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 “latches” the SH2 domain against the back of the kinase domain 1; 4
. Together, these unique interactions provide a regulatory clamp that allosterically holds the kinase domain in a tightly downregulated state.
Our experimental results suggest that in Abl constructs lacking the kinase domain, the preference of Hck for Abl SH3 phosphorylation is based on the length of the Abl SH2-kinase linker. Very little phosphorylation was detected in the SH32L1/3 protein but the addition of a few more residues (SH32L3/4) caused a major change in the phosphorylation ratio (). Addition of additional C-terminal linker residues shifted the phosphorylation towards double phosphorylation (more Tyr245 was phosphorylated, data not shown) and addition of the NCap forced almost all phosphorylation to entirely double with >95% of the molecules modified at both positions. Thus, Hck phosphorylates both Tyr89 and Tyr245 in c-Abl proteins that lack the kinase domain.
A different situation occurs in versions of Abl that contain both the regulatory and kinase domains. Recombinant purified c-Abl (2) (identical in sequence to the downregulated crystal structure of Nagar, et al. 4
; see ) was not detectably phosphorylated by Hck even after 24 hours, suggesting that this form of c-Abl is tightly downregulated and thus resistant to Hck-mediated phosphorylation of the regulatory apparatus. However, another form of c-Abl tested here, c-Abl (1), which was not myristoylated at the N-terminus, was rapidly phosphorylated on three tyrosine residues (loop Tyr412, linker Tyr245 and SH3 Tyr89). These observations are consistent with a previous report on phosphorylation of a form of recombinant Abl lacking N-terminal myristoylation 20
and suggest that the intramolecular restraints for c-Abl(1) may be disrupted as a result of the unmyristoylated NCap not being able to engage the pocket on the kinase domain. Such a form of c-Abl would be much better poised for phosphorylation by other kinases on sites that are normally buried in the downregulated core. In the Bcr-Abl fusion protein, the N-terminal myristoylation site is also removed; therefore Bcr-Abl may more closely resemble c-Abl(1) in solution than it does the tightly downregulated c-Abl(2). We speculate that this is one contributing factor that allows Bcr-Abl to be phosphorylated by Hck and other SFKs on the SH3 domain, SH2-kinase linker and other possible regulatory tyrosines in leukemia cell lines and in vitro 9
Based on biological data 6; 9
, we hypothesized that phosphorylation physically disrupts downregulatory SH3:linker interactions in c-Abl, perhaps by a mechanism similar to that observed upon mutation of residues in the SH3:linker interface 21–23
. To test this biophysically, we used HX MS to probe protein unfolding and dynamics in different constructs of Abl to determine whether phosphorylation destabilizes the unfolding of the SH3 domain in the absence of the kinase domain. Our data (summarized in ) showed that trans
binding (to the exogenous ligand BP1) was significantly reduced upon Tyr89 phosphorylation as was intramolecular binding of SH3 to the SH2-kinase linker in SH32L and NCapSH32L constructs. In addition, trans
-binding of the regulatory protein Abi-1 to the SH3 domain was also abolished by Hck-mediated phosphorylation of the SH3 domain (). Site-directed mutagenesis and HX MS showed that Tyr89 is the key residue necessary for preventing regulation after phosphorylation. Our findings are consistent with the observation that mutations at SH3 Tyr89 result in Bcr-Abl imatinib resistance in four independent isolates from a random mutagenesis screen 24
. Because imatinib prefers the inactive conformer of the Abl kinase domain, these mutations were presumed to destabilize the negative regulatory influence of SH3 on the linker in the context of Bcr-Abl. Our data imply that phosphorylation of Tyr89 by Hck may also favor the active conformation of Bcr-Abl and contribute to sustained kinase activity and imatinib resistance. The inhibition of binding, both in trans
and in cis
, likely only occurs in forms of the kinase that are not in the most downregulated state, such as Bcr-Abl.
Although Bcr-Abl exhibits constitutive tyrosine kinase activity, Hantschel and Superti-Furga have proposed that Bcr-Abl may retain some of the regulatory features observed in the c-Abl core 1
. Compared to c-Abl core, Bcr-Abl lacks the regulatory impact of myristoylation, but the interaction between SH3 domain and the linker are suggested to remain. Smith et al. 25
showed that an important requirement for Bcr-Abl activity is oligomerization mediated by the N-terminal coiled-coil region of Bcr-Abl, and that mutations in the N-terminal coiled-coil that block transformation are overcome by mutations that disrupt SH3 domain interaction with the linker. This observation supports the idea that the SH3 domain still exerts some negative regulatory influence over Bcr-Abl tyrosine kinase activity. Work presented here provides new biophysical evidence that phosphorylation of the SH3 domain at Tyr89 by Hck, and perhaps other kinases as shown by Meyn et al. 9
, has a similar destabilizing effect. It appears that in the Bcr-Abl fusion protein, partial relaxation of the downregulated core conformation due to loss of the myristoylated NCap may allow other kinases to be recruited that phosphorylate Tyr89, an action that blocks the negative regulatory influence of SH3:linker interaction as well as interaction with trans
-regulatory factors such as Abi-1. Selective inhibition of this phosphorylation activity, in combination with Abl-targeted drugs such as imatinib, may prove useful in CML therapy. Indeed, the dual Src/Abl inhibitor dasatinib is much more potent against CML cells than imatinib, and is active against most forms of imatinib-resistant CML 26