Characterization of a novel MK3 splice variant from murine ventricular myocardium
| Autor: | Nada Farhat, Louis Villeneuve, Aida M. Mamarbachi, Dharmendra Dingar, Nadège Moïse, Bruce G. Allen, Matthias Gaestel, Maya Khairallah |
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| Rok vydání: | 2010 |
| Předmět: |
MAPK/ERK pathway
Heart Ventricles p38 mitogen-activated protein kinases Molecular Sequence Data Protein Serine-Threonine Kinases p38 Mitogen-Activated Protein Kinases Article Mice 03 medical and health sciences Animals Amino Acid Sequence Cloning Molecular Extracellular Signal-Regulated MAP Kinases Protein kinase A 030304 developmental biology 0303 health sciences Messenger RNA biology Myocardium 030302 biochemistry & molecular biology Alternative splicing Intracellular Signaling Peptides and Proteins Cell Biology Molecular biology Recombinant Proteins Alternative Splicing Cytoplasm Mitogen-activated protein kinase biology.protein Phosphorylation |
| Zdroj: | Cellular Signalling. 22:1502-1512 |
| ISSN: | 0898-6568 |
| DOI: | 10.1016/j.cellsig.2010.05.019 |
| Popis: | p38 MAP kinase (MAPK) isoforms alpha, beta, and gamma, are expressed in the heart. p38alpha appears pro-apoptotic whereas p38beta is pro-hypertrophic. The mechanisms mediating these divergent effects are unknown; hence elucidating the downstream signaling of p38 should further our understanding. Downstream effectors include MAPK-activated protein kinase (MK)-3, which is expressed in many tissues including skeletal muscles and heart. We cloned full-length MK3 (MK3.1, 384 aa) and a novel splice variant (MK3.2, 266 aa) from murine heart. For MK3.2, skipping of exons 8 and 9 resulted in a frame-shift in translation of the first 85 base pairs of exon 10 followed by an in-frame stop codon. Of 3 putative phosphorylation sites for p38 MAPK, only Thr-203 remained functional in MK3.2. In addition, MK3.2 lacked nuclear localization and export signals. Quantitative real-time PCR confirmed the presence of these mRNA species in heart and skeletal muscle; however, the relative abundance of MK3.2 differed. Furthermore, whereas total MK3 mRNA was increased, the relative abundance of MK3.2 mRNA decreased in MK2(-/-) mice. Immunoblotting revealed 2 bands of MK3 immunoreactivity in ventricular lysates. Ectopically expressed MK3.1 localized to the nucleus whereas MK3.2 was distributed throughout the cell; however, whereas MK3.1 translocated to the cytoplasm in response to osmotic stress, MK3.2 was degraded. The p38alpha/beta inhibitor SB203580 prevented the degradation of MK3.2. Furthermore, replacing Thr-203 with alanine prevented the loss of MK3.2 following osmotic stress, as did pretreatment with the proteosome inhibitor MG132. In vitro, GST-MK3.1 was strongly phosphorylated by p38alpha and p38beta, but a poor substrate for p38delta and p38gamma. GST-MK3.2 was poorly phosphorylated by p38alpha and p38beta and not phosphorylated by p38delta and p38gamma. Hence, differential regulation of MKs may, in part, explain diverse downstream effects mediated by p38 signaling. |
| Databáze: | OpenAIRE |
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