Abstract A04: Biophysical and biochemical characterization of Src-phosphorylated KRas
Bibliographic record
Abstract
Abstract Tyrosine phosphorylation of several small GTPase proteins including Ras has been reported; however, investigations to understand the biologic role of tyrosine phosphorylated small GTPases have been limited. We will present the detailed characterization of the impact of tyrosyl phosphorylation of KRas on each stage of the GTPase cycle, and on the binding to the RBD of BRaf. Using NMR and MS analysis we demonstrate that a catalytic amount of Src (1 mole of Src into 250 moles of KRas) specifically phosphorylates KRas at two of the eight tyrosine residues (Y32 and Y64) in vitro. In HEK293 cells, ectopic expression of Src with KRas results in tyrosyl phosphorylation of the same sites in KRas. Tyrosyl phosphorylation of KRas perturbs the chemical shifts of residues in the two switch regions. Using our real-time NMR-based small GTPase assay, we illustrate that tyrosyl phosphorylation alters regulation of the KRas GTPase cycle by guanine exchange factor (SOScat) and GTPase activating protein (p120GAP), rendering KRas resistant to both activities. Furthermore, using an Octet BLI binding assay, we demonstrated that tyrosyl phosphorylation of KRas strongly impairs BRaf-RBD binding. In vitro, Src-phosphorylated KRas can be completely dephosphorylated by the phosphatase SHP2. In cells, we were able to detect endogenous tyrosyl phosphorylated Ras upon SHP2 CRISPR knockout. Together, our findings demonstrate that the GTPase cycle and effector binding affinity of KRas can be regulated by the balance of Src and SHP2 activities. Citation Format: Teklab Gebregiworgis, Christopher B. Marshall, Yoshihito Kano, Michael Ohh, Mitsuhiko Ikura. Biophysical and biochemical characterization of Src-phosphorylated KRas [abstract]. In: Proceedings of the AACR Special Conference on Targeting RAS-Driven Cancers; 2018 Dec 9-12; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2020;18(5_Suppl):Abstract nr A04.
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How this classification was reachedexpand
Full frame distilled prediction
Teacher imitationNot calibrated prevalence, not ground truth. Human validation pending. Learned from the 10,348 direct Codex labels and 10,348 direct Gemma labels. Candidate is the union of thresholded teacher heads; consensus is their intersection. These outputs are machine_predicted_unvalidated and are not human labels or direct frontier model labels.
Codex and Gemma teacher scores by category
| Category | Codex | Gemma |
|---|---|---|
| Metaresearch | 0.000 | 0.000 |
| Meta-epidemiology (narrow) | 0.000 | 0.000 |
| Meta-epidemiology (broad) | 0.000 | 0.000 |
| Bibliometrics | 0.000 | 0.000 |
| Science and technology studies | 0.000 | 0.000 |
| Scholarly communication | 0.000 | 0.000 |
| Open science | 0.000 | 0.000 |
| Research integrity | 0.000 | 0.000 |
| Insufficient payload (model declined to judge) | 0.000 | 0.000 |
Machine scores (provisional)
The two teacher heads of the student model, read on this work. A score orders the frame for review; it never asserts a category, and the validation status ships verbatim with every row.
Baseline scores from an immature model (maturity gate not passed, 7 training rounds). Scores rank; they never assert a category.
score_only:v0-immature-baseline · verbatim from the scoring run: score_only means the number may rank works, and no category label ships from itClassification
machine, unvalidatedMachine predicted; a candidate call from one teacher head, not a consensus.
How this classification was reached, model by model and score by score, is at the end of the page under "How this classification was reached".