Transcriptional, Post‐transcriptional, and Post‐translational Regulation of MK5 in Cardiac Cells
Bibliographic record
Abstract
Background MAP kinase‐activated protein kinase‐5 (MK5) is a protein serine/threonine kinase identified as a putative substrate of both p38α/β MAPKs and atypical MAPKs ERK3 and ERK4. MK5 mRNA is detected in adult cardiac ventricular fibroblasts and myocytes whereas MK5 immunoreactivity is detected in fibroblasts but not myocytes.Little is known about the molecular mechanisms regulating MK5 in the heart. This study was to investigate the regulation of MK5 activity in cardiac fibroblasts and to determine if pro‐hypertrophic agonists such as angiotensin II (Ang II), endothelin 1 (ET1) and norepinephrine (NE) stimulate the translation of MK5 in cardiac myocytes. Methods Cardiac ventricular fibroblasts and myocytes were isolated from MK5 +/+ mice and Sprague‐Dawley rats. MK5 was visualized by immunoblot assay following separation by SDS‐PAGE, 2‐dimensional gel electrophoresis, and Phos‐tag PAGE. mRNA abundance was determined by droplet digital PCR and qPCR. The subcellular localization of ERK3 and MK5 was determined by confocal immunocytofluorescence. Results The copy number of MK5 mRNA was similar in both adult cardiac fibroblasts and myocytes. However, the relative expression of MK5 mRNA splice variants differed between the cell types. In both, MK5.1 was the most abundant variant whereas MK5.2 was the least. MK5.2+MK5.5 represented about 35.9% of total MK5 mRNA in cardiac fibroblasts as compared to 22.9% in myocytes. However, the variant MK5.3 was 0.1% in fibroblasts as compared to 1.4% in myocytes. Considering the variants MK5.4+MK5.5, it was 2.9% in fibroblasts and 5.7% in the myocyte. In cardiac myocytes, inhibition of the proteasome inhibition using MG132 failed to rescue MK5 immunoreactivity. Similarly, incubation with hypertrophic agonists Ang II, ET1, or NE did not result in detection of MK5 immunoreactivity. In serum‐starved cardiac myofibroblasts, MK5 immunoreactivity was primarily localized to the nucleus. Activation of MK5 involves phosphorylation at threonine‐182 (Thr182). Following serum stimulation, phospho‐MK5(Thr182) immunoreactivity was observed in the cytoplasm: phospho‐MK5(Thr182)immunoreactivity appeared to be associated with the cytoskeleton and pseudopodia. No phospho‐MK5(Thr182) immunoreactivity was detected in serum‐stimulated myofibroblasts following incubation with an inhibitor of p38αβMAPK activity, SB203580. In actively dividing fibroblasts, Phos‐tag PAGE in the presence of Mn 2+ , but not in the presence of EDTA, revealed multiple phospho‐forms of MK5. In addition, the Phos‐tag profile of MK5 immunoreactivity in mouse ventricular fibroblasts differed from that of rat fibroblasts. Conclusions 1) The absence of MK5 immunoreactivity in cardiac ventricular myocytes was not due to rapid degradation by the proteasome, indicating differences in post‐transcriptional regulation of MK5 expression involve cell type‐specific translation. 2) In cardiac ventricular fibroblasts, phosphorylation of MK5 at threonine‐182 in response to serum stimulation requires p38a/bactivity. 3) Threonine‐182 is not the only site at which MK5 is phosphorylated in vivo. Support or Funding Information This study was supported by a Grant‐In‐Aid from the Heart and Stroke Foundation of Canada.
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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) | 0.001 | 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 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".