Journal of Molecular Biology
Volume 383, Issue 2, 7 November 2008, Pages 414-423
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Tyrosine Phosphorylation in the SH3 Domain Disrupts Negative Regulatory Interactions within the c-Abl Kinase Core

https://doi.org/10.1016/j.jmb.2008.08.040Get rights and content

Abstract

Recent studies have shown that trans-phosphorylation of the Abl SH3 domain at Tyr89 by Src-family kinases is required for the full transforming activity of Bcr-Abl. Tyr89 localizes to a binding surface of the SH3 domain that engages the SH2-kinase linker in the crystal structure of the c-Abl core. Displacement of SH3 from the linker is likely to influence efficient downregulation of c-Abl. Hydrogen-deuterium exchange (HX) and mass spectrometry (MS) were used to investigate whether Tyr89 phosphorylation affects the ability of the SH3 domain to interact intramolecularly with the SH2-kinase linker in cis as well as other peptide ligands in trans. HX MS analysis of SH3 binding showed that when various Abl constructs were phosphorylated at Tyr89 by the Src-family kinase Hck, SH3 was unable to engage a high-affinity ligand in trans and that interaction with the linker in cis was reduced dramatically in a construct containing the SH3 and SH2 domains plus the linker. Phosphorylation of the Abl SH3 domain on Tyr89 also interfered with binding to the negative regulatory protein Abi-1 in trans. Site-directed mutagenesis of Tyr89 and Tyr245, another tyrosine phosphorylation site located in the linker that may also influence SH3 binding, implicated Tyr89 as the key residue necessary for disrupting regulation after phosphorylation. These results imply that phosphorylation at Tyr89 by Src-family kinases prevents engagement of the Abl SH3 domain with its intramolecular binding partner leading to enhanced Abl kinase activity and cellular signaling.

Introduction

The Abelson (c-abl) proto-oncogene encodes a non-receptor protein-tyrosine kinase (c-Abl) that is tightly downregulated in cells.1 In contrast, the oncoprotein Bcr-Abl, which results from a chromosomal translocation that fuses Bcr sequences to the N-terminal region of c-Abl, is constitutively active.2, 3 The enhanced tyrosine kinase activity of Bcr-Abl fusion proteins is linked to chronic myelogenous leukemia (CML) and other forms of leukemia.3 Interestingly, almost all of the c-Abl protein sequence is retained in the context of Bcr-Abl. However, the molecular mechanisms of Abl kinase upregulation in Bcr-Abl are not completely understood.

The tyrosine kinase core of c-Abl consists of an N-terminal cap (NCap) region, an SH3 domain, an SH2 domain, and a kinase domain (see Fig. 1a). Multiple intramolecular interactions involving these regions have been observed in the crystal structures of the downregulated c-Abl core.4, 5 The SH3 domain binds the SH2-kinase linker, an interaction necessary to suppress kinase activity.6 The NCap region is immediately N-terminal to the SH3 domain and is essential for c-Abl downregulation.4, 5, 7, 8 The glycine residue at position 2 in NCap is myristoylated and binds to a deep pocket in the C-lobe of the kinase domain, thereby latching SH2 and SH3 in their downregulatory positions at the back of the kinase domain and stabilizing the intramolecular interactions between SH3/SH2 and the kinase domain.1, 4

Recent work has shown that the Src-family tyrosine kinases Hck, Lyn, and Fyn phosphorylate Bcr-Abl within the Abl-derived SH3 and SH2 domains.9 Tyr89 (Abl 1b numbering) in the Abl SH3 domain was found to be the most prominent phosphorylation site in vitro and was also highly phosphorylated by Src-family kinases within Bcr-Abl in CML cells.9 Phosphorylation of Tyr89 was shown to be necessary for the full biological activity of Bcr-Abl, as substitution of this tyrosine residue with phenylalanine reduced the transforming potential of Bcr-Abl in a cytokine-dependent myeloid cell line.10 The crystal structure of the c-Abl core (Fig. 1a) shows that Tyr89 localizes to the binding surface between the SH3 domain and the SH2-kinase linker, a region important for maintaining the inactive, down-regulated state.1 Phosphorylation of this site by Src-family kinases may disrupt the conformation of the downregulated form of Abl and thereby contribute to its transforming activity.

In the present study, hydrogen-exchange (HX) mass spectrometry (MS) was used to investigate whether phosphorylation at Tyr89 affects SH3 interactions with binding partners both in cis and in trans. We show that phosphorylation at Tyr89 by the Src-family kinase Hck inhibits SH3 binding both in trans to a peptide ligand and protein binding partner and in cis to the SH2-kinase linker, an interaction essential to negative regulation. Site-directed mutagenesis indicates that phosphorylation of Tyr245 in the SH2-kinase linker, which is also strongly phosphorylated by Hck, has little impact on the ability of SH3 to interact with the SH2-kinase linker. Overall, our results provide direct biophysical evidence that phosphorylation of Abl SH3 domain Tyr89 disrupts SH3:linker interaction and efficient downregulation of kinase activity. Phosphorylation of this site in the context of both c-Abl and Bcr-Abl may contribute to Abl kinase activation in vivo.

Section snippets

Tyrosine phosphorylation of Abl by Hck

To characterize the structural consequences of Abl phosphorylation by Hck, we expressed and purified a number of different recombinant Abl proteins, many of which have been described in detail.11,12 These constructs contained the Abl SH3 domain either alone or together with the SH2 domain, the NCap and various lengths of the SH2-kinase linker (Fig. 1b). Some of the proteins contained one site of known and heavy phosphorylation (SH3 Tyr89), others contained two sites (Tyr89 and linker Tyr245),

Discussion and Conclusions

In the downregulated state of the c-Abl core, intramolecular interactions are crucial for maintaining an inactive conformation. The crystal structures show that the c-Abl SH3 domain engages the polyproline type II helix formed by the SH2-kinase linker.4, 5 In addition, the SH2 domain docks onto the back of the C-terminal lobe of the Abl kinase domain. This interaction is stabilized further by the NCap when the myristoyl group at Gly2 binds to a deep pocket in the C-lobe of the kinase domain and

DNA constructs and protein purification

The human c-Abl SH3, SH32, SH32L, NCap3, NCap32, and NCap32L proteins were over-expressed and purified as described.12 The c-Abl(2) protein was purified from Sf9 insect cells upon co-expression with YopH, as described.12 The c-Abl(1) form, which was purified from Escherichia coli and contains residues 65-534 (Abl 1b numbering), was a gift from Nathanael Gray at DFCI/HMS. The BP1 peptide was synthesized as described.11 All other chemicals and solvents were obtained from Sigma and were used

Acknowledgements

We thank Nathanael Gray and Jianming Zhang of the Dana Farber Cancer Institute, Harvard Medical School for supplying the c-Abl(1) protein, and Jiong Wu at Cell Signaling Technology for the phosphospecific antibodies. We acknowledge the contributions of Rowena Mak, University of Pittsburgh, for construction of the GST-Abl expression plasmids used in Fig. 6. We are pleased to acknowledge generous financial support from the NIH: GM070590 (to J.R.E.) and CA101828 (to T.E.S.). This work is

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