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Characterization of a novel MK3 splice variant from murine ventricular myocardium
Nadège Moïse1, Dharmendra Dingar, Aida M Mamarbachi
1Montreal Heart Institute, Université de Montréal, Montreal, Quebec, Canada H3C 3J7.
Abstract:
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.
Insights
Differential regulation of MAPK-activated protein kinase (MK)-3 isoforms by p38 MAP kinase (MAPK) signaling may explain distinct cellular responses. This study identifies a novel MK3 splice variant (MK3.2) with altered signaling properties.
Area of Science:
- Cellular signaling
- Molecular biology
- Cardiovascular research
Background:
- p38 MAP kinase (MAPK) signaling pathways are crucial in cardiac function, with isoforms like p38alpha promoting apoptosis and p38beta promoting hypertrophy.
- The downstream effectors and mechanisms responsible for these divergent p38 MAPK effects remain largely unknown.
- MAPK-activated protein kinase (MK)-3 is a downstream target of p38 MAPK, expressed in cardiac and skeletal muscle.
Purpose of the Study:
- To investigate the downstream signaling of p38 MAPK by characterizing MK-3 isoforms and their regulation.
- To identify and functionally analyze novel splice variants of MK-3 in the murine heart.
- To elucidate how differential regulation of MK-3 isoforms contributes to the diverse cellular outcomes mediated by p38 MAPK.
Main Methods:
- Cloning and characterization of full-length MK3 (MK3.1) and a novel splice variant (MK3.2) from murine heart.
- Quantitative real-time PCR to assess mRNA expression of MK3 isoforms in different tissues and mouse models.
- Immunoblotting, ectopic expression studies, and in vitro kinase assays to analyze MK3 protein localization, stability, and phosphorylation by p38 MAPK isoforms.
Main Results:
- A novel MK3 splice variant, MK3.2, was identified, differing from MK3.1 by the skipping of exons 8 and 9, resulting in altered phosphorylation sites and lack of nuclear localization signals.
- MK3.1 localized to the nucleus and translocated upon osmotic stress, while MK3.2 localized throughout the cell and was degraded, a process inhibited by p38alpha/beta inhibitors and proteasome inhibitors.
- MK3.1 was efficiently phosphorylated by p38alpha and p38beta, whereas MK3.2 was poorly phosphorylated by these isoforms, and not by p38delta or p38gamma.
Conclusions:
- Differential expression and regulation of MK3 isoforms (MK3.1 and MK3.2) by p38 MAPK are demonstrated.
- The novel MK3.2 variant exhibits distinct cellular localization and stability compared to MK3.1, influenced by p38 MAPK activity and phosphorylation.
- These findings suggest that the differential signaling and regulation of MK3 isoforms contribute to the diverse cellular effects mediated by p38 MAPK pathways in the heart and other tissues.
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