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. 2012 May;3(5-6):402-13.
doi: 10.1177/1947601912460051.

Crk and ABI1: Binary Molecular Switches That Regulate Abl Tyrosine Kinase and Signaling to the Cytoskeleton

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Free PMC article

Crk and ABI1: Binary Molecular Switches That Regulate Abl Tyrosine Kinase and Signaling to the Cytoskeleton

Sajjad Hossain et al. Genes Cancer. .
Free PMC article

Abstract

The nonreceptor tyrosine kinases Abl and Arg are among the most well-characterized tyrosine kinases in the human genome. The activation of Abl by N-terminal fusions with Bcr (Bcr-Abl) or Gag (v-Abl) is responsible for chronic myeloid leukemia or Ph+ acute lymphoblastic leukemia and mouse leukemia virus, respectively. In addition, aberrant Abl and Arg activation downstream of several oncogenic growth factor receptors contributes to the development and progression of a variety of human cancers, often associated with poor clinical outcome, drug resistance, and tumor invasion and metastasis. Abl activation can occur by a variety of mechanisms that include domain interactions involving structural remodeling of autoinhibited conformations as well as direct phosphorylation by upstream kinases and phosphatases. Constitutive activation of Abl plays a significant role in regulating the actin cytoskeleton by modulating cell adhesion, motility, and invadopodia. This review addresses the role of Abl and Arg in tumor progression with particular emphasis on the roles of Crk and Abi1 adapter proteins as distinct molecular switches for Abl transactivation. These insights, combined with new insights into the structure of these kinases, provide the rationale to envision that Crk and Abi1 fine-tune Abl regulation to control signaling to the cytoskeleton.

Keywords: Abi1; Abl kinase; Crk; WAVE complex; cytoskeleton.

Conflict of interest statement

Declaration of Conflicting Interests: The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Figures

Figure 1.
Figure 1.
Structural differences of myristoylated versus nonmyristoylated Abl kinase. (A) Regulated structure of myristoylated c-Abl with major elements of autoinhibition as determined by the crystal structure,: The SH3 domain interacts with the SH2–catalytic domain (CD) linker and CD; the SH2 domain interacts with CD. In the myristoylated form of the kinase, the C-terminal helix of CD, αI′, forms a binding pocket for myristates. Activation of c-Abl by several mechanisms, which include the disassociation of myristates,, leads to phosphorylation of the SH2–CD linker and activation loop. These events result in uncoupling of SH2 and SH3 domains from the backside of CD. The C-terminal helix (αI) partially occludes the SH2 phosphotyrosine binding site. In this regard, Hantschel et al. propose the activation of myristoylated Abl by phosphotyrosine peptides binding to the SH2 domain. The “closed” structure of myristoylated Abl is incompatible with inhibition by Abi1. (B and C) Nonmyristoylated Abl 1a is likely to be less stable than myristoylated Abl (depicted by red arrows) because of the lack of the stabilizing interaction of the myristate with the CD (as this interaction provides an additional “lock,” maintaining the autoinhibitory interactions of SH3-SH2 domains with CD). Hence, the nonmyristoylated Abl is more prone than the myristoylated Abl isoform to assume the “open” (C) elongated conformation proposed by Nagar et al. and later studied by Filippakopoulos et al. The possibility of a ligand binding (such as containing proline motif consensus, PXXP, and phosphotyrosine -pY) to the open confirmation is indicated.
Figure 2.
Figure 2.
Diagrams depicting structural determinants of Abi1 and Crk: functional domains of Abi1 and Crk that regulate c-Abl tyrosine kinase. (Top) Two major isoforms of Abi1, isoform 2 and isoform 3,, and key determinants for Abl kinase regulation are depicted within the highlighted boxes: the c-Abl–SH3 domain binding region 185-PPSPP-189; pY213 that binds to the SH2 domain, and the SH3 domain that binds to the Abl proline-rich linker. Phosphotyrosine residues pY421, pY435, and pY506 that bind to the p85 regulatory subunit of PI3 kinase through SH2 domain binding of p85 are depicted above the boxes. pY421 also targets Src family tyrosine kinases, and pY213 targets the C-terminal SH2 domain of p85. (Bottom) Primary structure of CrkI and Crk II, with the SH2, SH3N, and SH3C domains depicted within boxes. Phosphotyrosine residues implicated in Abl kinase regulation: pY221 and pY251., Phosphotyrosine pY207 that binds to the Crk SH2 domain to form an autoinhibited structure in CrkL (not shown). Abl kinase regulatory domain binding regions are indicated in brackets.
Figure 3.
Figure 3.
Abi1 competes with Crk at the SH3 domain interaction site. (A) Determination of Abi1 Km for Abl kinases. Km is an important value that characterizes the relative substrate affinity for a given enzyme. Moreover, Km allows the comparison of different substrates for the same enzyme. Km of purified GST Abi1 isoform 2 was determined using recombinant c-Abl (47aa-end) purified from baculovirus culture treated with Gleevec (STI-571, Novartis Pharmaceuticals, East Hanover, NJ) as described and using reaction conditions as described by Tanis et al. The Km of GST Abi1 was 0.33 ± 0.5 µM, and the Km of GST Crk was 0.32 ± 0.02 µM (see below); hence, they are comparable. (B) Abi1 competes with Crk at its Abl SH3 domain binding site. For the competition assay with GST Crk, the purified Abi1 SH3 domain was cleaved from GST and used at 2 µM. Reduction of the Km from 0.32 ± 0.02 µM to 0.17 ± 0.01 µM and reduction of the Vmax by 24% indicated mixed competition. Crk associates with Abl through binding of its SH3 domain to the Abl proline-rich domain (PRD); the Abl PRD also binds Abi1 and Abi2. (Left) Kinase activity plots. (Right) Double reciprocal Lineweaver-Burk plots of the kinase activity data.
Figure 4.
Figure 4.
Cooperativity and allostery in Abl kinase-ligand interactions: candidate binding modes of Abi1 and Crk. Protein modules of Abi1 and Crk have the potential to interact with different Abl kinase conformations and to engage all 3 regulatory domains of Abl. (Top) Potential interactions with the Abl autoinhibitory/closed structure. PXXP-pY peptides cannot engage the Abl SH3-SH2 dual domain in the closed conformation because the Abl SH3 domain interacts tightly with the proline consensus of the SH2–catalytic domain (CD) linker and with the CD interface (left). The SH2 domain, however, might bind phosphopeptides as suggested by modeling data using the autoinhibited Abl structure and Src structure pY527 (not shown). Abi1 pY213 and published Crk pY221/207 data (REF) (middle). In addition, the closed structure might engage the proline-rich region in Abl (PRD) and an SH3 domain in a ligand such as Crk and Abi1 (far right). (Bottom) Potential interactions of ligands with an open/active structure of Abl. PXXP-pY–containing peptides such as Abi1 181-PPSPP-185; pY213 might interact with the Abl SH3-SH2 dual domain (left) and with the SH2 domain (middle). It is not yet known what the effect of such interactions is on the Abl SH2 interface residue I116, which is critical in maintaining Abl kinase activity. Moreover, proteins such as Crk or Abi1 might in addition engage the proline-rich linker (PRD) that immediately follows the CD of Abl. In such cases, there is a potential for an additional effect on the CD itself through a possible steric effect.
Figure 5.
Figure 5.
Abi1 and Crk might regulate Abl kinase activity by affecting the stability of the SH2–catalytic domain (CD) interaction in the open conformation of Abl kinase. In our article, we propose that Abi1 Pro-pY213 peptide inhibits Abl kinase activity through binding to Abl SH3-SH2 in an elongated conformation as proposed by small-angle X-ray scattering measurements and affecting the integrity of the SH2-CD interaction. pY213 peptide might exert its inhibition by binding to the Abl SH2 domain only. Based on our cell studies indicating that Abi1 inhibits c-Abl kinase, we propose that the mechanism involves multiple interactions of Abi1 and c-Abl at multiple domains starting from Abi1 PPSPP and pY213 sequences through the middle region of the Abi1 protein and including the SH3 domain at the C-terminus. The Abi1 SH3 domain was previously demonstrated to interact with the Abl proline-rich domain (PRD); homologous Abi2 SH3 was also demonstrated to interact with the Abl PRD., In fact, the “open” elongated conformation of Abl is compatible with the positioning of Abl and Abi1 binding sites alongside each other with the SH3 domain of Abi1 binding to the proline-rich region of Abl located C-terminally to the CD. Crk is proposed to interact in a similar fashion. The role of Abi1 or Crk on Abl kinase activity will depend on the effect on SH2-CD interaction. The critical I164 is indicated in yellow. Brackets indicate the alternatively spliced region of Abi1.
Figure 6.
Figure 6.
Dysregulated stoichiometry of Crk-Abl and Abi1-Abl complexes leads to an invasive phenotype of the tumor-associated actin cytoskeleton. Biochemical data,,, indicate that Abi1 and Crk target the same regulatory domains of c-Abl, thus suggesting binary regulation. We propose that Crk binding to Abl would lead to increased levels of Abi1 in actin regulatory complexes such as WAVE and/or NWASP, which would likely result in a tumorigenic phenotype (far right). Abi1 is also capable of association with the Src SH2 domain and p85 SH2 domain. The latter association would likely result in an enhanced, or dysregulated PI3 kinase activity, as suggested by Abi1 knockout cell studies.

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