Abstract 5154: Regulatory roles of conserved phosphorylation sites in the activation loop of extracellular signal-regulated kinase 1.
Bibliographic record
Abstract
Abstract The catalytic domains of most eukaryotic protein kinases are highly conserved in their primary structures. Phosphorylation of the activation loop, a variable region between kinase catalytic subdomains VII and VIII, is a common mechanism for regulating protein phosphotransferase activity. Our investigation of the distribution and evolutionary conservation of phosphorylation sites in 492 typical human protein kinase catalytic domains revealed that about 75% of the 303 known activating phosphosites are located within the activation loop. Extracellular signal-regulated kinase 1 (ERK1) belongs to the mitogen-activated protein kinase (MAPK) family, which is one of the most intensively studied protein kinase families and serves as a paradigm for regulation of protein kinases in signaling modules. ERK1 homologues play fundamental roles in regulation of cell growth and proliferation from yeast to humans. In addition to the well-documented TEY activating site in the activation loop of ERK1 and its closest relative ERK2, three flanking phosphosites have been confirmed (T198, T207 and Y210 from ERK1) by high throughput mass spectrometry. We noticed that T207 and Y210 correspond to conserved phosphosites (T/S-XX-Y) located just prior to the subdomain VIII APE motif in many other protein-serine/threonine kinases including CDKs, CAMKs, PKB/Akts and PKCs, which may implicate a common regulatory role for of these phosphosites in a wide range of kinases. To investigate the roles of these three flanking phosphosites, we mutated T198, T207 and Y210 of ERK1 individually and in combinations. In vitro kinase assays with myelin basic protein (MBP) using purified mutants indicated the functional importance of T207 and Y210, but not T198 in regulating ERK1 catalytic activity. By single substitution of the T207 to alanine, the activity of ERK1 towards MBP decreased dramatically without affecting the phosphorylation on TEY motif by MEK1. An additional mutation at Y210 caused the loss of most ERK1 phosphotransferase activity. However, neither of the T207E nor Y210E mutants had the ability to mimic the phosphorylation state and produce the full activation of ERK1. In addition, Y210 could be important for ERK1 to be recognized by MEK1, since the substitution of this residue blocked most of the phosphorylation on TEY sites by this MAPK kinase. Preliminary data also indicated the possibility of autophosphorylation on T207 and Y210, which probably happens after the phosphorylation of the TEY sites by MEK1. The phosphorylation of the flanking sites may help to stabilize the active state of the kinase. Our findings contribute to an improved understanding of MAPK activation and regulation. Hyper-phosphorylation within the kinase activation loop may serve as a general mechanism for protein kinases to achieve full stimulation by autophosphorylation following the initial activation by other upstream kinases. Citation Format: Shenshen Lai, Steven Pelech. Regulatory roles of conserved phosphorylation sites in the activation loop of extracellular signal-regulated kinase 1. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5154. doi:10.1158/1538-7445.AM2013-5154
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How this classification was reachedexpand
Full frame machine prediction
Teacher imitationNot calibrated prevalence, not ground truth. Human validation pending. The Gemma side is a direct model label for every work in the frame, read from the title-only record. The Codex side is a classifier learned from the 10,348 direct Codex labels and calibrated to design-weighted sample rates; fields without enough sample support carry no Codex call. Candidate is the union of the two sides; consensus is their intersection. These outputs are machine_predicted_unvalidated and are not human labels.
Distilled classifier scores by category (both heads)
| 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) | 0.004 | 0.002 |
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 source (direct Gemma or distilled Codex), 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".