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Characterization of the expression and regulation of MK5 in the murine ventricular myocardium
Dharmendra Dingar1, Marie-Josée Benoit, Aida M Mamarbachi
1Montreal Heart Institute, 5000 Belanger St., Montréal, Québec, Canada.
Abstract:
MK5, a member of the MAPK-activated protein kinase family, is highly expressed in the heart. Whereas MK2 and MK3 are activated by p38 MAPK, MK5 has also been shown to be activated by ERK3 and ERK4. We studied the regulation of MK5 in mouse heart. mRNA for 5 splice variants (MK5.1-5.5), including the original form (MK5.1), was detected. MK5 comprises 14 exons: exon 12 splicing was modified in MK5.2, MK5.3, and MK5.5. MK5.2 and MK5.5 lacked 6 bases at the 3'-end of exon 12, whereas MK5.3 lacked exon 12, resulting in a frame shift and premature termination of translation at codon 3 of exon 13. MK5.4 and MK5.5 lacked exons 2-6, encoding kinase subdomains I-VI, and were kinase-dead. All 5 MK5 variants were detected at the mRNA level in all mouse tissues examined; however, their relative abundance was tissue-specific. Furthermore, the relative abundance of variant mRNA was altered both during hypertrophy and postnatal cardiac development, suggesting that the generation or the stability of MK5 variant mRNAs is subject to regulation. When expressed in HEK293 cells, MK5.1, MK5.2 and MK5.3 were nuclear whereas MK5.4 and MK5.5 were cytoplasmic. A p38 MAPK activator, anisomycin, induced the redistribution of each variant. In contrast, MK5 co-immunoprecipitated ERK3, but not ERK4 or p38 alpha, in control and hypertrophying hearts. GST pull-down assays revealed unbound ERK4 and p38 alpha but no free MK5 or ERK3 in heart lysates. Hence, 1) in heart MK5 complexes with ERK3 and 2) MK5 splice variants may mediate distinct effects thus increasing the functional diversity of ERK3-MK5 signaling.
Insights
This study reveals five MK5 splice variants in mouse hearts, with their expression and localization regulated by development and hypertrophy. MK5 forms complexes with ERK3, suggesting diverse signaling roles.
Area of Science:
- Molecular Biology
- Cardiovascular Research
- Signal Transduction
Background:
- Mitogen-activated protein kinase kinase 5 (MK5) is a key enzyme in the MAPK-activated protein kinase family, highly expressed in cardiac tissue.
- While MK2 and MK3 are activated by p38 MAPK, MK5 is also activated by ERK3 and ERK4, indicating complex regulatory pathways.
- Understanding MK5 regulation is crucial for comprehending cardiac function and disease.
Purpose of the Study:
- To investigate the regulation of MK5 in mouse hearts, focusing on its splice variants and interactions.
- To determine the tissue-specific expression and developmental changes of MK5 splice variants.
- To elucidate the role of ERK3 and p38 MAPK in MK5 activation and localization within the heart.
Main Methods:
- Analysis of mRNA expression for five MK5 splice variants (MK5.1-5.5) in mouse tissues using RT-PCR.
- Investigation of variant-specific localization in HEK293 cells and response to p38 MAPK activation.
- Co-immunoprecipitation and GST pull-down assays to identify protein-protein interactions in heart lysates.
Main Results:
- Five MK5 splice variants were detected, with differential mRNA abundance across tissues and altered levels during cardiac development and hypertrophy.
- MK5 variants exhibited distinct subcellular localizations (nuclear vs. cytoplasmic), which were modulated by p38 MAPK activation.
- MK5 was found to complex with ERK3 in heart lysates, but not with ERK4 or p38 alpha, suggesting a specific interaction.
Conclusions:
- MK5 splice variants contribute to functional diversity within the ERK3-MK5 signaling pathway in the heart.
- The regulation of MK5 variant generation and stability is influenced by cardiac development and hypertrophic conditions.
- Specific interactions between MK5 and ERK3 highlight a critical signaling axis in cardiac physiology.
