Phosphorylation of Crk on tyrosine 251 in the RT loop of the SH3C domain promotes Abl kinase transactivation (original) (raw)
Related papers
Oncogene, 2005
To gain a better understanding of how Crk II regulates the function of the Abl tyrosine kinase, we explored the function of the C-terminal linker and SH3 domain, a region of Crk II that is still poorly understood. Molecular modeling, tryptophan fluorescence, and covariation sequence alignment indicate that the Crk-SH3-C has a unique binding groove and RT loop not observed in typical SH3 domains. Based on these models, we made a series of mutations in the linker and in residues predicted to destabilize the putative binding pocket and RT loop. In Abl transactivation assays, Y222F and P225A mutations in the linker resulted in strong transactivation of Abl by Crk II. However, mutations predicted to be at the surface of the Crk SH3-C were not activators of Abl. Interestingly, combinations of activating mutations of Crk II with mutations in the highly conserved PNAY sequence in the SH3-C inactivated the activating mutations, suggesting that the SH3-C is necessary for activation. Our data provide insight into the role of highly conserved residues in the Crk-SH3-C, suggesting a mechanism for how the linker and the Crk-SH3-C function in the transactivation of the Abl tyrosine kinase.
Advances in Brief Cellular Interactions of CRKL , an SH 2-SH 3 Adaptor Protein
2006
Chronic myelogenous leukemia is characterized by a specific chromo somal translocation, t(9;22), in which the ABL protooncogene and the BCR gene become juxtaposed. The chlmenc BCR/ABL gene produces a P210 fusion protein with deregulated tyrosine kinase activity. We have recently isolated a complementary DNA, CRKL, which could code for an adaptor protein consisting of one SH2 and two SH3 domains and lacking any catalytic domain. In the current study, we show that CRKL is highly phosphorylated in the chronic myelogenous leukemia cell line 1362 and that it is a substrate for the p2lO BCR/ABL and p145 ABL kinases. BCR/ABL and ABL are coimmunoprecipitated with CRKL in vivo, dem onstrating that relatively stable complexes are formed. In addition, the nucleotide exchange factor mSOS1 was found to be coimmunoprecipitated with CRKL These findings establish a putative signal transduction path way through which BCR/ABL mediates its oncogenic activity.
Crk family adaptor proteins trans -activate c-Abl kinase
Genes to Cells, 2001
Background: c-Abl kinase is activated in response to a variety of biological stimuli. Crk family adaptor proteins can interact physically with c-Abl and be involved in the activation of c-Abl kinase. Results: We report that the Crk family of adaptor proteins act as transacting activators of c-Abl kinase. The interaction of the amino-terminal Src-homology (SH) 3 domain of c-Crk and the proline-rich motifs of c-Abl is an essential step for the phosphorylation of c-Crk by c-Abl, as well as the activation of c-Abl by c-Crk. The activation of c-Abl by c-Crk is negatively regulated by phosphorylation of the tyrosine 221 of c-Crk. Our data suggest that, in the absence of phosphorylation of the tyrosine Y221, the SH2 domain of c-Crk becomes free to bind to target molecules while the carboxyl-terminal SH3 domain of c-Crk binds to the proline-rich region of c-Abl, inducing the activation of c-Abl by c-Crk. Conclusions: This study suggests that the Crk family functions as transacting activators of c-Abl kinase. The phosphorylation of c-Crk may regulate c-Abl kinase.
Tyrosine 207 in CRKL is the BCR/ABL phosphorylation site
Oncogene, 1997
BCR/ABL has a causal role in Philadelphia (Ph)chromosome positive leukemia. The SH2/SH3 adapter protein CRKL is a major substrate of the deregulated BCR/ABL tyrosine kinase and is aberrantly tyrosinephosphorylated in Ph-positive leukemia cells. In this study, experiments were pursued to identify and analyse the CRKL phosphorylation site(s). In an in vitro kinase assay, CRKL phosphorylation by the abl kinase was limited to a small region between the two CRKL SH3 domains. Within this region, mutation of tyrosine residue 207 yielded a mutant CRKL which could not be phosphorylated by BCR/ABL. Stable overexpression of CRKL or CRKL-Y207F did not transform NIH3T3 cells, while the Y207F mutation eliminated tyrosinephosphorylation of CRKL. These studies indicate that Y207 in CRKL represents the major in vivo phosphorylation site. Phosphorylation of Y207 provides a binding site for the CRKL SH2 domain and potentially for other SH2-containing proteins. The Y207F mutation in CRKL did not enhance or decrease association with various target signalling proteins, including SOS or C3G, which interact speci®cally with the CRKL N-SH3 domain. These ®ndings suggest that complex formation with cellular targets is not modulated by CRKL tyrosinephosphorylation.
Cellular interactions of CRKL, and SH2-SH3 adaptor protein
Cancer research, 1994
Chronic myelogenous leukemia is characterized by a specific chromosomal translocation, t(9;22), in which the ABL protooncogene and the BCR gene become juxtaposed. The chimeric BCR/ABL gene produces a P210 fusion protein with deregulated tyrosine kinase activity. We have recently isolated a complementary DNA, CRKL, which could code for an adaptor protein consisting of one SH2 and two SH3 domains and lacking any catalytic domain. In the current study, we show that CRKL is highly phosphorylated in the chronic myelogenous leukemia cell line K562 and that it is a substrate for the p210 BCR/ABL and p145 ABL kinases. BCR/ABL and ABL are coimmunoprecipitated with CRKL in vivo, demonstrating that relatively stable complexes are formed. In addition, the nucleotide exchange factor mSOS1 was found to be coimmunoprecipitated with CRKL. These findings establish a putative signal transduction pathway way through which BCR/ABL mediates its oncogenic activity.
A Src-Like Inactive Conformation in the Abl Tyrosine Kinase Domain
PLOS Biology, 2006
The improper activation of the Abl tyrosine kinase results in chronic myeloid leukemia (CML). The recognition of an inactive conformation of Abl, in which a catalytically important Asp-Phe-Gly (DFG) motif is flipped by approximately 1808 with respect to the active conformation, underlies the specificity of the cancer drug imatinib, which is used to treat CML. The DFG motif is not flipped in crystal structures of inactive forms of the closely related Src kinases, and imatinib does not inhibit c-Src. We present a structure of the kinase domain of Abl, determined in complex with an ATP-peptide conjugate, in which the protein adopts an inactive conformation that resembles closely that of the Src kinases. An interesting aspect of the Src-like inactive structure, suggested by molecular dynamics simulations and additional crystal structures, is the presence of features that might facilitate the flip of the DFG motif by providing room for the phenylalanine to move and by coordinating the aspartate side chain as it leaves the active site. One class of mutations in BCR-Abl that confers resistance to imatinib appears more likely to destabilize the inactive Src-like conformation than the active or imatinib-bound conformations. Our results suggest that interconversion between distinctly different inactive conformations is a characteristic feature of the Abl kinase domain. Citation: Levinson NM, Kuchment O, Shen K, Young MA, Koldobskiy M, et al. (2006) A Src-like inactive conformation in the Abl tyrosine kinase domain. PLoS Biol 4(5): e144.
Oncogene, 2001
c-Abl is a nuclear and cytoplasmic tyrosine kinase involved in a variety of cellular growth and dierentiation processes. In contrast to its oncogenic counterparts, like BCR-Abl, c-Abl is not constitutively tyrosine phosphorylated and its catalytic activity is very low. Here we report tyrosine phosphorylation of endogenous c-Abl and a concomitant increase in catalytic activity. Using Abl 7/7 cells reconstituted with mutated c-Abl forms, we show that phosphorylation and activity depend on Tyr412 in the activation loop. Tyr412 is also required for stimulation by PDGF or by cotransfection of active Src. Phosphorylation of Tyr412 can occur autocatalytically by a trans-mechanism and cause activation of otherwise inactive c-Abl, suggesting a positive feedback loop on c-Abl activity. In the recent structure of the Abl catalytic domain bound to the STI-571 inhibitor, unphosphorylated Tyr412 in the activation loop points inward and appears to interfere with catalysis. We mutated residues involved in stabilizing this inhibited form of the activation loop and in positioning Tyr412. These mutations resulted in tyrosine phosphorylation and activation of c-Abl, as if relieving c-Abl from inhibition. Tyr412 is therefore necessary both for activity and for regulation of c-Abl, by stabilizing the inactive or the active conformation of the enzyme in a phosphorylationdependent manner. Oncogene (2001) 20, 8075 ± 8084.
Genes & cancer, 2012
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...