Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2011 Apr;15(4):928-37.
doi: 10.1111/j.1582-4934.2010.01093.x.

Mitogen activated protein kinase at the nuclear pore complex

Affiliations

Mitogen activated protein kinase at the nuclear pore complex

Randolph S Faustino et al. J Cell Mol Med. 2011 Apr.

Abstract

Mitogen activated protein (MAP) kinases control eukaryotic proliferation, and import of kinases into the nucleus through the nuclear pore complex (NPC) can influence gene expression to affect cellular growth, cell viability and homeostatic function. The NPC is a critical regulatory checkpoint for nucleocytoplasmic traffic that regulates gene expression and cell growth, and MAP kinases may be physically associated with the NPC to modulate transport. In the present study, highly enriched NPC fractions were isolated and investigated for associated kinases and/or activity. Endogenous kinase activity was identified within the NPC fraction, which phosphorylated a 30 kD nuclear pore protein. Phosphomodification of this nucleoporin, here termed Nup30, was inhibited by apigenin and PD-98059, two MAP kinase antagonists as well as with SB-202190, a pharmacological blocker of p38. Furthermore, high throughput profiling of enriched NPCs revealed constitutive presence of all members of the MAP kinase family, extracellular regulated kinases (ERK), p38 and Jun N-terminal kinase. The NPC thus contains a spectrum of associated MAP kinases that suggests an intimate role for ERK and p38 in regulation of nuclear pore function.

PubMed Disclaimer

Figures

Fig 1
Fig 1
ERK distribution before and after growth stimulation. Cultured smooth muscle cells were stained to visualize the distribution of MAP kinases within quiescent and proliferating smooth muscle cell. (A) Quiescent cells exhibited diffuse, cytosolic localization of non-activated ERK-2 that was concentrated within the nucleus in proliferating cells (B). Activated ERK-2 demonstrated a distribution pattern reminiscent of actin staining in quiescent cells (C) and with robust nuclear localization upon proliferation (D). Images representative of results obtained from sample size of n= 3.
Fig 2
Fig 2
Broad phosphoprofile of putative ERK substrates in quiescent and proliferating smooth muscle cells. Smooth muscle cells were stained using antibodies against phosphoserine and phosphothreonine. (A) In quiescent cells, phosphoserine containing proteins localized primarily to the nucleus with faint cytosolic staining. (B) Proliferation caused moderate increases in cytosolic distribution of phosphoserine, but maintained nuclear presence. (C) Quiescent cells stained for phosphothreonine diffuse throughout cytosolic and nuclear compartments, but exhibits even distribution among both in proliferating cells (D). Images representative of results obtained from sample size of n= 3.
Fig 3
Fig 3
Isolation of a purified NPC fraction. (A) NPC isolation workflow, illustrating sequential enrichment procedure. S1–S4 indicate supernatant fractions. (B) Fractions from the NPC isolation procedure were loaded onto SDS-PAGE (50 μg per lane) and immunoblotted using mAb414. Crude nuclei (N) show staining of p62 (arrow), a nucleoporin recognized by mAb414, with subsequent enrichment in the final NPC-rich fraction (eNPC). Lane assignments: N: crude nuclei; S1: supernatant from nuclease digested nuclei; S2: supernatant from detergent extracted nuclei; S3: supernatant from first salt wash; S4: supernatant from second salt wash; eNPC: purified NPC/lamina fraction. Numbers to the right indicate molecular mass markers in kD. (C) Removal of lamins from NPC fraction. NPC fractions were immunodepleted of nuclear lamins prior to use in the phosphorylation assays. Illustrated are representative immunoblots of NPC fractions that were depleted, then probed for lamins A/C or B1. In both cases, lamins were effectively removed. Pre-immunodepletion samples were loaded on the right for comparison. NPC: nuclear pore complex; iNPC: immunodepleted NPC.
Fig 4
Fig 4
Calcium-independent endogenous phosphorylation of enriched NPC fraction. The purified NPC fraction was incubated in kinase reaction buffer in the presence or absence of 5 mM calcium for different times before stopping with 2% SDS. Typical reaction blots are shown in (A). Endogenous phosphorylation of a 30 kD protein within the purified NPC fraction does not show any calcium dependency. Incubation of the reaction mixture in the presence of 5 mM EGTA, a chelating agent, does not show any attenuation in phosphorylation after 30 min. The results from several independent experiments are shown in (B) (n= 4).
Fig 5
Fig 5
Endogenous phosphorylation antagonized by MAP kinase inhibitors. Various pharmacological antagonists were incubated with the final, purified NPC fraction in the presence and absence of EGTA. Endogenous phosphorylation of a protein within the NPC fraction was significantly inhibited by apigenin, a broad range MAP kinase inhibitor. Controls contained no inhibitor and consisted solely of purified NPCs. Inhibitors used – AIP (30 nM); CKI (40 μM), apigenin (50 μM), PD-98059 (40 μM). Results representative of independent samples (n= 3).
Fig 6
Fig 6
Dose-dependent inhibition of endogenous MAP kinase activity. The 30 kD protein within the NPC fraction was inhibited in a concentration-dependent manner using increasing amounts of apigenin. Molecular weight is given on the side in kD. Below: Graphical analysis of a number of experiments (n= 3) reveals potent inhibition of endogenous kinase activity with increasing doses. Data were reported as mean ± S.E.M. *P < 0.05 versus control. Controls were taken to be band intensity of the protein in the absence of apigenin.
Fig 7
Fig 7
Involvement of p38 MAP kinase. (A) Activated p38 showed diffuse cytosolic, and speckled nuclear, staining in quiescent cells (upper panel) which localized the nucleus in proliferating cells (lower panel). (B) Dose-dependent inhibition of endogenous NPC phosphorylation by SB-202190. Incubation of phosphorylation reaction mixture with SB-202190 attenuated endogenous phosphorylation of the 30 kD band in the NPC fraction. Data were reported as mean ± S.E.M. from several experiments (n= 3). *P < 0.05 versus control. Controls were taken to be phosphorylation intensity of protein in the absence of SB-202190.

Similar articles

Cited by

References

    1. Pearson G, Robinson F, Gibson TB, et al. Mitogen-activated protein (MAP) kinase pathways: regulation and physiological functions. Endocr Rev. 2001;22:153–83. - PubMed
    1. Buehr M, Smith A. Genesis of embryonic stem cells. Philos Trans R Soc Lond B Biol Sci. 2003;358:1397–402. - PMC - PubMed
    1. Yao Y, Li W, Wu J, et al. Extracellular signal-regulated kinase 2 is necessary for mesoderm differentiation. Proc Natl Acad Sci USA. 2003;100:12759–64. - PMC - PubMed
    1. Burdon T, Smith A, Savatier P. Signalling, cell cycle and pluripotency in embryonic stem cells. Trends Cell Biol. 2002;12:432–8. - PubMed
    1. Jirmanova L, Afanassieff M, Gobert-Gosse S, et al. Differential contributions of ERK and PI3-kinase to the regulation of cyclin D1 expression and to the control of the G1/S transition in mouse embryonic stem cells. Oncogene. 2002;21:5515–28. - PubMed

Publication types

MeSH terms

Substances