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. 2012 May 13;8(6):590-6.
doi: 10.1038/nchembio.954.

Domain Organization Differences Explain Bcr-Abl's Preference for CrkL Over CrkII

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Free PMC article

Domain Organization Differences Explain Bcr-Abl's Preference for CrkL Over CrkII

Wojciech Jankowski et al. Nat Chem Biol. .
Free PMC article

Abstract

CrkL is a key signaling protein that mediates the leukemogenic activity of Bcr-Abl. CrkL is thought to adopt a structure that is similar to that of its CrkII homolog. The two proteins share high sequence identity and indistinguishable ligand binding preferences, yet they have distinct physiological roles. Here we show that the structures of CrkL and phosphorylated CrkL are markedly different than the corresponding structures of CrkII. As a result, the binding activities of the Src homology 2 and Src homology 3 domains in the two proteins are regulated in a distinct manner and to a different extent. The different structural architecture of CrkL and CrkII may account for their distinct functional roles. The data show that CrkL forms a constitutive complex with Abl, thus explaining the strong preference of Bcr-Abl for CrkL. The results also highlight how the structural organization of the modular domains in adaptor proteins can control signaling outcome.

Conflict of interest statement

Competing financial interests

The authors declare no competing financial interests.

Figures

Figure 1
Figure 1. Structural and dynamic properties of CrkL
(a) Structure of CrkL. The SH2, SH3N and SH3C domains are colored green, magenta, and blue, respectively. The linker regions are colored grey. The SH3C domain does not interact with the other domains. (b) Close-up view of the SH2–SH3N interface in CrkL. Only polar/charged residues mediate the interaction between the two domains. (c) Plot of the ratio of R2 over R1 15N relaxation rates of the CrkL backbone as a function of residue number. The R2/R1 ratio provides information about the tumbling of the molecule with higher values indicating slower tumbling. (d) Correlation times (τC) for the tumbling of CrkL. The SH2–SH3N module tumbles as a rigid unit whereas the SH3C domain tumbles much faster and independently of the other domains. (e) Residues undergoing substantial μs-ms time scale motions, as denoted by enhanced Rex values, are mapped on the structure of CrkL. Almost all residues located at the interface between the SH2 and SH3N domains exhibit relatively high Rex values indicating that the binding interface is dynamic.
Figure 2
Figure 2. Binding of pTyr- and PPII-peptide ligands to CrkL and CrkII
(a, b) Structure of the SH2–SH3N module in (a) CrkL and (b) CrkII. The pTyr-peptide and PPII-peptide are shown as they have been previously determined to bind to the isolated SH2 (PDB 1JU5) and SH3N domains (PDB 1CKA), respectively. The pTyr-peptide binding site in CrkL is partially masked but is completely accessible in CrkII. Conversely, the PPII-peptide binding site in CrkL is completely accessible but is entirely masked in CrkII. (c) Dissociation constants (Kd) of pTyr-peptide and PPII-peptide complexes with CrkL (Supplementary Fig. 8a) and CrkII. The Kd values of PPII-peptide binding to CrkII were obtained from ref. .
Figure 3
Figure 3. Structural and dynamic properties of pCrkL
(a) Structure of pCrkL. Phosphorylated Tyr207 (pTyr207) is shown as orange sticks. (b) Close-up view of the pTyr207-binding site. The SH2–SH3N interface adjusts slightly to accommodate the binding of the linker to SH2. (c) Plot of the ratio of R2 over R1 relaxation rates of pCrkL as a function of residue number. As in CrkL, the SH2–SH3N module in pCrkL tumbles as a unit whereas the SH3C domain tumbles much faster and independently of the other domains. (d) Kd values of PPII-peptide complexes with CrkL (Supplementary Fig. 8a) and CrkII variants. (e) Pull-down of CrkL and pCrkL with DOCK1, an SH3N binding physiological partner of CrkL (Supplementary Fig. 12).
Figure 4
Figure 4. Effect of Tyr207 phosphorylation on CrkL folding and its association with Abl kinase
(a) 1H-15N HSQC NMR spectra of the linker region of CrkL containing the phosphorylated Tyr207 (pTyr-linker) in the presence of CrkL (orange) and after the addition of catalytic amounts of AblKD and ATP-Mg+2 (blue). The pTyr-linker is 15N-labeled whereas CrkL and AblKD are unlabeled. (b) Analysis of the NMR experiments in (a) shows that the pTyr-linker binds to the SH2 domain of CrkL. Phosphorylation of Tyr207 in CrkL induces the intramolecular association of pTyr207 and SH2. As a result, the pTyr-linker is displaced. (c) Pull-down of CrkL and pCrkL with paxillin, an SH2 binding physiological partner of CrkL (Supplementary Fig. 12). (d) 1H-15N HSQC NMR spectra of free CrkL (blue), in complex with AblPxxP (orange) and after adding ATP-Mg+2 (magenta). AblPxxP is a construct that encompasses the kinase domain and the first PxxP motif that binds to CrkL. (e) Analysis of the NMR experiments in (d) shows that CrkL forms a 1:1 complex with AblPxxP using its SH3N domain. Phosphorylation of Tyr207 elicits the intramolecular association of pTyr207 and SH2 but the intramolecular folding in CrkL has no effect on the CrkL–AblPxxP complex, which remains tightly associated. (f) Pull-down of CrkL and pCrkL with full-length Abl (form 1b) (Supplementary Fig. 12).
Figure 5
Figure 5. CrkL versus CrkII in integrin signaling
(i) Integrin activation elicits p130CAS phosphorylation by tyrosine kinases (TK) and as a result CrkL and CrkII are recruited. (ii) GEFs (for example, DOCK1 and C3G) associate with CrkL and CrkII via their SH3N domain giving rise to efficient localized activation (iii) of small GTPases (for example, Rac and Rap) at the membrane. (iv) Abl-induced phosphorylation of CrkL and CrkII forces their dissociation from p130CAS and thus results in signaling suppression. The distinct structural organization of CrkL and CrkII modulates the interactions with their physiological partners to a different extent. The blue and orange shaded regions in SH2 and SH3N denote the pTyr- and PPII-binding sites, respectively. See text for details.

Comment in

  • Structural biology: CrkL is not Crk-like.
    Kobashigawa Y, Inagaki F. Kobashigawa Y, et al. Nat Chem Biol. 2012 May 17;8(6):504-5. doi: 10.1038/nchembio.963. Nat Chem Biol. 2012. PMID: 22596199 No abstract available.

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