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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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Subcellular localization of MK2 and MK2/mk5 hybrids. a EGFP-MK2 resides predominantly in the nucleus of untreated cells and this subcellular localization is not changed upon activation of the cAMP/PKA pathway by forskolin. Cells were treated with 50 μM forskolin (FSK) for 30 min. b Alignment of the region spanning Ser-115 in MK5 with the corresponding sequence in MK2. The insertion of Asp (d) is highlighted in grey. c Insertion of a phosphomimicking Asp in MK2 that corresponds to Ser-115 in MK5 does not affect the subcellular localization of this MK2insD mutant. d Swapping the MK2 domain with the MK5 motif encompassing Ser-115 changes the subcellular localization of the MK2 from almost exclusively nuclear to both cytoplasmic and nuclear for the MK2/mk5 hybrid protein (top panels). Forskolin triggers nuclear exclusion of MK2/mk5 (middle panels). The MK2/mk5 S115D mutant resides predominantly in the cytoplasm of untreated cells (bottom panels). The EGFP fusion proteins were 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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Fig5: Subcellular localization of MK2 and MK2/mk5 hybrids. a EGFP-MK2 resides predominantly in the nucleus of untreated cells and this subcellular localization is not changed upon activation of the cAMP/PKA pathway by forskolin. Cells were treated with 50 μM forskolin (FSK) for 30 min. b Alignment of the region spanning Ser-115 in MK5 with the corresponding sequence in MK2. The insertion of Asp (d) is highlighted in grey. c Insertion of a phosphomimicking Asp in MK2 that corresponds to Ser-115 in MK5 does not affect the subcellular localization of this MK2insD mutant. d Swapping the MK2 domain with the MK5 motif encompassing Ser-115 changes the subcellular localization of the MK2 from almost exclusively nuclear to both cytoplasmic and nuclear for the MK2/mk5 hybrid protein (top panels). Forskolin triggers nuclear exclusion of MK2/mk5 (middle panels). The MK2/mk5 S115D mutant resides predominantly in the cytoplasm of untreated cells (bottom panels). The EGFP fusion proteins were 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: Both MK2 and MK5 reside predominantly in the nucleus of untreated cells [19, 22, 27, 35]. However, we have previously shown that PKA does not interact with nor phosphorylate MK2 [22]. Moreover, we found that forskolin did not induce nuclear export of the EGFP-MK2 fusion protein (Fig. 5a). Interestingly, Ser-115 in MK5 is encompassed in the motif RISQ, while the corresponding sequence in MK2 and MK3 is RIQ, lacking an equivalent of Ser-115 (Fig. 5b). To evaluate the importance of this Ser-115 in PKA-induced nuclear export of MK5, we reasoned that a MK2 mutant with an insertion of Asp as a phosphomimicking Ser-115 in the RIQ motif (= MK2insD) may behave identically to MK5 S115D, i.e. with both nuclear and cytoplasmic localizations in untreated cells. Contrary to MK5 S115D, the EGFP-MK2insD mutant remained exclusively in the nucleus of HeLa cells (Fig. 5c) and HEK293 cells (results not shown). Alignment of human and mouse MK5, MK2, and MK3 revealed differences in residues C-terminal to Ser-115 (Fig. 5b). To examine the possible contribution of these residues in PKA-triggered nuclear export, we constructed chimeric MK2 containing the wild-type MK5, and MK5 S115D sequences, to generate the fusion proteins EGFP-MK2/mk5 and EGFP-MK2/mk5 S115D, respectively. EGFP-MK2/mk5 S115D behaved as EGFP-MK5 S115D because it was detected in both the nucleus and cytoplasm of untreated cells (Fig. 5d). Surprisingly, EGFP-MK2/mk5 was found in both the nucleus and the cytoplasm, but was most obvious in the cytoplasm. However, forskolin treatment caused complete nuclear exclusion of EGFP-MK2/mk5 (Fig. 5d). Taken together, these results indicate that Ser-115 is necessary but not sufficient to control subcellular distribution of MK5 in response to an activated PKA pathway. Amino acids C-terminal to Ser-115 also contribute to PKA-regulated subcellular distribution of MK5.Fig. 5


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)

Subcellular localization of MK2 and MK2/mk5 hybrids. a EGFP-MK2 resides predominantly in the nucleus of untreated cells and this subcellular localization is not changed upon activation of the cAMP/PKA pathway by forskolin. Cells were treated with 50 μM forskolin (FSK) for 30 min. b Alignment of the region spanning Ser-115 in MK5 with the corresponding sequence in MK2. The insertion of Asp (d) is highlighted in grey. c Insertion of a phosphomimicking Asp in MK2 that corresponds to Ser-115 in MK5 does not affect the subcellular localization of this MK2insD mutant. d Swapping the MK2 domain with the MK5 motif encompassing Ser-115 changes the subcellular localization of the MK2 from almost exclusively nuclear to both cytoplasmic and nuclear for the MK2/mk5 hybrid protein (top panels). Forskolin triggers nuclear exclusion of MK2/mk5 (middle panels). The MK2/mk5 S115D mutant resides predominantly in the cytoplasm of untreated cells (bottom panels). The EGFP fusion proteins were 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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Fig5: Subcellular localization of MK2 and MK2/mk5 hybrids. a EGFP-MK2 resides predominantly in the nucleus of untreated cells and this subcellular localization is not changed upon activation of the cAMP/PKA pathway by forskolin. Cells were treated with 50 μM forskolin (FSK) for 30 min. b Alignment of the region spanning Ser-115 in MK5 with the corresponding sequence in MK2. The insertion of Asp (d) is highlighted in grey. c Insertion of a phosphomimicking Asp in MK2 that corresponds to Ser-115 in MK5 does not affect the subcellular localization of this MK2insD mutant. d Swapping the MK2 domain with the MK5 motif encompassing Ser-115 changes the subcellular localization of the MK2 from almost exclusively nuclear to both cytoplasmic and nuclear for the MK2/mk5 hybrid protein (top panels). Forskolin triggers nuclear exclusion of MK2/mk5 (middle panels). The MK2/mk5 S115D mutant resides predominantly in the cytoplasm of untreated cells (bottom panels). The EGFP fusion proteins were 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: Both MK2 and MK5 reside predominantly in the nucleus of untreated cells [19, 22, 27, 35]. However, we have previously shown that PKA does not interact with nor phosphorylate MK2 [22]. Moreover, we found that forskolin did not induce nuclear export of the EGFP-MK2 fusion protein (Fig. 5a). Interestingly, Ser-115 in MK5 is encompassed in the motif RISQ, while the corresponding sequence in MK2 and MK3 is RIQ, lacking an equivalent of Ser-115 (Fig. 5b). To evaluate the importance of this Ser-115 in PKA-induced nuclear export of MK5, we reasoned that a MK2 mutant with an insertion of Asp as a phosphomimicking Ser-115 in the RIQ motif (= MK2insD) may behave identically to MK5 S115D, i.e. with both nuclear and cytoplasmic localizations in untreated cells. Contrary to MK5 S115D, the EGFP-MK2insD mutant remained exclusively in the nucleus of HeLa cells (Fig. 5c) and HEK293 cells (results not shown). Alignment of human and mouse MK5, MK2, and MK3 revealed differences in residues C-terminal to Ser-115 (Fig. 5b). To examine the possible contribution of these residues in PKA-triggered nuclear export, we constructed chimeric MK2 containing the wild-type MK5, and MK5 S115D sequences, to generate the fusion proteins EGFP-MK2/mk5 and EGFP-MK2/mk5 S115D, respectively. EGFP-MK2/mk5 S115D behaved as EGFP-MK5 S115D because it was detected in both the nucleus and cytoplasm of untreated cells (Fig. 5d). Surprisingly, EGFP-MK2/mk5 was found in both the nucleus and the cytoplasm, but was most obvious in the cytoplasm. However, forskolin treatment caused complete nuclear exclusion of EGFP-MK2/mk5 (Fig. 5d). Taken together, these results indicate that Ser-115 is necessary but not sufficient to control subcellular distribution of MK5 in response to an activated PKA pathway. Amino acids C-terminal to Ser-115 also contribute to PKA-regulated subcellular distribution of MK5.Fig. 5

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