scholarly journals Crystal structure of an SH2–kinase construct of c-Abl and effect of the SH2 domain on kinase activity

2015 ◽  
Vol 468 (2) ◽  
pp. 283-291 ◽  
Author(s):  
Sonja Lorenz ◽  
Patricia Deng ◽  
Oliver Hantschel ◽  
Giulio Superti-Furga ◽  
John Kuriyan
Keyword(s):  
Crystal Structure ◽  
Functional Role ◽  
Kinase Activity ◽  
Sh2 Domain ◽  
Constitutive Activation ◽  
Src Homology 2 ◽  
Kinase C ◽  
Src Homology

Constitutive activation of the tyrosine kinase c-Abl in the cell involves interactions between the SH2 (Src-homology 2) and kinase domains (KDs). We present a crystal structure of a c-Abl construct comprising these domains and analyse the functional role of their interface in vitro.

Triazolopyrimidinium salts: discovery of a new class of agents for cancer therapy

2020 ◽  
Vol 12 (5) ◽  
pp. 387-402
Author(s):  
Mariateresa Badolato ◽  
Fabrizio Manetti ◽  
Antonio Garofalo ◽  
Francesca Aiello
Keyword(s):  
Anticancer Agents ◽  
Docking Studies ◽  
In Vitro Models ◽  
Sh2 Domain ◽  
Activity Profile ◽  
Cancer Cell Growth ◽  
New Class ◽  
Src Homology 2 ◽  
Src Homology

Aim: The [1,2,4]triazolo[1,5- a]pyrimidine core is highly privileged in medicinal chemistry due to its versatile pharmacological activity profile. Recently, the search for novel anticancer agents has focused on [1,2,4]triazolo[1,5- a]pyrimidine derivatives. Results: Our hit functionalization has led to the discovery of new [1,2,4]triazolo[1,5- a]pyrimidinium salts with potential anticancer activity. Among a small library of molecules, compound 9 significantly inhibits cancer cell growth in a panel of in vitro models. Molecular docking studies and preliminary binding assay have displayed that 9 could directly bind the Src homology 2 (SH2) domain of STAT3 protein. Conclusion: Compound 9 is a novel promising lead compound that motivates additional evaluation of [1,2,4]triazolo[1,5- a]pyrimidinium salts as novel potential chemotherapeutics.

scholarly journals Multiple cytokines stimulate the binding of a common 145-kilodalton protein to Shc at the Grb2 recognition site of Shc.

1994 ◽  
Vol 14 (10) ◽  
pp. 6926-6935 ◽  
Author(s):  
L Liu ◽  
J E Damen ◽  
R L Cutler ◽  
G Krystal
Keyword(s):  
Sodium Dodecyl ◽  
Sh2 Domain ◽  
Hemopoietic Cell ◽  
Interleukin 3 ◽  
Src Homology 2 ◽  
Phosphorylated Form ◽  
Isoelectric Points ◽  
Src Homology ◽  
Cytokine Stimulation

We recently reported that interleukin-3, Steel factor, and erythropoietin all induce the tyrosine phosphorylation of Shc and its association with Grb2 in hemopoietic cell lines. We have now further characterized the proteins that become associated with Shc following stimulation with these cytokines and found that, in response to all three, the tyrosine-phosphorylated form of Shc binds to common 145- and 52-kDa proteins which also become tyrosine phosphorylated in response to these growth factors. The 145-kDa protein, which appears, from antiphosphotyrosine blots of two-dimensional O'Farrell gels, to exist in four different phosphorylation states following cytokine stimulation (with isoelectric points ranging from 7.2 to 7.8), does not appear to be immunologically related to the beta subunit of the interleukin-3 receptor, c-Kit, BCR, ABL, JAK1, JAK2, Sos1, eps15, or insulin receptor substrate 1 protein. Silver-stained sodium dodecyl sulfate gels indicate that the association of the 145-kDa protein with Shc occurs only after cytokine stimulation and that it can bind to the tyrosine-phosphorylated form of Shc in its non-tyrosine-phosphorylated state. The latter finding, in conjunction with the observations that p145 does not bind, in vitro, to the Src homology 2 (SH2) domain of Shc, that it is not present in anti-Grb2 immunoprecipitates, and that a phosphopeptide which blocks the binding of Shc to the SH2 domain of Grb2 also blocks the binding of Shc to p145, suggests that p145 contains an SH2 domain and competes with Grb2 for the same tyrosine-phosphorylated site on Shc. This implicates p145 as a potential regulator of Ras activity and, perhaps, of other as yet unidentified functions of Shc.

scholarly journals p56lck interacts via its src homology 2 domain with the ZAP-70 kinase.

1994 ◽  
Vol 179 (4) ◽  
pp. 1163-1172 ◽  
Author(s):  
P Duplay ◽  
M Thome ◽  
F Hervé ◽  
O Acuto
Keyword(s):  
Tyrosine Kinases ◽  
Intracellular Signaling ◽  
Specific Binding ◽  
Sh2 Domain ◽  
Jurkat Cells ◽  
Vitro Assay ◽  
Phosphorylated Proteins ◽  
Src Homology 2 ◽  
Src Homology

p56lck, a member of the src family of protein tyrosine kinases, is an essential component in T cell receptor (TCR) signal transduction. p56lck contains a src homology 2 (SH2) domain found in a number of proteins involved in intracellular signaling. SH2 domains have been implicated in protein-protein interactions by binding to sequences in target proteins containing phosphorylated tyrosine. Using an in vitro assay, we have studied specific binding of tyrosine-phosphorylated proteins to a recombinant p56lck SH2 domain. In nonactivated Jurkat cells, two tyrosine-phosphorylated proteins were detected. Stimulation with anti-CD3 monoclonal antibodies induced the binding of seven additional tyrosine-phosphorylated proteins to the SH2 domain of p56lck. We have identified the zeta-associated tyrosine kinase, ZAP-70, as one of these proteins. Evidence suggests that binding of ZAP-70 to p56lck SH2 is direct and not mediated by zeta. The significance of this interaction was further investigated in vivo. p56lck could be coprecipitated with the zeta/ZAP-70 complex and conversely, ZAP-70 was detected in p56lck immunoprecipitates of activated Jurkat cells. The physical association of p56lck and ZAP-70 during activation supports the recently proposed functional cooperation of these two tyrosine kinases in TCR signaling.

Molecular Mechanisms of JAK2V617F Oncogenic Activation: The JAK2- V617F Mutant Utilizes the SH2 Domain for Constitutive Kinase Activity in the Presence of Unstimulated Heterodimeric Cytokine Receptor.

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 175-175
Author(s):  
Sivahari P Gorantla ◽  
Tobias Dechow ◽  
Rebekka Grundler ◽  
Christian Peschel ◽  
Justus Duyster
Keyword(s):  
Molecular Mechanisms ◽  
Kinase Activity ◽  
Jak2 V617f ◽  
Sh2 Domain ◽  
Cytokine Receptor ◽  
Cytokine Receptors ◽  
Type I ◽  
Constitutive Activation ◽  
Downstream Signaling

Abstract The JAK2-V617F mutation has been reported in the majority of MPDs including PV, ET, and IMF. This mutation leads to the constitutive activation of the JAK2 tyrosine kinase activity and overexpression of JAK2V617F renders hematopoietic cell lines growth factor-independent. However, the molecular mechanism leading to constitutive activation of JAK2V617F is largely unclear and the requirement of homodimeric or heterodimeric cytokine receptors needs to be determined. Here we show that oncogenic JAK2-V617F requires an intact SH2 domain for constitutive kinase activity. To this end we mutated the conserved arginine 426 within the SH2 domain to a lysine. Ba/F3 cells expressing JAK2V617F grew IL-3-independent and showed constitutive activation of JAK2, STAT5, and ERK1/2. In contrast, introduction of the SH2 mutation in JAK2V617F abrogated both transformation as well as constitutive activation of downstream signaling pathways. Accordingly, reconstitution of JAK2 mutants in a JAK2-negative cell line with IL-3R co-expression revealed reduced activation of JAK2 when the SH2 domain was mutated. It has been reported that JAK2 binding to homodimeric type I cytokine receptor may facilitate JAK2V617F-mediated transformation. Interestingly, co-expression of the homodomeric EpoR with SH2 mutated JAK2V617F rescues the phenotype indicating that the SH2 domain is required for JAK2 signaling in the presence of heterodimeric but not homodimeric cytokine receptors. Membrane localization studies showed equal membrane distribution of SH2-mutated and unmutated JAK2-V617F indicating that the SH2 domain mutation does not affect subcellular distribution of JAK2. However, co-IP experiments revealed a possible role for the SH2 domain in the dimerization and transphosphorylation of JAK2. Consequently, reduced transphosphorylation was seen in IL-3R- but not in EpoR-expressing cells. In a BM transplantation model we found that an intact SH2 domain in JAK2V617F was required for the induction of a MPD-like disease. Thus, our results points to an important role of the SH2 domain for the constitutive activation of JAK2V617F in cells expressing heterodimeric cytokine receptors.

scholarly journals Rapid and efficient purification of Src homology 2 domain-containing proteins: Fyn, Csk and phosphatidylinositol 3-kinase p85

1994 ◽  
Vol 302 (3) ◽  
pp. 737-744 ◽  
Author(s):  
M Koegl ◽  
R M Kypta ◽  
M Bergman ◽  
K Alitalo ◽  
S A Courtneidge
Keyword(s):  
Tyrosine Kinases ◽  
Intramolecular Interaction ◽  
Affinity Purification ◽  
Sh2 Domain ◽  
Exchange Chromatography ◽  
Phosphatidylinositol 3 Kinase ◽  
Src Homology 2 ◽  
Src Homology ◽  
Phosphatidylinositol 3

To analyse the regulation of Src family tyrosine kinases in vitro, we have purified Fyn and Csk, a kinase capable of regulating Fyn activity by phosphorylation, from baculovirus-infected insect cells. The proteins were purified by affinity purification over a phosphotyrosine column. Highly purified proteins were eluted from the resin by a salt gradient and further purified by ion-exchange chromatography. This purification scheme was successfully applied to a third, unrelated protein that also contains the Src homology 2 (SH2) domain, namely the 85 kDa subunit of phosphatidylinositol 3-kinase, indicating that this method is versatile and should prove applicable to any protein with an accessible SH2 domain. The binding of Csk to different phosphopeptides was tested, and specificity for the autophosphorylation site of Fyn was demonstrated. Pure Csk was used to phosphorylate Fyn and down-regulate its kinase activity, and the kinetic parameters of both the active and the repressed forms of Fyn were determined. Repression of Fyn activity by Csk reduced binding of Fyn to phosphopeptides to undetectable levels, supporting the model that predicts an intramolecular interaction of the Fyn SH2 domain with a C-terminal phosphotyrosine residue.

scholarly journals pp60v-src transformation of rat cells but not chicken cells strongly correlates with low-affinity phosphopeptide binding by the SH2 domain.

1997 ◽  
Vol 8 (5) ◽  
pp. 843-854 ◽  
Author(s):  
M F Verderame
Keyword(s):  
Structural Integrity ◽  
Cell Transformation ◽  
Binding Pocket ◽  
Sh2 Domain ◽  
Site Directed Mutagenesis ◽  
Morphological Transformation ◽  
Sh2 Domains ◽  
Src Homology

Substrates critical for transformation by pp60v-src remain unknown, as does the precise role of the src homology 2 (SH2) domain in this process. To continue exploring the role of the SH2 domain in pp60v-src-mediated transformation, site-directed mutagenesis was used to create mutant v-src alleles predicted to encode proteins with overall structural integrity intact but with reduced ability to bind phosphotyrosine-containing peptides. Arginine-175, which makes critical contacts in the phosphotyrosine-binding pocket, was mutated to lysine or alanine. Unexpectedly, both mutations created v-src alleles that transform chicken cells with wild-type (wt) efficiency and are reduced for transformation of rat cells; these alleles are host dependent for transformation. Additionally, these alleles resulted in a round morphological transformation of chicken cells, unlike 12 of the 13 known host-dependent src SH2 mutations that result in a fusiform morphology. Analysis of phosphopeptide binding by the mutant SH2 domains reveal that the in vitro ability to bind phosphopeptides known to have a high affinity for wt src SH2 correlates with wt (round) morphological transformation in chicken cells and in vitro ability to bind phosphopeptides known to have a low affinity for wt src SH2 correlates with rat cell transformation. These results suggest that the search for critical substrates in rat cells should be among proteins that interact with pp60v-src with low affinity.

Multiple cytokines stimulate the binding of a common 145-kilodalton protein to Shc at the Grb2 recognition site of Shc

1994 ◽  
Vol 14 (10) ◽  
pp. 6926-6935
Author(s):  
L Liu ◽  
J E Damen ◽  
R L Cutler ◽  
G Krystal
Keyword(s):  
Sodium Dodecyl ◽  
Sh2 Domain ◽  
Hemopoietic Cell ◽  
Interleukin 3 ◽  
Src Homology 2 ◽  
Phosphorylated Form ◽  
Isoelectric Points ◽  
Src Homology ◽  
Cytokine Stimulation

We recently reported that interleukin-3, Steel factor, and erythropoietin all induce the tyrosine phosphorylation of Shc and its association with Grb2 in hemopoietic cell lines. We have now further characterized the proteins that become associated with Shc following stimulation with these cytokines and found that, in response to all three, the tyrosine-phosphorylated form of Shc binds to common 145- and 52-kDa proteins which also become tyrosine phosphorylated in response to these growth factors. The 145-kDa protein, which appears, from antiphosphotyrosine blots of two-dimensional O'Farrell gels, to exist in four different phosphorylation states following cytokine stimulation (with isoelectric points ranging from 7.2 to 7.8), does not appear to be immunologically related to the beta subunit of the interleukin-3 receptor, c-Kit, BCR, ABL, JAK1, JAK2, Sos1, eps15, or insulin receptor substrate 1 protein. Silver-stained sodium dodecyl sulfate gels indicate that the association of the 145-kDa protein with Shc occurs only after cytokine stimulation and that it can bind to the tyrosine-phosphorylated form of Shc in its non-tyrosine-phosphorylated state. The latter finding, in conjunction with the observations that p145 does not bind, in vitro, to the Src homology 2 (SH2) domain of Shc, that it is not present in anti-Grb2 immunoprecipitates, and that a phosphopeptide which blocks the binding of Shc to the SH2 domain of Grb2 also blocks the binding of Shc to p145, suggests that p145 contains an SH2 domain and competes with Grb2 for the same tyrosine-phosphorylated site on Shc. This implicates p145 as a potential regulator of Ras activity and, perhaps, of other as yet unidentified functions of Shc.

scholarly journals Role of Src homology 2 domain-mediated PTK signaling in mouse zygotic development

Reproduction ◽  
2006 ◽  
Vol 132 (3) ◽  
pp. 413-421 ◽  
Author(s):  
Li Meng ◽  
JinPing Luo ◽  
Chunhua Li ◽  
William H Kinsey
Keyword(s):  
Marine Invertebrates ◽  
Sh2 Domain ◽  
Calcium Oscillations ◽  
Src Family Kinases ◽  
Developmental Potential ◽  
Fyn Kinase ◽  
Src Homology 2 ◽  
Src Homology ◽  
Src Homology 2 Domain

Fyn and other Src-family kinases play an essential role at several steps during egg activation following fertilization of externally fertilizing species, such as marine invertebrates, fish, and frogs. Recent studies demonstrate that the requirement for Src-family kinases in activation of the mammalian egg is different from lower species, and the objective of this study was to test the role of the Fyn kinase in the mouse egg activated by intracytoplasmic sperm injection (ICSI). An Src homology 2 (SH2) domain containing fusion protein was used to suppress Fyn function in the mouse zygote following ICSI. Eggs injected with the Fyn SH2 domain at an intracellular concentration of 4–8 μM exhibited reduced developmental potential with 100% of the zygotes being arrested following the first or the second cleavage. At higher concentrations, the protein blocked pronuclear congression and the zygotes remained at the pronuclear stage. The SH2 domain had no effect on sperm-induced calcium oscillations in distinct contrast to its effect on the eggs of lower species. The results indicate that the SH2 domain of Fyn kinase plays an important role in pronuclear congression as well as early cleavage events and that this effect appears not to involve disruption of calcium oscillations.

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