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Serine residue 115 of MAPK-activated protein kinase MK5 is crucial for its PKA-regulated nuclear export and biological function.

Kostenko S, Shiryaev A, Gerits N, Dumitriu G, Klenow H, Johannessen M, Moens U - Cell. Mol. Life Sci. (2010)

Bottom Line: While p38(MAPK), ERK3 and ERK4 fail to trigger nuclear export of the kinase dead T182A and K51E MK5 mutants, S115D/T182A and K51E/S115D mutants were able to enter the cytoplasm of resting cells.Finally, we demonstrated that mutations in Ser-115 affect the biological properties of MK5.Taken together, our results suggest that Ser-115 plays an essential role in PKA-regulated nuclear export of MK5, and that it also may regulate the biological functions of MK5.

View Article: PubMed Central - PubMed

Affiliation: Faculty of Health Sciences, Institute of Medical Biology, University of Tromsø, Tromsø, Norway.

ABSTRACT
The mitogen-activated protein kinase-activated protein kinase-5 (MK5) resides predominantly in the nucleus of resting cells, but p38(MAPK), extracellular signal-regulated kinases-3 and -4 (ERK3 and ERK4), and protein kinase A (PKA) induce nucleocytoplasmic redistribution of MK5. The mechanism by which PKA causes nuclear export remains unsolved. In the study reported here we demonstrated that Ser-115 is an in vitro PKA phosphoacceptor site, and that PKA, but not p38(MAPK), ERK3 or ERK4, is unable to redistribute MK5 S115A to the cytoplasm. However, the phospho-mimicking MK5 S115D mutant resides in the cytoplasm in untreated cells. While p38(MAPK), ERK3 and ERK4 fail to trigger nuclear export of the kinase dead T182A and K51E MK5 mutants, S115D/T182A and K51E/S115D mutants were able to enter the cytoplasm of resting cells. Finally, we demonstrated that mutations in Ser-115 affect the biological properties of MK5. Taken together, our results suggest that Ser-115 plays an essential role in PKA-regulated nuclear export of MK5, and that it also may regulate the biological functions of MK5.

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Ser-115 is not implicated in p38MAPK-induced nuclear export of MK5. a HeLa cells transfected with EGFP-MK5 or EGFP-MK5 S115A plasmids were left untreated or exposed to sodium arsenite (250 μM for 120 min). b As in a, but cells were contransfected with plasmids encoding activated MKK6 (MKK6 E/E) and p38MAPK. The subcellular localization of the ectopically expressed MK5 variants was visualized as described in the legend for Fig. 3. On average 50 cells expressing EGFP-MK5 were analysed and representative images are shown. Similar results were obtained in independent experiments
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Fig8: Ser-115 is not implicated in p38MAPK-induced nuclear export of MK5. a HeLa cells transfected with EGFP-MK5 or EGFP-MK5 S115A plasmids were left untreated or exposed to sodium arsenite (250 μM for 120 min). b As in a, but cells were contransfected with plasmids encoding activated MKK6 (MKK6 E/E) and p38MAPK. The subcellular localization of the ectopically expressed MK5 variants was visualized as described in the legend for Fig. 3. On average 50 cells expressing EGFP-MK5 were analysed and representative images are shown. Similar results were obtained in independent experiments

Mentions: Studies by us and others have shown that stimuli that activate the p38MAPK pathway or activated p38MAPK mutants trigger cytoplasmic redistribution of MK5 [19, 24, 30]. This encouraged us to investigate whether the phosphorylation state of Ser-115 can interfere with p38MAPK-mediated subcellular localization of MK5. Exposure of cells to sodium arsenite, a potent activator of the p38MAPK pathway, resulted in nuclear export of EGFP-MK5 S115A (Fig. 8a). Similarly, coexpression of the constitutive active MKK6 E/E mutant and p38MAPK also caused an increase in cytoplasmic residence of EGFP-MK5 S115A (Fig. 8b). These findings strongly suggest that p38MAPK-regulated subcellular localization of MK5 is independent of Ser-115 phosphorylation.Fig. 8


Serine residue 115 of MAPK-activated protein kinase MK5 is crucial for its PKA-regulated nuclear export and biological function.

Kostenko S, Shiryaev A, Gerits N, Dumitriu G, Klenow H, Johannessen M, Moens U - Cell. Mol. Life Sci. (2010)

Ser-115 is not implicated in p38MAPK-induced nuclear export of MK5. a HeLa cells transfected with EGFP-MK5 or EGFP-MK5 S115A plasmids were left untreated or exposed to sodium arsenite (250 μM for 120 min). b As in a, but cells were contransfected with plasmids encoding activated MKK6 (MKK6 E/E) and p38MAPK. The subcellular localization of the ectopically expressed MK5 variants was visualized as described in the legend for Fig. 3. On average 50 cells expressing EGFP-MK5 were analysed and representative images are shown. Similar results were obtained in independent experiments
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Related In: Results  -  Collection

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Fig8: Ser-115 is not implicated in p38MAPK-induced nuclear export of MK5. a HeLa cells transfected with EGFP-MK5 or EGFP-MK5 S115A plasmids were left untreated or exposed to sodium arsenite (250 μM for 120 min). b As in a, but cells were contransfected with plasmids encoding activated MKK6 (MKK6 E/E) and p38MAPK. The subcellular localization of the ectopically expressed MK5 variants was visualized as described in the legend for Fig. 3. On average 50 cells expressing EGFP-MK5 were analysed and representative images are shown. Similar results were obtained in independent experiments
Mentions: Studies by us and others have shown that stimuli that activate the p38MAPK pathway or activated p38MAPK mutants trigger cytoplasmic redistribution of MK5 [19, 24, 30]. This encouraged us to investigate whether the phosphorylation state of Ser-115 can interfere with p38MAPK-mediated subcellular localization of MK5. Exposure of cells to sodium arsenite, a potent activator of the p38MAPK pathway, resulted in nuclear export of EGFP-MK5 S115A (Fig. 8a). Similarly, coexpression of the constitutive active MKK6 E/E mutant and p38MAPK also caused an increase in cytoplasmic residence of EGFP-MK5 S115A (Fig. 8b). These findings strongly suggest that p38MAPK-regulated subcellular localization of MK5 is independent of Ser-115 phosphorylation.Fig. 8

Bottom Line: While p38(MAPK), ERK3 and ERK4 fail to trigger nuclear export of the kinase dead T182A and K51E MK5 mutants, S115D/T182A and K51E/S115D mutants were able to enter the cytoplasm of resting cells.Finally, we demonstrated that mutations in Ser-115 affect the biological properties of MK5.Taken together, our results suggest that Ser-115 plays an essential role in PKA-regulated nuclear export of MK5, and that it also may regulate the biological functions of MK5.

View Article: PubMed Central - PubMed

Affiliation: Faculty of Health Sciences, Institute of Medical Biology, University of Tromsø, Tromsø, Norway.

ABSTRACT
The mitogen-activated protein kinase-activated protein kinase-5 (MK5) resides predominantly in the nucleus of resting cells, but p38(MAPK), extracellular signal-regulated kinases-3 and -4 (ERK3 and ERK4), and protein kinase A (PKA) induce nucleocytoplasmic redistribution of MK5. The mechanism by which PKA causes nuclear export remains unsolved. In the study reported here we demonstrated that Ser-115 is an in vitro PKA phosphoacceptor site, and that PKA, but not p38(MAPK), ERK3 or ERK4, is unable to redistribute MK5 S115A to the cytoplasm. However, the phospho-mimicking MK5 S115D mutant resides in the cytoplasm in untreated cells. While p38(MAPK), ERK3 and ERK4 fail to trigger nuclear export of the kinase dead T182A and K51E MK5 mutants, S115D/T182A and K51E/S115D mutants were able to enter the cytoplasm of resting cells. Finally, we demonstrated that mutations in Ser-115 affect the biological properties of MK5. Taken together, our results suggest that Ser-115 plays an essential role in PKA-regulated nuclear export of MK5, and that it also may regulate the biological functions of MK5.

Show MeSH