Journal of Molecular Biology
Tyrosine Phosphorylation in the SH3 Domain Disrupts Negative Regulatory Interactions within the c-Abl Kinase Core
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
References (30)
- et al.
Organization of the SH3-SH2 unit in active and inactive forms of the c-Abl tyrosine kinase
Mol. Cell
(2006) - et al.
Structural Basis for the Autoinhibition of c-Abl Tyrosine Kinase
Cell
(2003) - et al.
c-Abl has high intrinsic tyrosine kinase activity that is stimulated by mutation of the Src homology 3 domain and by autophosphorylation at two distinct regulatory tyrosines
J. Biol. Chem.
(2000) - et al.
Autoinhibition of c-Abl
Cell
(2002) - et al.
A myristoyl/phosphotyrosine switch regulates c-Abl
Cell
(2003) - et al.
SRC family kinases phosphorylate the BRC-ABL SH3-SH2 region and modulate BCR-ABL transforming activity
J. Biol. Chem.
(2006) - et al.
The Src family kinase Hck interacts with Bcr-Abl by a kinase-independent mechanism and phosphorylates the Grb2-binding site of Bcr
J. Biol. Chem.
(1997) - et al.
Partial unfolding of diverse SH3 domains on a wide timescale
J. Mol. Biol.
(2006) - et al.
Identification and characterization of EX1 kinetics in H/D exchange mass spectrometry by peak width analysis
J. Am. Soc. Mass Spectrom.
(2006) - et al.
Allosteric inhibition of the nonmyristoylated c-Abl tyrosine kinase by phosphopeptides derived from Abi1/Hssh3 bp1
Biochim. Biophys. Acta
(2008)