Based on these results we postulate that phosphorylation of Y213 of Abi1 by c-Abl kinase is followed by binding of Abi1 to the Abl SH2 domain with subsequent inhibition of c-Abl kinase activity. If verified, this would be the first demonstration of inhibition of c-Abl kinase by a phosphopeptide located in trans in another protein, in this case, Abi1.
We propose that Abi1 phosphopeptides inhibit c-Abl kinase through an allosteric mechanism. This mechanism involves binding of the phosphorylated Y213 to the Abl SH2 domain. An observed decrease of the Vmax
, with no change of the Km
is consistent with a noncompetitive mechanism of inhibition of kinase activity by the phosphopeptide containing pY213. However, the effect of pY213 on Abl kinase activity is relatively weak (decrease of Vmax of about 38% at peptide concentration of 1 mM). This is in contrast to high binding affinity data obtained from surface plasmon resonance studies using the GST tagged Abl SH2 domain. The binding data obtained from intrinsic fluorescence quenching experiments, obtained with the untagged protein, or from overlay binding assays [35
] (K. Machida, B.J. Mayer and L. Kotula, unpublished results), indicate much lower binding affinity of pY213, i.e. in the micromolar range. These results suggest a strong effect of the GST tag, most likely due to its dimerization effect, on SH2 binding affinities obtained using SPR as previously suggested [33
]. Our binding data for pY213-SH2, obtained with untagged protein, is consistent with the relatively weak effect on Abl kinase activity in vitro
The in vitro
kinase data demonstrated here pertains to the nonmyristoylated, partially capped, partially activated form of c-Abl kinase. However, there is no crystal structural information on the active kinase in the context of the SH3 and SH2 domains. Recent SAXS [18
] studies indicate that the active kinase is likely to exist in the elongated form. Thus it is possible that pY213 decreases Abl kinase activity by increasing the rigidity of the kinase domain through the interactions of the SH2 domain and the N-lobe of the kinase domain, hence the noncompetitive mechanism of inhibition. The noncompetitive nature of the inhibition is demonstrated by Lineweaver-Burke plot despite a relatively high concentration of the peptide used in kinase assay. Supporting the hypothesis that pY213 regulates c-Abl tyrosine kinase activity through interactions with the c-Abl SH2 domain, is the fact that pY213 regulates physical association of Abi1. This is demonstrated here by binding assays showing interaction of pY213 with the Abl SH2 domain () as well as by immunoprecipitation results indicating that Abi1-pY213 interacts with the active Abl kinase in LNCaP cells (). pY213 phosphorylation, and consequently the strength of Abi1-Abl kinase interaction, is STI-571-dependent as indicated by co-transfection experiments. Interestingly, treatment of K562 cells with STI-571 reduced pY213 levels as compared to untreated cells [36
] suggesting the possibility that Abi1 is a substrate of, and functions downstream of, BCR-Abl.
The lack of a crystal structure of the nonmyristoylated kinase makes it difficult to interpret the effects of Pro-pY213 peptide on kinase activity. We base our interpretation on the following facts: 1) Pro-pY213 represents the region from Abi1 that regulates c-Abl kinase activity in vitro
. At higher concentrations the peptide inhibits Abl kinase activity. 2) Mutated or truncated peptides either inhibit to a lesser extent than Pro-pY213 or do not inhibit at all. These data permit identification of three critical elements affecting activity: 181
(representing the core Abl SH3 domain binding PXXP motif); pY213 (representing the critical SH2 domain binding phosphotyrosine); and Y198, which works together with the 181
motif as demonstrated by Pro-Y198 peptide. 3) Multiple conformations of the autoinhibited (inactive) and active Abl kinase are possible based on recent findings on Abl [18
] that include SAXS studies as well as Src kinase [37
]. These studies indicate significant rearrangements of the SH3-SH2 dual domain around the catalytic domain of Src-like family kinases. Therefore, we hypothesize that different effects of the pY213 and Pro-pY213 and Abl kinase activity might be a result of different peptide affinities to transient Abl conformations. For example, the presence of residual autoinhibitory interactions of the SH3 and SH2 domain assembly with the catalytic domain of the kinase [16
] in the nonmyristoylated Abl kinase might explain the lack of effect of Pro-pY213 on the kinase activity at lower peptide concentrations. 4) The fact that the binding affinity of the dual domain-binding Pro-pY213 with Abl SH2 and SH3 domains is much greater than the sum of the binding affinities of single SH3 and SH2 ligands suggests that there is a requirement for concurrent interaction of SH2 and SH3 domains with a consolidated ligand for regulation of Abl kinase activity.
obtained from the kinetic analysis of Abi1 peptides (not shown) are higher and do not correlate with corresponding KD
obtained from binding assays. One possible explanation may include the fact that the binding assays were performed with Abl domain(s) purified from recombinant bacteria, which would be nonphosphorylated, whereas kinase assays were performed with partially active, tyrosine phosphorylated kinase obtained from baculovirus. In this regard, a low level of pY412 and PY-99 immunoreactivity was confirmed in baculovirus-purified kinase (not shown). Importantly, these types of kinase preparations are extremely prone to activation due to autophosphorylation leading to observed differences in basal kinase activity (compare ). Tyrosine phosphorylation of Abl [10
] due to autophosphorylation at or near Abi1 peptide binding regions may significantly influence their binding affinities.
The Pro-pY213 region of Abi1 represents an important element that regulates Abl kinase activity in vivo as demonstrated in LNCaP cells. Abi1 Y213F or 181AESEA185 mutants did not inhibit Abl kinase activation, which indicates that concurrent binding of Abi1 to both SH3 and SH2 domain of Abl is critical for regulation. This is consistent with in vitro binding data demonstrating significant enhancement of the binding affinity of the consolidated Abi1 ligand over single-site ligands. It is possible that the 181AESEA185 mutant, despite having a higher affinity binding site for Abl, is incapable of Abl inhibition in LNCaP cells because of lower expression of total Abi1 in comparison to the clone that expresses wild type Abi1-Ha. LNCaP cells express both isoforms of Abl i.e. myristoylated and nonmyristoylated as determined by mRNA analysis (data not shown). Thus, the effects of the recombinant Abi1 on both isoforms of kinase cannot be excluded in these cells.
The hypothesis that Abi1 acts on the nonmyristoylated isoform of Abl is suggested by inhibition of the kinase in co-transfection experiments in Cos7 cells. Apparently, the nonmyristoylated kinase is constitutively active upon transfection into cells, while Abl kinase must be activated with pervanadate in LNCaP cells in order to demonstrate regulation by Abi1. As pervanadate is considered a general tyrosine phosphatase inhibitor, the action of Abi1 on Abl may be through an allosteric effect, as we postulate, or through a “shielding” effect on the catalytic domain by Abi1 SH3 domain interacting with the proline-rich region of c-Abl [22
]. Thus, steric hindrance caused by tagging of Abi1 at the C-terminal may decrease its inhibitory effect on Abl kinase as demonstrated here. We cannot exclude the possibility that Abi1 is also a competitive inhibitor in vivo
in addition to its allosteric inhibition. Importantly, Abi1 must be phosphorylated at pY213, presumably by Abl, for the proposed regulation to occur. Consequently, in intact cells, Abi1 is both a candidate substrate and the candidate regulator of Abl kinase activity.
The proposed mechanism of regulation of c-Abl by Abi1 includes the possibility that Abi1 plays a role in the initial activation of c-Abl as proposed [38
]. This hypothesis would most likely apply to the myristoylated, autoinhibited kinase. Structural studies of c-Abl [15
] indicate that the phosphotyrosine binding site is partially occluded in the crystal structure of the myristoylated c-Abl fragment containing the SH3-SH2-catalytic domain assembly. Thus, Pro-pY213 could potentially activate the myristoylated kinase through the SH2 domain interaction as proposed [15
]. It is also possible that Abi1 may downregulate c-Abl that has been activated by phosphopeptides [15
]. This might occur, for example, by competing off the activating phosphopeptide by Abi1-pY213.
Conservation of the regulatory sequences suggests that other members of Abi/Hssh3bp1 family of proteins also regulate Abl. The region containing the regulatory tyrosine 213 is highly conserved between Abi1 and Abi2 from Xenopus through human, and is present in Drosophila Abi (). The conserved sequences also include the PXXP motif, 181
, which binds to the c-Abl SH3 domain, and tyrosine 198. All isoforms of Abi1 [22
], or Abi2 [21
] contain the regulatory sequence indicating the conservation of c-Abl regulation in all Abi isoforms. The conserved region of Abi1 apparently plays a role in the regulation of c-Abl kinase activity in cells; here we addressed the role of the regulatory sequences in the context of Abi1 isoform 2. The fact that multiple isoforms are expressed from the Abi1/Hssh3bp1 gene [24
] suggests the possibility of differential effects on Abl kinase activity as well as multiple downstream effects on actin cytoskeleton and Wave complex regulation [40
Figure 5 c-Abl kinase regulatory region of the Abi protein family. The region of Abi1 containing the Abl SH3 domain binding site containing the core PXXP consensus, PPSPP, Y198, and the SH2 domain binding site (underlined, with regulatory phosphotyrosine, pY) (more ...)
In summary, we identified a candidate molecular mechanism of regulation of nonmyristoylated Abl kinase. Nonmyristoylated, mutated forms of Abl, such as BCR-Abl, are implicated in chronic myelogenous leukemia and in some forms of acute lymphocytic leukemia [3
]. Although the use of STI-571 has brought great promise for the treatment of these diseases, some patients have become resistant to the drug following long-term treatment. STI-571-resistance mutations are found in Abl SH3 and SH2 domains [42
] suggesting the possibility that in these cases BCR-Abl escapes possible residual regulation by other interactions including that with Abi1. The studies described here may improve our understanding of the mechanisms of Abl regulation, and may directly impact studies of BCR-Abl.