle progression. A nine-amino acid peptide motif called the destruction box (D box) is present at the N terminus of the yeast mitotic cyclins. This short sequence is required for cyclin ubiquitination and subsequent proteolysis. The anaphase-promoting complex/cyclosome (APC/C) is a multisubunit E3 required for cyclin ubiquitination. We have tested the D box of five mitotic cyclins for interaction with six APC/C subunits. The APC/C subunit Cdc23, but not five other subunits tested, interacted by two-hybrid analysis with the N terminus of wild-type Clb2. None of these subunits interacted with the N termini of the cyclins Clb1, Clb3, or Clb5. Mutations in the D box sequences of Clb2 inhibited interaction with Cdc23 both in vivo and in vitro. Our results provide the first evidence for a direct interaction between an APC/C substrate (Clb2) and an APC/C subunit (Cdc23).