Disease relevance of CRKL

• Identification of CRKL as the constitutively phosphorylated 39-kD tyrosine phosphoprotein in chronic myelogenous leukemia cells [1].
• The viral Crk oncogene (v-Crk) is known to induce sarcomas in chicken and its cellular homologs c-Crk I, c-Crk II, and Crk-like (CRKL) have been implicated in many signal transduction events [2].
• Mice lacking the homologue of the human 22q11.2 gene CRKL phenocopy neurocristopathies of DiGeorge syndrome [3].
• In contrast, all BCR/ABL+ CML and acute lymphoblastic leukemia patient samples examined showed clear tyrosine-phosphorylation of CRKL [4].
• Inhibition of Grb2 and Crkl proteins results in growth inhibition of Philadelphia chromosome positive leukemic cells [5].

High impact information on CRKL

• The similarity between the Crkol-/- phenotype and the clinical manifestations of DGS/VCFS implicate defects in CRKL-mediated signaling pathways as part of the molecular mechanism underlying this syndrome [3].
• In a trk-met-Gab1-specific branching morphogenesis assay, association of Gab1 with Shp2, but not PI(3)K, CRKL, or Shc was essential to induce a biological response in MDCK cells [6].
• IC50(imatinib) is defined as the in vitro concentration of drug required to reduce phosphorylation of the adaptor protein Crkl by 50% [7].
• IC50imatinib was determined in pretherapy blood samples by measuring the in vitro imatinib-induced reduction of the phosphorylated form of the adaptor protein Crkl (CT10 regulator of kinase like) [8].
• Colocalization of DOCK2 with Crk-like (CrkL) and F-actin was shown by immunocytochemical analysis with the use of Jurkat cells [9].

Biological context of CRKL

• In primary neutrophils from patients with CML, the major novel tyrosine-phosphorylated protein is CRKL, an SH2-SH3-SH3 linker protein which has an overall homology of 60% to CRK, the human homologue of the v-crk oncogene product [10].
• Besides binding motifs for phosphatidylinositol 3-kinase and Grb2, Gab 1 contains multiple Tyr-X-X-Pro (YXXP) motifs which, when phosphorylated, are potential binding sites for the adapter proteins c-Crk and Crk-like (CRKL) [11].
• Constitutive phosphorylation of CRKL is unique to CML, indicating that it may be a useful target for therapeutic intervention [1].
• Knockdown of RIN1 in epithelial-cell lines blocked the induction of CRKL phosphorylation, confirming that RIN1 normally functions as an inhibitor of cell motility [12].
• We identified one SH2 and two SH3 domains within the 303 amino acid open reading frame of this crk-like gene, CRKL [13].

Anatomical context of CRKL

• However, in the lymphoid H9 cell line, the major tyrosine-phosphorylated CRKL-binding protein was p110(HEF1) [14].
• Several other phosphoproteins were also detected in anti-CRKL immunoprecipitates, one of which has been identified as paxillin, a 68-kDa focal adhesion protein which we have previously shown to be phosphorylated by p210BCR/ABL [10].
• Additionally, CRKL is involved in signaling initiated by crosslinking of beta integrins, and B cell or T cell receptors [15].
• In Ph- peripheral blood cells, CRKL is present only in the nonphosphorylated form [4].
• Here we report that CRKL is phosphorylated when overexpressed, activates RAS and JUN kinase signaling pathways, and transforms fibroblasts in a RAS-dependent fashion [16].

Associations of CRKL with chemical compounds

• Differential signaling after beta1 integrin ligation is mediated through binding of CRKL to p120(CBL) and p110(HEF1) [14].
• In primary leukemic neutrophils from patients with CML, the major tyrosine phosphorylated protein is CRKL, an SH2-SH3-SH3 adapter protein which has an overall homology of 60% to CRK, the human homologue of the v-crk oncogene [17].
• Far Western blotting with a CRKL-SH3 glutathione S-transferase fusion protein showed that CRKL binds directly to p85(PI3K )in vitro [18].
• In cells expressing the proline deletion mutation of BCR-ABL, CRKL is still tyrosine phosphorylated and forms a complex with BCR-ABL as demonstrated by coimmunoprecipitation [19].
• Thus, we suggest that del22q11 is a contiguous gene syndrome involving dose-sensitive interaction of CRKL and TBX1 and locally aberrant RA signaling [20].

Physical interactions of CRKL

• In both cell types, CRKL is constitutively complexed to C3G, SOS, and c-ABL through its SH3 domains, and the stoichiometry of these complexes does not change upon integrin ligation [14].
• The distal C terminus of CBL (residues 671-906) binds the CRKL adapter protein [21].
• Steel factor induces tyrosine phosphorylation of CRKL and binding of CRKL to a complex containing c-kit, phosphatidylinositol 3-kinase, and p120(CBL) [18].
• When phosphorylated, Crkl binds through its Src homology 2 (SH2) domain to other tyrosine phosphoproteins such as paxillin and Cbl [22].

Enzymatic interactions of CRKL

• Within this region, mutation of tyrosine residue 207 yielded a mutant CRKL which could not be phosphorylated by BCR/ABL [23].
• In addition, c-Crk II and CRKL are tyrosine phosphorylated and complexed with numerous other tyrosine phosphorylated proteins in Tel-Abl expressing Ba/F3 cells [24].

Other interactions of CRKL

• Anti-CRKL immunoprecipitates from CML cells, but not normal cells, were found to contain p210BCR/ABL and c-ABL [10].
• These results suggest that the p210BCR/ABL oncogene may be physically linked to the focal adhesion-associated protein paxillin in hematopoietic cells by CRKL [10].
• CRKL has one SH2 and two SH3 domains, with 60% homology to CRK II [15].
• In two hematopoietic cell lines, MO7e and H9, we found that CRKL rapidly associates with tyrosine-phosphorylated proteins after cross-linking of beta1 integrins with fibronectin or anti-beta1 integrin monoclonal antibodies [14].
• Stimulation of human embryonic kidney cells (HEK293) with HGF leads to Gab1 association with CRKL [11].

Analytical, diagnostic and therapeutic context of CRKL

• CRKL binds via its first SH3 domain to several downstream signal transducers, including C3G an activator of the small GTPase Rap1 [11].
• In vitro complexes were highly stable and in vivo complexes of c-Crk and CRKL with HPK1 were detectable by co-immunoprecipitation with transiently transfected cells but also with endogenous proteins [25].
• Immunoblotting with anti-CRKL antibodies showed the presence of CRKL protein in CML cells and cell lines as well as in antiphosphotyrosine immunoprecipitates from CML cells [1].
• Our data suggest that the interaction between CRKL and the proline deletion mutant of BCR-ABL is an indirect interaction as CRKL does not interact directly with the proline deletion mutant of BCR-ABL in a gel overlay assay or in a yeast two-hybrid assay [19].

References