Abstract
The catalytic activity of the c-Abl tyrosine kinase is tightly regulated by its Src homology 3 (SH3) domain through a complex mechanism that may involve intramolecular binding to Pro242 in the linker region between the SH2 and catalytic domains as well as interactions with a trans-inhibitor. We analysed the effect of mutation or replacement of SH3 on c-Abl tyrosine kinase activity and transformation. Random mutagenesis of SH3 identified several novel point mutations that dysregulated c-Abl kinase activity in vivo, but the RT loop was insensitive to mutational activation. Activating SH3 mutations abolished binding of proline-rich SH3 ligands in vitro, while mutations at Ser140 in the connector between the SH3 and SH2 domains activated Abl kinase activity in vivo and in vitro but did not impair SH3 ligand-binding. Abl was regulated efficiently when its SH3 domain was replaced with a heterologous SH3 from c-Src that binds a different spectrum of proline-rich ligands, but not by substitution of a modular WW domain with similar ligand-binding specificity. These results suggest that the SH3 domain regulates Abl principally by binding to the atypical intramolecular ligand Pro242 rather than a canonical PxxP ligand. Coordination between the SH3 and SH2 domains mediated by the connector region may be required for regulation of Abl even in the absence of SH2 ligand binding.
Highlights
Introduction cAbl is a large, non-receptor tyrosine kinase of metazoans whose precise functions are unknown, but roles for c-Abl in growth factor and integrin signaling, cell cycle regulation, neurogenesis, and responses to DNA damage and oxidative stress haveother evidence suggests that c-Abl kinase activity is suppressed in vivo through binding of a cellular inhibitor (Pendergast et al, 1991)
We demonstrate here that the Src homology 3 (SH3) domain in c-Abl maintains a majority of its negative regulatory function in the presence of mutations that signi®cantly modify its PxxP ligand speci®city. c-Abl kinase activity is dysregulated when its SH3 is replaced by a modular WW domain that binds a similar spectrum of proteins, yet is fairly eciently regulated upon substitution with the Src SH3 domain, which has quite dierent PxxP ligand-binding speci®city
An intermolecular salt bridge between Glu117 in the Abl SH3 domain and Lys313 in the catalytic domain that has been implicated in suppression of c-Abl kinase activity (Barila and SupertiFurga, 1998) is preserved in the Abl+Src SH3 chimera, because the Src SH3 contains an aspartate at position 117 (Figure 1a)
Summary
Other evidence suggests that c-Abl kinase activity is suppressed in vivo through binding of a cellular inhibitor (Pendergast et al, 1991). C-Abl is constitutively active when expressed in S. pombe (Walkenhorst et al, 1996), but not in S. cerevisiae (Brasher and Van Etten, unpublished observations) or Xenopus oocytes (Dorey et al, 1999). These observations suggest that c-Abl may require an inhibitor to completely suppress its activity in vivo. Pag/ Msp has been shown to inhibit c-Abl kinase activity upon co-expression in vivo (Wen and Van Etten, 1997)
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